“De discussie van het nut van fundamenteel onderzoek …€œDe discussie van het nut van...

260
“De discussie van het nut van fundamenteel onderzoek is er één van alle tijden. Het waren niet voor niets monniken en kluizenaars die zich aan de wetenschap wijdden. De meeste mensen hadden letterlijk wel wat beters te doen.” Gerard ’t Hooft Nobelprijswinnaar Natuurkunde 1999 Voor Katrien en Helene, De vrouwen van mijn leven

Transcript of “De discussie van het nut van fundamenteel onderzoek …€œDe discussie van het nut van...

“De discussie van het nut van fundamenteel onderzoek

is er één van alle tijden.

Het waren niet voor niets monniken en kluizenaars

die zich aan de wetenschap wijdden.

De meeste mensen hadden letterlijk wel

wat beters te doen.”

Gerard ’t Hooft

Nobelprijswinnaar Natuurkunde 1999

Voor Katrien en Helene, De vrouwen van mijn leven

Metabolic changes in high producing dairy cows and the consequences on oocyte and embryo quality. Jo Leo Moniek René LEROY, Doctor in Veterinary Medicine Funding: This research was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen), grant n° 1236. Cover: “Wie staat daar naar mij te kijken?” Foto Sam Miller Printing: Plot-it, Merelbeke

ISBN: 90-5864-083-3

EAN: 9789058640833

Metabolic changes in high producing dairy cows and

the consequences on oocyte and embryo quality

De gevolgen van de metabole veranderingen bij hoogproductieve melkkoeien voor de eicel- en embryokwaliteit

(Met een Nederlandstalige samenvatting)

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Diergeneeskundige Wetenschappen aan de Faculteit Diergeneeskunde, Universiteit Gent,

Maandag 21 november, 2005

door

Jo LMR Leroy

Vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde Faculteit Diergeneeskunde

Universiteit Gent

Department of Reproduction, Obstetrics, and Herd Health Faculty of Veterinary Medicine,

Ghent University

[email protected]

Promotoren: Prof. Dr. Ann Van Soom Prof. Dr. Geert Opsomer

Copromotor: Prof. Dr. Dr. h. c. Aart de Kruif

Table of Contents List of Abbreviations

Chapter 1 General Introduction 1

Chapter 2 Aims of the Study 13

Chapter 3 Reduced Fertility in High Yielding Dairy Cows: Are the Oocyte and the Embryo in Danger? – A Review 17

Chapter 4 The Intrafollicular Environment in High Yielding Dairy Cows 55Chapter 4A Metabolite and Ionic Composition of Follicular Fluid from

different-sized Follicles and their Relationship to Serum Concentrations in Dairy Cows 57

Chapter 4B Metabolic Changes in Follicular Fluid of the Dominant Follicle in High Yielding Dairy Cows Early Post Partum 75

Chapter 5 Negative Energy Balance in High Yielding Dairy Cows and the Consequences for Oocyte Quality 95

Chapter 5A Non-esterified Fatty Acids in Follicular Fluid of the Dominant Follicle in High Yielding Dairy Cows and their Effect on the Developmental Capacity of Bovine Oocytes in vitro 97

Chapter 5B The in vitro Development of Bovine Oocytes after Maturation in Glucose and β-Hydroxybutyrate Concentrations associated with Negative Energy Balance in Dairy Cows 125

Chapter 6 A New Technique to Evaluate the Lipid Content of Single Oocytes and Embryos 141

Chapter 6A The Use of a Fluorescent Dye, Nile Red, to Evaluate the Lipid Content of Single Mammalian Oocytes 143

Chapter 6B Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach 165

Chapter 7 Comparison of Embryo Quality in High Yielding Dairy Cows, in Dairy Heifers and in Beef Cows 175

Chapter 8 General Discussion and Conclusions 197

Summary 219

Samenvatting 229

Acknowledgments- Dankwoord 239

Curriculum Vitae - Publicaties 247

List of Abbreviations

AI artificial insemination ANOVA analysis of variance BB Belgian Blue beef cows BCS body condition score β-OHB β-hydroxybutyrate BSA bovine serum albumine COC cumulus oocyte complex DMEM dulbecco’s modified eagle medium DMSO dimethyl sulfoxide E estradiol 17β EGF epidermal growth factor ER embryo recovery session FCS foetal calf serum FF follicular fluid HDL high density lipoproteins IFN-tau interferon tau IGF insulin like growth factor IGF-BP insulin like growth factor binding protein IVM in vitro maturation LA linoleic acid (C18:2) LDL low density lipoproteins LHFC lactating Holstein Friesian cows NEB negative energy balance NEFA non-esterified fatty acids NLHFH non-lactating (nulliparous) Holstein Friesian heifers OA oleic acid (C18:1) P4 progesterone PA palmitic acid (C16:0) pp post partum RIA radio immunoassay SA stearic acid (C18:0) SD standard deviation SEM standard error of the mean SOF synthetic oviduct fluid TC total cholesterol TCM tissue culture medium TG triglycerides TP total protein VLDL very low density lipoproteins

Chapter 1

General Introduction

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

General Introduction

2

General Introduction

3

Bovine milk and dairy products have always been an appreciated part of man’s diet. A

cow can process feed (e.g. grass), that is useless for human consumption, into highly

nutritious milk (or meat). It was due to this specific and valuable feature that cattle became

among the first domesticated animals, albeit some time after goats and sheep. Since their

domestication, several cattle breeds have been developed and further selected for the

production of beef, as draft animals or for the production of milk. During the last decades,

significant genetic improvements, combined with increased nutritional management, have

allowed the modern dairy industry to create highly sophisticated dairy breeds producing

enormous amounts of milk. However, it has frequently been reported that, along with

continuously increasing milk production, dairy cow fertility has been declining (Lean et al.,

1989; Royal et al., 2000; Lucy, 2001; Butler, 2003; López-Gatius, 2003; Bousquet et al.,

2004; Mee et al., 2004). In order to maintain profitability, modern dairy cows should conceive

within the first three months after parturition, which is, metabolically speaking, their most

demanding period. Furthermore, disappointing reproductive performance plays a predominant

role in culling decisions (Rajala-Schultz and Gröhn, 2001). Maintaining dairy cows healthy

and highly fertile, without a decrease in milk yield, is the ultimate goal of the modern dairy

industry in an attempt to meet the increasing demands of a rapidly expanding human

population in an economically and ecologically acceptable way.

Shortly after giving birth, dairy cows’ milk production increases tremendously and

thus they encounter huge energy losses. This drain of energy is impossible to be sufficiently

compensated for by energy uptake through feed, and gives rise to a period of negative energy

balance (Rukkwamsuk et al., 1999). Figure 1 shows how dairy cows physiologically adapt to

this period of negative energy balance. The overall function of a dairy cow’s adaptation

during a period of NEB is to shift the body’s fuel supply away from glucose (which is

necessary for milk production) and towards the use of lipid derived energy sources (Herdt,

2000; Vernon, 2002). However, because of the high milk production, more and more

frequently modern dairy cows experience a maladaptation. Their physiological feedback

mechanisms fail, leading to pathological situations such as fatty liver and (sub)clinical

ketosis. Particularly overconditioned cows, but also cows of high genetic merit for milk, or

animals with suboptimal health have difficulties to adapt and hence are extremely vulnerable

during this transition period (Herdt, 2000; Jorritsma et al., 2003). Typically, these cows

display a reduced appetite early post partum, leading to an even higher lipid mobilization and

liver triglyceride infiltration which may result in high plasma ketone levels (Rukkwamsuk et

General Introduction

4

al., 1999). The rapid mobilization of body reserves, reflected in the loss of body condition (up

to 10% of the body weight at calving), may aggravate the already depressed dry matter intake

(McMillan, 1998; Vernon, 2002). Primiparous cows typically show an even more negative

energy balance (NEB) since they still need extra energy for body growth (Cavestany et al.,

2005). It has been shown that such metabolic stress, associated with several endocrine

malfunctions, is hard to reconcile with a satisfying reproductive performance.

Figure 1. Feedback mechanism during negative energy balance resulting in reduced glucose use in peripheral tissue. The three predominant metabolites, on which further experiments (Chapter 4 and 5) concentrate, are indicated with red circles: glucose, non-esterified fatty acids (NEFA) and β-hydroxybutyrate (ketone body).

From a biological point of view, it makes sense for the dam to favour milk production

over fertility, which is referred to as ‘nutrient prioritization’ (Lucy, 2003). Since the available

nutrients are scarce, it is more important for the dam to invest those limited nutrient resources

in the survival of the current offspring in stead of gambling on the health and survival of the

oocyte that is yet to be fertilized and gives rise to a healthy offspring (Silvia, 2003). Over the

GLUCOSE

GLUCOSE

Insulin

Insulin

LIPOGENESIS

LIPOLYSIS

Adipose tissue

NEFA

Alternative energy source in peripheral tissue

SAVE ON GLUCOSE

Acetyl CoA

KETONE BODIES

Fatty Acids

Triglycerides VLDL Lipid

accumulation

Oxaloacetate Krebs Cycle

GLUCONEOGENESIS +

+

DIET: Proprionate

Liver

LACTOSE

General Introduction

5

passed decades, the dairy industry exploited this prioritization to maximize milk yield,

creating a ‘nutrient high-way’ from the digestive tract and body reserves directly to the udder.

The metaphor of this energy high-way is highly applicable to the specific metabolic situation

of our high producing dairy cows. Other exits of this high-way, providing for example energy

to the reproductive system, are passed by or even closed during the first weeks post partum.

On the other hand, the energy required to grow and ovulate a follicle, to form a corpus luteum

and to maintain early pregnancy is negligible compared to the energy demands for production

and maintenance. So it is more rational to assume that the ‘pollution’ caused by the heavy

energy traffic towards the udder, rather than a net energy shortage, is responsible for the

hampered reproductive functions.

Reproductive failure is certainly a multifactorial problem in which the amount of

produced milk as such only plays a minor role compared with the importance of negative

energy balance, body condition and postpartum diseases (Loeffler et al., 1999; de Vries and

Veerkamp, 2000; Snijders et al., 2000; Lucy, 2001). Daily milk yield is not an appropriate

indicator of negative energy balance because feed intake and management practices both

confound the association between yield and energy balance (Villa-Godoy et al., 1988;

McMillan, 1998; de Vries and Veerkamp, 2000; Kruip et al., 2000).

Other factors, such as the high energy and protein-rich rations typically fed to modern dairy

cows to sustain the high level of milk production together with the increased herd size, have

been associated with the disappointing fertility outcomes (Butler, 1998; Lucy, 2001; Fahey et

al., 2002; Lucy, 2003). Finally, the genetic selection for high milk production as such may

also be a cause of reduced fertility (Snijders et al.,2000; Snijders et al., 2001).

Specific pathways linking the above mentioned factors with the disturbed reproductive

funtions in metabolically compromised postpartum dairy cows are complex and have been

intensively investigated for many years (reviewed by Butler, 2003). Much of the effort has

been focused on alterations in endocrine signalling (hypothalamus-pituitary-ovary axis) and

ovarian dysfunction. The effects on follicular development and the subsequent indicators of

impaired fertility such as reduced oestrous symptoms or anoestrus, cyst formation, delayed

first ovulation, and prolonged calving to first insemination intervals have been extensively

documented (Harrison et al., 1990; Opsomer et al., 1998; Beam and Butler, 1997; de Vries

and Veerkamp, 2000; Diskin et al., 2003; Vanholder et al., 2002; Lopez et al., 2004).

However, even when a positive energy balance and a correct endocrine signalling are re-

General Introduction

6

established which ultimately results in an ovulation, reproduction is not guaranteed. As has

been reviewed by Bousquet et al. (2004), the success rate of artificial insemination showed a

dramatic drop in almost all countries housing high yielding dairy cows without an obvious

reduction in sperm quality. Furthermore, early embryonic mortality is proposed to be a

significant cause of reproductive failure in ruminants (Dunne et al., 1999; Mann and

Lamming, 2001; Bilodeau-Goeseels and Kastelic, 2003). Driven by these observations it is

only recently that some studies began to focus on the oocyte and subsequent embryo quality

as potentially important factors, which are, physiologically spoken, most closely linked with

conception rate and hence fertility (Boland et al., 2001). O’Callaghan and Boland (1999)

stated that:

“The observed decline in fertility in high producing dairy cattle is mostly a problem of

bad oocyte- and hence embryo quality, rather than being an endocrine disruption.”

Oocytes and embryos are suggested to be highly sensitive to any disruption in their

environment caused by metabolic, dietary or other factors, thereby having fatal consequences

for the final fertility (McEvoy et al., 2001). The knowledge about the oocyte’s micro-

environment and the quality of the oocyte or the embryo proper in high yielding dairy cows is

extremely limited. First of all, it is not known whether metabolic alterations in the peripheral

circulation, such as high non-esterified fatty acids, urea or β-hydroxybutyrate concentrations

and low glucose concentrations, have an impact on the follicular fluid composition. Secondly,

assumed that such metabolic changes in the follicular fluid occur, do they have any impact on

oocyte metabolism and its developmental capacity? After all, there is scientific evidence that

for example high non-esterified fatty acid concentrations are toxic for different kind of cell

types, including bovine (Vanholder et al., 2005) and human granulosa cells (Mu et al., 2001),

Leydig cells (Lu et al., 2003) and pancreatic β-cells (Maedler et al., 2001). Similar adverse

effects have been described for urea (Ocon and Hansen, 2003) and ketone bodies (Franklin et

al., 1991). It is not inconceivable that also the oocyte is vulnerable to these critical

metabolites. Exploring such untrodden research field could reveal crucial knowledge in the

pathogenesis of the widely reported failure of conception.

In the case an embryo is formed, it is not known whether this early embryo displays an

inferior quality caused by a carry-over effect via the oocyte or due to direct effects of altered

energy, protein or lipid metabolism in the modern dairy cow. Based on the results of ample in

vitro studies, it is generally accepted that the post-fertilization micro-environment is

determinant for embryo quality in terms of morphology, lipid content, metabolism and gene

General Introduction

7

expression (Wrenzycki et al., 2000; Abe et al., 2002; Rizos et al., 2002; Rizos et al., 2003).

Whether the knowledge of these in vitro models is also applicable on the specific in vivo

situation in modern dairy cows, is food for further research.

Finding answers to all these questions is of capital importance to substantiate that not

only endocrine signalling and ovarian activity is disturbed but that also the oocyte and the

embryo could be directly affected in high yielding dairy cows early post partum. The

subfertility problem can only be solved when all possible factors in the pathogenesis are

unravelled.

General Introduction

8

References

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Beam SW, Butler WR. 1997. Energy balance and ovarian follicle development prior to first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biology of Reproduction 56: 133-142.

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Boland MP, Lonergan P, O’Callaghan D. 2001. Effect of nutrition on endocrine parameters, ovarian physiology, and oocyte and embryo development. Theriogenology 55: 1323-1340.

Bousquet D, Bouchard E, DuTremblay D. 2004. Decreasing fertility in dairy cows: myth or reality? Le Médecin Vétérinaire 34: 59-61.

Butler WR. 1998. Effect of protein nutrition on ovarian and uterine physiology in dairy cattle. Journal of Dairy Science 81: 2533-2539.

Butler WR. 2003. Energy balance realtionships with follicular development, ovulation and fertility in pp dairy cows. Livestock Production Science 83: 211-218.

Cavestany D, Blanc JE, Kulcsar M, Uriarte G, Chilibroste P, Meikle A, Febel H, Ferraris A, Krall E. 2005. Studies of the transition cow under a pasture-based milk production system: metabolic profiles. Journal of Veterinary Medicine Series A 52: 1-7.

de Vries MJ, Veerkamp RF. 2000. Energy balance of dairy cattle in relation to milk production variables and fertility. Journal of Dairy Science 83: 62-69.

Diskin MG, Mackey DR, Roche JF, Sreenan JM. 2003. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Animal Reproduction Science 78: 345-370.

Dunne LD, Diskin MG, Boland MP, O’Farrell KJ, Sreenan JM. 1999. The effect of pre- and post-insemination plane of nutrition on embryo survival in beef heifers. Animal Science 69: 411-417.

Fahey J, O’Sullivan K, Crilly J, Mee JF. 2002. The effect of feeding and management practices on calving rate in dairy herds. Animal Reproduction Science 74: 133-150.

Franklin ST, Young JW, Nonnecke BJ. 1991. Effects of ketones, acetate, butyrate, and glucose on bovine lymphocyte proliferation. Journal of Dairy Science 74: 2507-2514.

Harrison RO, Ford SP, Young JW, Conley AJ, Freeman AE. 1990. Increased milk production versus reproductive and energy status of high producing dairy cows. Journal of Dairy Science 73: 2749-2758.

Herdt TH. 2000. Ruminant adaptation to negative energy balance. Veterinary Clinics of North America: Food Animal Practice 16: 215-230.

Jorritsma R, Wensing T, Kruip TA, Vos PL, Noordhuizen JP. 2003. Metabolic changes in early lactation and impaired reproductive performance in dairy cows. Veterinary Research 34: 11-26.

Kruip TAM, Stefanowska J, Ouweltjes W. 2000. Robot milking and effect on reproduction in dairy cows: a preliminary study. Animal Reproduction Science 60-61: 443-447.

General Introduction

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Lean IJ, Galland JC, Scott JL. 1989. Relationships between fertility, peak milk yields and lactational persistency in dairy cows. Theriogenology 31: 1093-1103.

Loeffler SH, de Vries MJ, Schukken YH. 1999. The effects of timing of disease occurance, milk yield, and body condition on fertility of dairy cows. J Dairy Sci 82: 2589-2604.

Lopez H, Satter LD, Wiltbank MC. 2004. Relationship between level of milk production and estrous behavior of lactating dairy cows. Animal Reproduction Science 81: 209-223.

López-Gatius F. 2003. Is fertility declining in dairy cattle? A retrospective study in northeastern Spain. Theriogenology 60: 89-99.

Lu ZH, Mu Y, Wang BA, Li XL, Lu JM, Li JY, Pan CY, Yanase T, Nawata H. 2003. Saturated free fatty acids, palmitic acid and stearic acid, induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cells. Biochemical and Biophysical Research Communications 303: 1002-1007.

Lucy MC. 2001. Reproductive loss in high-producing dairy cattle: where will it end? Journal of Dairy Science 84: 1277– 1293.

Lucy MC. 2003. Mechanisms linking nutrition and reproduction in postpartum cows. Reproduction Supplement 61: 415–427.

Maedler K, Spinas GA, Dyntar D, Moritz W, Kaiser N, Donath MY. 2001. Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes 50: 69-76.

Mann GE, Lamming GE. 2001. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 121: 175-180.

McEvoy TG, Robinson JJ, Ashworth CJ, Rooke JA, Sinclair KD. 2001. Feed and forage toxicants affecting embryo survival and fetal development. Theriogenology 55: 113-129.

McMillan WH. 1998. Statistical models predicting embryo survival to term in cattle after embryo transfer. Theriogenology 50: 1053-1070.

Mee J, Evans R, Dillon P. 2004. Is Irish dairy herd fertility declining? Proceedings 23rd World Buiatrics Congress, July 11-16, 2004, Québec, Canada. (Abstr.).

Mu Y-M, Yanase T, Nishi Y, Tanaka A, Saito M, Jin CH, Mukasa C, Okabe T, Nomura M, Goto K, Nawata H. 2001. Saturated FFAs, palmitic acid and stearic acid, induce apoptosis in human granulosa cells. Endocrinology 142: 3590-3597.

O’Callaghan D, Boland MP. 1999. Nutritional effects on ovulation. Animal Science 68: 299–314. Ocon OM, Hansen PJ. 2003. Disruption of bovine oocytes and preimplantation embryos by urea and

acidic pH. Journal of Dairy Science 86: 1194-2000. Opsomer G, Coryn M, Deluyker H, de Kruif A. 1998. An analysis of ovarian dysfunction in high

yielding dairy cows after calving based on progesterone profiles. Reproduction in Domestic Animals 33: 193-204.

Rajala-Schultz PJ, Gröhn YT. 2001. Comparison of economically optimized culling recommandations and actual culling decisions of Finnish Ayrshire cows. Preventive Veterinary Medicine 49: 29-39.

Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. 2002. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Molecular Reproduction and Development 61: 234-48.

General Introduction

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Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP, Lonergan P. 2003. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biology of Reproduction 68: 236-243.

Royal M, Mann GE, Flint APF. 2000. Strategies for reversing the trend towards subfertility in dairy cattle. The Veterinary Journal 160: 53-60.

Rukkwamsuk T, Wensing T, Kruip TAM. 1999. Relationship between triacylglycerol concentrations in the liver and first ovulation in postpartum dairy cows. Theriogenology 51: 1133-1142.

Silvia WJ. 2003. Addressing the decline in reproductive performance of lactating dairy cows: a researcher’s perspective. Veterinary Science Tomorrow 3: 1-5.

Snijders SE, Dillon P, O'Callaghan D, Boland MP. 2000. Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows. Theriogenology 53: 981-989.

Snijders SEM, Dillon PG, O’Farrell KJ, Diskin M, Wylie ARG, O’Callaghan D, Rath M, Boland MP. 2001. Genetic merit for milk production and reproductive success in dairy cows. Animal Reproduction Science 65: 17-31.

Vanholder T, Leroy JLMR, Van Soom A, Opsomer G, Maes D, Coryn M, de Kruif A. 2005 . Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro. Animal Reproduction Science 87: 33-44.

Vanholder T, Opsomer G, Govaere JL, Coryn M, de Kruif A. 2002. Cystic ovarian disease in dairy cattle: etiology, pathogenesis, and risk factors. Tijdschrift voor Diergeneeskunde 127: 146-155.

Vernon RG. 2002. Nutrient partitioning, lipid metabolism and relevant imbalances. Proceedings of the 12th World Buiatrics Congress, 18-23 August, 2002; Hannover, Germany.

Villa-Godoy A, Hughes TL, Emery RS, Chapin LT, Fogwell RL. 1988. Association between energy balance and luteal function in lactating dairy cows. Journal of Dairy Science 71: 1063-1072.

Wrenzycki C, De Sousa P, Overström EW, Duby RT, Herrmann D, Watson AJ, Niemann H, O’Callaghan D, Boland MP. 2000. Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos. Journal of Reproduction and Fertility 118: 69-78.

Chapter 2

Aims of the Study

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Aims of the Study

14

Aims of the Study

15

Our main hypothesis is that inferior oocyte and/or embryo quality plays a major role in

the pathogenesis of reduced fertility in high yielding dairy cows. Therefore, it is important to

investigate to which extent oocytes are exposed to the typical metabolic changes in high

yielding dairy cows early post partum and whether this could ultimately lead to a hampered

oocyte developmental competence. The more, also embryo quality could be adversely

affected and should therefore be scrutinized.

To test this hypothesis, the specific aims of the present thesis are:

1. to investigate the intrafollicular environment of the oocyte by determining the

chemical composition of follicular fluid of differently sized follicles and to compare

and correlate these results with the serum composition in dairy cows post mortem

(Chapter 4A)

2. to assess to what extent metabolic adaptations that typically occur in high yielding

cows early post partum are reflected in the follicular fluid of the dominant follicle

(Chapter 4B)

3. to study the concentration and composition of the non-esterified fatty acid fraction in

the fluid of the dominant follicle early post partum and to investigate its effect on the

developmental capacity of bovine oocytes in an in vitro maturation model

(Chapter 5A)

4. to examine the effect of β-hydroxybutyrate and glucose concentrations, associated

with negative energy balance, during in vitro maturation on the developmental

competence of bovine oocytes in vitro

(Chapter 5B)

5. to develop a new technique to evaluate the lipid content of single bovine oocytes and

embryos as it can be used as a possible quality parameter

(Chapter 6)

6. to investigate embryo quality in high yielding dairy cows, compared to embryos from

maiden dairy heifers and non-lactating beef cows; and to identify factors associated

with quality and lipid content of embryos in dairy cows and heifers

(Chapter 7)

Chapter 3

Reduced Fertility in High Yielding Dairy Cows:

Are the Oocyte and the Embryo in Danger?

A Review

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Chapter 3: Reduced Fertility in Dairy Cows – A Review

18

Chapter 3: Reduced Fertility in Dairy Cows – A Review

19

Introduction

Reproductive failure in high producing dairy cattle is a multifactorial problem. The

pathogenesis of this subfertility is complex and especially the interactions between the

negative energy balance (NEB) early post partum and the hypothalamus-pituitary-ovary-

uterus axis have been studied thoroughly (Ducker et al., 1985; Lucy, 2001; Butler, 2003). The

disturbed endocrine signalling leads to a retarded resumption of ovarian cyclicity post partum

which has been recognized as a major factor in dairy cow reproductive failure (Opsomer,

1998). However, attention has recently been shifting towards the ubiquitously reported

disappointing conception rates (Bousquet et al., 2004) and towards a remarkably high

incidence of early embryonic mortality (Dunne et al., 1999; Mann and Lamming, 2001;

Bilodeau-Goeseels and Kastelic, 2003). Therefore, it is of crucial significance to concentrate

on the quality of the oocyte and the embryo proper in order to approach the problem of

subfertility adequately (O’Callaghan and Boland, 1999). Are the intrinsic quality of the

oocyte and the embryo, which are the most essential factors for living offspring, compromised

in modern high yielding dairy cows?

Recent studies confirmed that the female gamete and the embryo are probably in danger.

Snijders et al. (2000) studied the in vitro developmental competence of oocytes from dairy

cows with a high and a moderate genetic merit for milk production. Oocytes from high

genetic merit cows resulted in significantly lower blastocyst yields in vitro, irrespective of

milk production as such. This suggests possible adverse effects of the enforced genetic

selection towards milk production on fertility (see below). The results of similar studies

concerning oocyte quality in high yielding dairy cows are summarized in table 1.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

20

Table 1. Survey of 7 different studies concerning oocyte quality in high yielding dairy cows.

Author Reduced oocyte quality (yes/no)

Finding

Kruip et al., 1995

Yes After a more profound NEB oocyte’s developmental capacity is reduced compared to control cows (80-120 days pp).

Kendrick et al., 1999

Yes Morphological oocyte quality declines after day 30 pp.

Gwazdauskas et al., 2000

Yes Higher morphological oocyte quality at day 28 pp than at day 117 pp.

Snijders et al., 2000

Yes Higher genetic merit for milk production cows have a reduced oocyte’s developmental competence compared to low genetic merit cows.

Wiltbank et al., 2001

Yes Better morphological oocyte quality in non-lactating than lactating dairy cows.

Walters et al., 2002b

Yes Morphological oocyte quality declines after day 70 pp

Argov et al., 2004

No No differences in morphological oocyte quality and developmental capacity at day 73 and at day 263 pp.

Apart from oocyte quality, Wiltbank et al. (2001) demonstrated that non-lactating

dairy cows yielded significantly more good quality embryos than lactating ones. Sartori et al.

(2002) also focused on the quality of day 5 embryos at 2 to 3 months after calving. They

found that embryos from lactating dairy cows were remarkably inferior compared to embryos

from non-lactating cows or maiden heifers. The more, a high proportion of non-viable

embryos have been described in lactating cows (Sartori et al., 2002). More studies are needed

to get an informative and overall picture of average embryo quality in high yielding dairy

cows.

Conclusively, it can be stated that reduced oocyte and/or embryo quality has been

demonstrated in high yielding dairy cows. Therefore, it is assumed that inferior oocyte and/or

embryo quality is at least partly responsible for the reduced fertility in general and more

specifically for the low conception rates and higher prevalence of early embryonic mortality.

Poor oocyte quality and subsequently disappointing embryonic development may be the result

of a compromised follicular development and intrafollicular environment. A disrupted micro-

environment in the oviduct or the uterus may also lead to an inferior embryo quality.

In the present paper we will review possible mechanisms through which oocyte and

embryo developmental competence could be hampered in the specific situation of the modern

high yielding dairy cow. Firstly the effects of a NEB and the associated endocrine and

Chapter 3: Reduced Fertility in Dairy Cows – A Review

21

metabolic changes on oocyte quality will be discussed in detail. Secondly, attention will be

paid to the corpus luteum and the uterine environment supporting early embryo development.

Finally, the review will concentrate on possible consequences of milk yield stimulating

rations (high starch, fat and protein content) typically fed to dairy cows on the success rate of

an oocyte to become a healthy embryo, which should ultimately establish a normal

pregnancy. Figure 1 shows a diagrammatic representation and summarizes the major

mechanisms through which oocyte and/or embryo quality can be affected. It is important to

mention however, that quite some (possibly important) mechanisms have only been

investigated by means of in vitro models or by studies on non-lactating cows or heifers.

Extrapolations from these models to the specific situation in high yielding dairy cows should

therefore always be made with caution.

Oocyte and embryo quality: what does that mean?

The ultimate test (or the gold standard) for oocyte quality is its ability to be fertilized,

to develop to the blastocyst stage and finally to establish a pregnancy resulting in living

offspring (Lonergan et al., 2001). Unfortunately, from a practical point of view, it is

impossible to transfer all embryos of interest in living recipients and finally check them for

pregnancy. Therefore, other parameters, which are said to be well correlated with the actual

oocyte quality as described above, should be used. The most reliable and commonly used

parameter is the oocyte’s developmental competence in vitro and more specifically the timing

of the first cleavage of the zygote (Van Soom et al., 1992). In vivo, intrafollicular conditions

are determinant for oocyte quality. It is generally accepted that maternal mRNA and protein

molecules are synthesized and accumulated during oocyte growth and maturation (Lonergan,

2003a, Vassena et al. 2003; van den Hurk and Zhao, 2005). The latter is crucial to guarantee

the survival of the early embryo prior to embryonic genome activation which happens at the

8-16 cell stage. Not only the abundance of such developmentally important gene transcripts

(mRNA) in the oocytes but also the extent of the poly(A) tail and their methylation state is

related to the developmental competence of the oocyte and can be influenced by the

maturation (i.e. follicular) environment (Watson et al., 2000; Gandolfi and Gandolfi, 2001;

Lonergan et al., 2003b). Furthermore, other parameters to estimate oocyte quality such as the

morphological appearance of cumulus investment and ooplasm (de Loos et al., 1989; Hawk

and Wall, 1994), lipid content (Leroy et al., 2005), ultrastructural evaluation of the nuclear

Chapter 3: Reduced Fertility in Dairy Cows – A Review

22

stage and ooplasm (Revah and Butler, 1996; Yaakub et al., 1997; O’Callaghan et al., 2000),

presence of gene transcripts (Wrenzyki et al., 2000) and presence of apoptotic markers are

routinely used (Yuan et al., 2005).

Similarly, several invasive and non-invasive parameters (morphology, cell number,

developmental kinetics, apoptosis, genetic anomalies …) have been described to evaluate

embryo quality (reviewed by Van Soom et al., 2003). However, embryo quality is

predominantly determined by the culture environment and less by the oocyte’s origin

(Lonergan et al., 2001; Knijn et al., 2002). Thus, the post-fertilization micro-environment in

the oviduct and uterus is crucial and has a major impact on embryo quality (metabolism and

gene expression) (Wrenzycki et al., 2000; Rizos et al., 2002).

Applying this knowledge to the specific situation of high yielding dairy cows, it can be

assumed that both the oocyte and the embryo are vulnerable to possible adverse changes in

the environment at the level of the follicle and fallopian tube or uterus, respectively. Britt

(1992) hypothesized that the developmental competence of the oocyte and the steroidogenic

capacity of the follicle in high yielding dairy cows is determined during the long period (up to

80 days) of follicular growth prior to ovulation. Thus, primordial follicles exposed to adverse

conditions associated with the metabolic challenging period of NEB early post partum, are

less capable of producing adequate amounts of oestrogens and progesterone (after ovulation)

(Britt, 1992; Roth et al., 2001). The more, these follicles are doomed to contain an inferior

oocyte which will be ovulated around 60-80 days post partum. Finally, in the case an embryo

is formed, the microenvironment of the oviduct and uterus can be hostile preventing normal

embryo development (Boland et al., 2001; McEvoy et al., 2001; Kenny et al., 2002b).

Conclusively, the ‘Britt hypothesis’ affirms the idea that oocyte and embryo quality in high

producing dairy cows are really in danger. In the following paragraphs scientific evidence is

reviewed confirming or denying this important assumption.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

23

Figure 1. Diagrammatic presentation of the major mechanisms through which the negative energy balance or nutrition can influence oocyte and/or embryo quality. Δ stands for ‘changes’.

ovulation

OVARY OVIDUCT UTERUS

NEBMetabolic Δ Endocrine Δ

Elongated conceptus

Expanded blastocyst

Compact morula

Liver

NUTRITIONEnergy and Protein

Metabolic Δ Endocrine Δ

60 – 90 days

Carry over effects on oocyte and follicle quality

Carry over effects on CL function

High progesterone metabolism in the liver

PROGESTERONE

IFN-τ

PGF2α

FF

Affecting steroid production, FF and oocyte quality

Affecting health of primordial follicle

Affecting embryo quality, micro-environment and endometrium secretions

AFFECTED • Oocyte quality • Steroid secretion

CONCEPTION FAILURE LOW PROGESTERONE EARLY EMBRYO MORTALITY

0 d 1 d 2 d 4 d 8 d6 d 17d-2

Primordial follicle

Dominant follicle

Mature oocyte 2 cell embryo 32 cell embryo

Corpus luteum

-90-60 d

Chapter 3: Reduced Fertility in Dairy Cows – A Review

24

Adverse effects of a negative energy balance on oocyte quality

Endocrine link between negative energy balance and oocyte quality

Folliculogenesis is a very complex and finely tuned process in which endocrine and

paracrine signals play an important role (for review, see Webb et al., 2004). The

developmental capacity of the oocyte is intrinsically linked to the growth phase and the health

of the developing follicle (Bilodeau-Goeseels and Panich, 2002; Sutton et al., 2003; Lequarré

et al., 2005). Meanwhile, it is generally accepted that a NEB and concurrent weight loss can

hamper the well orchestrated process of follicular growth at the level of the hypothalamus-

pituitary-ovary axis (Beam and Butler, 1997; Lucy, 2000; Armstrong et al., 2002b; Gong,

2002; Lucy, 2003; Webb et al., 2004). First of all, lower insulin and IGF-I, increased growth

hormone, and probably also reduced leptin concentrations are major endocrine pathways

through which follicular growth can be directly (via influencing the sensitivity of the ovary

for gonadotrophins) or indirectly (via lower LH concentrations and pulsatility) hampered in

high yielding dairy cows (Gong, 2002; Lucy, 2003; Webb et al., 2004). Secondly, it has

recently been described that lactating cows, compared to non-lactating heifers, have less

oestrogenic dominant follicles. These follicles therefore require a prolonged growing phase up

to larger diameters in order to trigger an adequate LH pulse frequency and LH surge (Lopez et

al., 2004; Sartori et al., 2004). Apart from the reduced oestrogen production, an elevated

oestrogen metabolism in the liver during the period of NEB due to a higher plane of nutrition

could also account for the lower preovulatory oestrogen concentrations in lactating dairy cows

compared to non-lactating heifers (Sangsritavong et al., 2002). This may explain the tendency

towards prolonged follicular growth and thus towards delayed ovulation or even anovulation

in high yielding dairy cows (Lucy, 2003). The ultimate consequences of disturbed follicular

growth and function on the intrinsic oocyte quality involved is, however, unknown. Lucy

(2001) compared this specific situation with persistent follicles which frequently contain an

inferior oocyte due to premature nuclear maturation. This can be explained by a decreased

flow of meiosis-arresting substances from granulosa cells to the oocyte (Revah and Butler,

1996). Vos et al. (1996) on the other hand, did not find any adverse effect of a 16 h

postponement of the LH surge on the developmental competence of in vivo matured oocytes.

It is clear that major disruptions in follicular growth and function due to a NEB will

predominantly lead to anovulation and atresia of the dominant follicle rather than resulting in

Chapter 3: Reduced Fertility in Dairy Cows – A Review

25

the ovulation of an inferior oocyte. However, it has never been investigated whether the

endocrine imbalances during the first weeks post partum have long-term adverse effects on

the quality of the oocyte, which will be ovulated approximately two months later. As has been

mentioned above, this possibility has been raised for the first time by Britt (1992) and will be

referred to as the ‘Britt hypothesis’. In the following paragraphs it is suggested how typical

endocrine disruptions during the NEB in dairy cows theoretically may alter oocyte

developmental competence (Reis et al., 2002). It is furthermore important to mention already

that the interpretation of possible steroid effects on oocyte quality is further complicated by

the observation that steroid binding proteins are present in follicular fluid (FF) and that their

concentrations are related to the oocyte’s developmental competence (Yding-Anderson,

1990).

During normal follicular growth 17β-estradiol concentrations in the preovulatory

follicle decline sharply after the LH peak, paralleled by an increase in progesterone

concentration (Fortune and Hansel 1985). As described above, it has been demonstrated that

deviant oestrogen concentrations hamper a correct nuclear maturation probably by affecting

the spindle formation and microtubuli organisation (Beker et al., 2002; Beker-Van

Woudenberg et al., 2004).

High yielding dairy cows generally have lower progesterone concentrations after the

first ovulations post partum due to a less active CL associated with NEB (Villa-Godoy et al.,

1988; Sartori et al., 2004), and due to the high nutritional levels which increase the

progesterone metabolism in the liver (Vasconcelos et al., 2003). At 18 hours after the LH

surge, progesterone constitutes about 90% of the intrafollicular steroid content (Silva and

Knight, 2000). Adequate progesterone secretion by the granulosa cells is necessary to ensure

optimal maturation and postovulatory development of the oocyte probably through a direct

positive effect on the pre-ovulatory oocyte (Wehrman et al., 1993; McEvoy et al., 1995).

Progesterone is crucial for the correct down-regulation of the gap junctions in granulosa cells,

thereby isolating the cumulus oocyte complex (COC) and thus reducing the oestrogen

concentrations in the oocyte below the threshold to maintain meiotic arrest (McEvoy et al.,

1995). Furthermore, progesterone is said to be involved in the process of polyadenylation of

the maternal mRNA thereby regulating expression of developmentally important genes in the

oocyte (McEvoy et al., 1995).

Chapter 3: Reduced Fertility in Dairy Cows – A Review

26

Fat mobilization due to a NEB results in the release of lipid stored progesterone

(Hamudikuwanda et al., 1996; Rabiee et al., 2002). However, the consequences of these so

called suprabasal progesterone concentrations on oocyte maturation are not known. Both in

heifers (Båge, 2003) and in lactating dairy cows (Waldmann et al., 2001) it has been shown

that suprabasal (> 0.5 nmol/l) progesterone concentrations at the moment of AI resulted in

significantly lower conception rates.

One of the major side effects of a NEB is the disruption of a correct luteinizing

hormone (LH) pulse frequency and amplitude leading to prolonged parturition to first

ovulation intervals (Lucy, 2003; Webb et al., 2004). A perfect pulsatile LH secretion is also

considered extremely important for final oocyte growth and maturation (Hyttel et al., 1997).

The preovulatory LH surge is the critical signal for the resumption of meiotic progression

from metaphase I to II which ensures the developmental competence of the pre-ovulatory

oocyte (Hyttel et al., 1997; Rizos et al., 2002; Humblot et al., 2005). Since no LH receptors

are present in the oocyte proper, the signals triggering oocyte maturation are therefore likely

to originate in the surrounding cumulus cells (van den Hurk and Zhao, 2005). A well

established LH surge provokes a shift in steroid production by the granulosa cells from

predominantly oestrogenic to a progesterogenic environment which is necessary to resume

meiosis (van den Hurk and Zhao, 2005). Lindsey et al. (2002) however, demonstrated in

heifers treated with GnRH agonist implants, that both the pulsatile LH secretion and the

preovulatory LH surge are particularly indispensable to guarantee good cytoplasmatic oocyte

maturation rather than being essential for the nuclear maturation. The oocytes, that were

collected in the latter study, underwent cleavage after IVF but no blastocysts were formed.

Leptin or the obese gene product is a peptide that is secreted by the adipocytes and

acts as a direct or indirect messenger (via inhibition of neuropeptide Y), signalling the

nutritional status of the body (Keisler et al., 1999; Chilliard et al., 2005). Recently leptin has

been implicated in the interaction between nutrition and fertility (Keisler et al., 1999;

Denniston et al., 2003). Leptin concentrations are inversely correlated with the extent and the

duration of the NEB in high yielding dairy cows (Block et al., 2001; Liefers et al., 2003). It

can directly affect oocyte quality in mice (Swain et al., 2004). The exact role of leptin in the

subfertility problem in high yielding dairy cows and the possible direct effects on oocyte and

embryo quality need however further investigation.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

27

Insulin concentrations are typically decreased in high yielding dairy cows during the

period of NEB early post partum (Beam and Butler, 1997). Insulin is not only important for

an adequate follicular response to gonadotrophins (Frajblat and Butler, 2000) and for

follicular growth, but probably has also direct stimulatory effects on the oocyte (Butler and

Smith, 1989). Specific information on the direct influence or importance of low insulin

concentrations for oocyte growth and/or maturation in modern dairy cows, is almost absent.

Insulin-like growth factor I (IGF-I) concentrations in serum of dairy cows are

directly correlated with the energy status and are essential for normal follicular development

(Beam and Butler, 1997; McCaffery et al., 2000; van den Hurk and Zhao, 2005). Insulin-like

growth factor I receptors (Type 1) and IGF binding proteins (IGFBP) have been described in

bovine oocyte and cumulus cells in both preantral and antral follicles, suggesting that IGF-I

directly regulates oocyte growth and maturation (Armstrong et al., 2002a; Nuttinck et al.,

2004). Walters et al. (2002a) found a lower IGF-I concentration in FF early post partum

compared to mid-lactation, and those FF concentrations (150 ng/ml) were on average three

times higher than in serum (50 ng/ml) (Spicer et al., 1992; Comin et al., 2002). As stated

above, the bioactivity of this hormone is physiologically spoken more important than the

absolute IGF-I concentration as such. It has even been suggested that especially the binding

proteins and not the IGF-I concentrations as such are changed due to an altered energy

balance (Spicer et al., 1992; Comin et al., 2002). Based on in vitro studies, IGF-I has been

attributed stimulatory effects on oocyte maturation (Izadyar et al., 1997; Pawshe et al., 1998)

promoting embryo formation and quality (Sirisathien and Brackett, 2003). However, the latter

could not be confirmed by Quetglas et al. (2001). More research is certainly needed.

Postpartum growth hormone (GH) concentrations are elevated in high yielding dairy

cows due to low insulin concentrations (Kruip and Kemp, 1999; Tripp et al., 2000). Tripp et

al. (2000) did not find any effect of injected bovine somatotropin on the number or the

developmental capacity of the collected oocytes. In vitro, GH (100 ng/ml) is able to stimulate

the oocyte’s nuclear and cytoplasmatic maturation and can enhance cumulus cell health and

proliferation (Kölle et al., 2003). The in vitro used GH concentrations are however much

higher than the concentration in vivo during early lactation (around 4 ng/ml) (Beam and

Butler, 1997). Whether the elevated GH concentrations in dairy cows early post partum can

promote oocyte maturation or even compensate the negative effects of other described

endocrine or metabolic changes, is not known.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

28

Metabolic link between negative energy balance and oocyte quality

Much attention has already been paid to the effect of the NEB on endocrine changes in

serum and in the follicle, affecting follicular growth and health but probably also oocyte

quality (see above). However, only few studies concentrated on possible effects of the NEB

associated concentrations of glucose, β-hydroxybutyrate (β-OHB) or non-esterified fatty acids

(NEFA) on oocyte quality. Besides, little is known about the implications of these post

partum biochemical serum changes in the composition of the FF embedding the granulosa

cells and supporting the oocyte to undergo the finely tuned process of growth, pre- and final

maturation.

Quiescent follicles display a discontinuous and unilaminar wall due to multiple and

irregular gaps, only providing a partial isolation of the oocyte from the surrounding stroma,

resulting in an almost direct contact between oocyte and blood (Zamboni, 1974). Thus, in this

stage all kinds of metabolites should easily be able to gain access to the follicle probably

affecting the oocyte. This observation supports the ‘Britt hypothesis’. Along with follicular

growth, the oocyte gets more and more isolated from its surroundings due to the formation of

the zona pellucida and FF, several layers of granulosa cells and the basal lamina (blood-

follicle barrier) (Zamboni, 1974). When reaching the pre-ovulatory follicle size, the

permeability of this blood-follicle barrier seems to increase again (Edwards, 1974,

Bagavandoss et al., 1983). Edwards (1974) suggested already that agents capable of

interfering with oocyte health could be introduced in the preovulatory follicle and could also

exert their effects after ovulation, since relatively large quantities of FF remain present

between the cumulus cells. Hence, it can be hypothesized that the metabolic changes

associated with the NEB early post partum may alter the intrafollicular environment and

thereby affecting the oocyte quality. Scientific evidence for long-term (Britt hypothesis) or

even short-term effects of metabolic changes on oocyte quality in high producing dairy cows

is however scarce and future research should concentrate on this. In the studies reviewed

below, only short-term effects have been investigated since, according to our knowledge,

studies of long-term (60-80 days) effects on oocyte quality are impossible to be carried out

form a practical point of view.

Apart from the indirect effects of hypoglycaemia in the modern dairy cow early post

partum (via influencing LH secretion or ovarian responsiveness to gonadotrophins), it can be

hypothesized that low glucose concentrations may also affect oocyte quality directly. Landau

Chapter 3: Reduced Fertility in Dairy Cows – A Review

29

et al. (2000) demonstrated that FF glucose concentrations are influenced by nutritional status.

In the cumulus cells, glucose is primarily converted via the glycolytic pathway into pyruvate

and lactate being the oocyte’s preferred substrates for ATP production (Cetica et al., 2002;

reviewed by Sutton et al., 2003). In the oocyte on the other hand, glucose is predominantly

metabolized in the pentose phosphate pathway (PPP) for DNA and RNA synthesis (Sutton et

al., 2003). Despite the relatively low level of utilization, glucose is thus an indispensable

molecule during oocyte maturation since especially the PPP activity rather than the glycolysis

(ATP production) is involved in the meiotic progression and thus in the developmental

capacity of the oocyte (Downs and Utecht, 1999; Cetica et al., 2002; Sutton et al., 2003).

Addition of glucose to the in vitro maturation medium improves cumulus expansion, nuclear

maturation, embryo cleavage and blastocyst development (Krisher and Bavister, 1998;

Sutton-McDowall et al., 2004). Thus, it can be concluded that inadequate glucose supplies

may compromise the oocyte’s developmental capacity.

Early pp, all high producing dairy cows go through a period of NEB during which the

mobilization of body lipids is crucial to fulfil energy requirements for maintenance and milk

production (Chilliard et al., 1998). Especially the typically low insulin and glucose, and high

growth hormone concentrations in combination with stress (catecholamines) have been shown

to regulate the lipogenic and lipolytic enzymes in the adipose tissue (Vernon, 2002). The

characteristic NEFA rise in serum during that period of energy shortage is the net result of

lipolysis of adipocyte triglycerides by hormone sensitive lipase and re-esterification (acetyl

CoA carboxylase and fatty acid synthase) of these liberated fatty acids (Chilliard et al., 1998;

Vernon, 2002). Plasma NEFA concentrations are very well correlated with the energy balance

and may provide a potential signal of dietary status to the neural centra (Walters et al.,

2002a). The massive lipid mobilization can provoke liver steatosis (Vernon, 2002), resulting

in suboptimal liver function which can hamper fertility (Rukkwamsuk et al., 1999).

Furthermore, Kruip and Kemp (1999) suggested possible direct toxic effects of high NEFA

concentrations at the level of the ovary which may support the hypothesis of Britt (1992).

Parallel with low glucose concentrations, it can be hypothesized that elevated NEFA

concentrations may contribute to reduced fertility in high yielding dairy cows by exerting

detrimental effects on oocyte developmental competence. However, the knowledge about the

effect of this characteristic NEFA rise in serum on the NEFA concentration or composition in

FF is almost absent. Acute fasting results in a NEFA rise both in serum and in FF (Comin et

al., 2002; Jorritsma et al., 2003). Whether this is also the case during the period of NEB early

Chapter 3: Reduced Fertility in Dairy Cows – A Review

30

post partum, is not known. This knowledge is nevertheless of paramount importance in

substantiating the above stated hypothesis through in vitro maturation models. Jorritsma et al.

(2004) demonstrated toxic effects of supraphysiological concentrations of albumin bound

oleic acid on oocyte maturation. Homa and Brown (1992) described similar effects of linoleic

acid. The toxic effects of elevated NEFA concentrations have also been documented in bovine

(Vanholder et al., 2005) and human granulosa cells (Mu et al., 2001), in Leydig cells (Lu et

al., 2003) and pancreatic β-cells (Maedler et al., 2001). Further research should elucidate the

role of high NEFA concentrations at the level of the oocyte in the pathogenesis of reduced

fertility in high yielding dairy cows.

Elevated ketone concentrations, another important metabolic feature of a NEB, have

been associated with immunity depression through direct toxic effects on cells of the

immunity system (Franklin et al., 1991). High producing dairy cows typically are more

sensitive to all kinds of infections (eg. mastitis, endometritis) which in turn can suppress

fertility indirectly (see further). Information about the ketone body concentrations in the FF of

high yielding dairy cows early post partum as little as evidence of potential effects of ketotic

environments on oocyte developmental capacity is however absent. β-Hydroxybutyrate has

been shown to be teratogenic for young murine embryos (Horton et al., 1985).

Steroidogenic capacity of the corpus luteum, uterine function and embryo quality

Lonergan and coworkers (2001) demonstrated that oocyte quality is crucial to

ascertain whether an embryo will be formed or not. The post-fertilization period, however, is

of profound importance in determining the quality and thus the viability of the embryo

(Lonergan et al., 2001). Successful early embryonic development depends on a delicate and

synchronized balance between the establishment of the luteolytic signal in the uterus and the

production of an antiluteolytic factor, interferon tau, by the embryo. The secretion of this

protein depends on the normal development of a strong embryo and it allows the continued

secretion of adequate amounts of progesterone through an inhibition of PGF2α production in

the endometrium (Mann and Lamming, 2001; Goff, 2002). In turn, normal embryo growth

depends both on its inherent developmental competence and on the adequacy of the uterine

environment. Most embryo losses probably occur before day 14 of gestation and they can

account for up to 40% of all pregnancy failures (Dunne et al., 2000; Silke et al., 2002). Since

Chapter 3: Reduced Fertility in Dairy Cows – A Review

31

embryonic mortality is said to be a major cause of reduced fertility, it has been suggested that

the well balanced process of embryo growth and recognition is disturbed in high producing

dairy cows (Dunne et al., 2000; Silke et al., 2002). The key hormone, guaranteeing successful

embryo development, is progesterone (Mann et al., 2001; Mann and Lamming, 2001).

Adequate post-mating progesterone concentrations are crucial for ovum viability as

they modulate the endometrial secretions and thus the correct uterine receptivity (McEvoy et

al., 1995). Both the retarded onset of progesterone rise after ovulation and the suboptimal

progesterone concentrations during the luteal phase are suggested to partly account for the

reduced conception rates in high yielding dairy cows (Mann and Lamming, 2001). The typical

NEB early post partum can affect fertility later in lactation by reducing the number of ovarian

cycles. A sufficient number of ovulatory oestrus cycles preceding AI is crucial for adequate

priming of the uterus (Butler, 2003). Villa-Godoy et al. (1988) showed that cows that went

through a period of NEB post partum displayed significantly lower progesterone

concentrations up to 3 first ovarian cycles after calving. As has been mentioned above, the

maximal progesterone concentration in lactating cows is lower whereas the volume of the

luteal tissue is clearly larger compared with non-lactating heifers (Sartori et al., 2004). This

implies a hampered luteal capability of secreting progesterone.

Secondly, it is most likely that, comparable with the situation for oestrogen

concentrations, increased steroid metabolism in the liver could be held responsible for the

lower circulating progesterone concentrations (Sangsritavong et al., 2002; Vasconcelos et al.,

2003). Vasconcelos et al. (2003) reported a negative correlation between milk production and

progesterone concentrations, most likely through differences in plane of nutrition and thus

differences in liver metabolism (Rabiee et al., 2002; Butler 2003; Vasconcelos et al., 2003).

Peripheral concentrations of progesterone on day 0 and 1 after the LH peak are said to

be crucial for embryo survival in sheep, probably by modifying the final stages of oocyte

maturation (Ashworth et al., 1989; McEvoy et al., 1995). Other studies in the ewe however

demonstrated that the positive effect of progesterone is particularly acting at the level of the

uterus (Lozano et al., 1998). Embryos recovered from cows with higher circulating

progesterone concentrations were more developed and produced higher amounts of INF-tau

than embryos from cows with lower progesterone concentrations (Mann and Lamming,

2001). Finally, Butler et al. (1996) stressed the importance of elevated progesterone

Chapter 3: Reduced Fertility in Dairy Cows – A Review

32

concentrations both prior and after breeding. Besides, it is noteworthy to mention that there

exists almost no correlation between systemic progesterone concentrations in the ovarian vein

and those in the endometrium (Lozano et al., 1998).

Other hormones such as insulin and IGF-I are shown to stimulate progesterone

production by luteal cells (Schams et al., 2004). Like progesterone, also IGF-I could exert a

direct positive effect on embryo health (Moreira et al., 2000). A signalling function between

embryo and uterus is attributed to this growth factor as demonstrated by conceptus stimulated

expression of endometrial IGF-II and progesterone stimulated expression of endometrial

IGFBP-2 (Geisert et al., 1991; Watson et al., 1999). Furthermore, IGF is able to support

embryo production in vitro as has been mentioned earlier.

In conclusion, it can be stated that inadequate progesterone and probably also IGF

concentrations are responsible for a suboptimal microenvironment in the uterus, incapable of

supporting early embryonic life. This may partly account for the high incidence of embryonic

mortality in high yielding dairy cows. The specific effects of diet type (energy and protein

content) on uterine environment and thus embryo viability will be discussed later in this

review.

Mechanisms linking nutrition with oocyte and embryo quality

Dietary changes cause an immediate and rapid alteration in a range of humoral factors,

which can profoundly alter endocrine and metabolic signalling pathways (O’Callaghan et al.,

2000; Boland et al., 2001; Diskin et al., 2003). Modern dairy cows are typically fed starch

and protein rich diets to maximize milk production. In the following paragraphs, we will

review some possible clues through which such rations may interfere with oocyte and/or

embryo quality.

Most studies linking nutrition and fertility describe the influence of short-term changes

in feed intake on all kinds of fertility parameters. A major part of these studies searches for an

optimal diet to stimulate superovulatory responses and to increase the yield of good quality

oocytes or embryos. The results of these studies are therefore of limited value in connection

with the specific situation of modern high yielding dairy cows, since in their case, milk

Chapter 3: Reduced Fertility in Dairy Cows – A Review

33

stimulating diets are fed during months and superovulation treatments are generally not

applied. Furthermore, postpartum dairy cows are rarely used in such experimental set-ups

because milk production as such and the encountered NEB can confound possible effects of

nutrition on fertility. Literature focusing on long-term effects of milk yield stimulating diets

on fertility and more specifically on oocyte or embryo quality early post partum is very

scarce.

High energy intake through starch rich diets

In an attempt to reduce the extent and the duration of the NEB, dairy cows are fed high

energy diets (high starch content). In this respect, high energy diets are considered to be

beneficial for fertility since they stimulate the resumption of normal endocrine signalling

(insulinogenic diet) leading to the onset of ovarian activity. When cows are in positive energy

balance again, the amount of energy intake through feed can influence follicular dynamics

and circulating concentrations of steroids and growth factors (for reviews see: O’Callaghan

and Boland, 1999; Webb et al., 2004) which can affect oocyte and embryo quality in a direct

or indirect way (see above).

Firstly, high feeding levels are suggested on the one hand to reduce circulating

progesterone concentrations via an upregulation of the steroid catabolism in the liver

(Vasconcelos et al., 2003) but on the other hand are said to stimulate progesterone production

by the CL (Armstrong et al., 2001). Reports in literature about a possible causal link between

energy intake, peripheral progesterone concentrations and embryo viability are contradictory

(Abecia et al., 1997; Dunne et al., 1999; McEvoy et al., 2001; Lozano et al., 2003).

Secondly, Wrenzycki et al. (2000) demonstrated more specifically that both diet type

and quantity can have significant effects on the expression of developmentally important

genes in embryos such as Cu/Zn super oxide dismutase (SOD) (prevention of oxidative stress)

and on pyruvate utilization in day 6 bovine embryos. Ad libitum uptake of barley based

concentrates reduced pyruvate utilization and significantly enhanced the expression of Cu/Zn

SOD in embryos compared to embryos from the restricted group. Yaakub et al. (1999a) found

similar adverse effects on embryo quality of a barley based high concentrate-low fibre diet fed

before superovulation and embryo recovery. It is possible that a diet induced shift in the

volatile fatty acid profiles in the rumen (propionate versus acetate), affects embryo quality

indirectly via an altered energy metabolism (insulin and/or IGF-I concentrations) or directly

Chapter 3: Reduced Fertility in Dairy Cows – A Review

34

via compositional changes in follicular, tubal or uterine fluids (Wrenzycki et al., 2000). This

is an interesting finding because our modern dairy cows typically receive a high concentrate

and low fibre diet.

There is increasing evidence that possible adverse effects of high energy diets on early

embryonic development may be programmed even before fertilization, so during the

acquisition of oocyte developmental competence in the follicle (O’Callaghan and Boland,

1999; McEvoy et al., 2001; Lozano et al., 2003). Oocytes collected from heifers (McEvoy et

al., 1997; Nolan et al., 1998; Yaakub et al., 1999b) or ewes (Lozano et al., 2003) that were

fed a low energy diet showed a higher developmental competence in vitro. Whether changed

pre- and/or postovulatory progesterone concentrations may explain the altered oocyte quality

remains a point of discussion (McEvoy et al., 1995; Yaakub et al., 1999b).

Other studies, however, demonstrated that the amount of energy intake is positively

correlated with oocyte quality through elevated insulin and IGF-I concentrations in serum and

in FF. As explained above, adequate insulin and IGF-I concentrations are beneficial for

follicular growth and oocyte quality (Landau et al., 2000; Armstrong et al., 2002a).

Furthermore, the bioavailability of IGF-I is optimized because the follicular concentrations of

IGFBP-2 and -4 decrease in animals fed a high energy diet (Armstrong et al., 2001; Comin et

al., 2002). Finally, it is important to mention that the amount of energy intake stimulates

oestrogen secretion by granulosa cells, having beneficial effects on oocyte quality (Armstrong

et al., 2002b; Comin et al., 2002).

Conclusively it can be said that several pathways are proposed associating dietary

energy intake and oocyte or embryo quality. Although the net effects of the reported pathways

on final gamete and embryo viability are contradictory, they certainly can form an interesting

clue for further research in the field of subfertility in high producing dairy cows.

High energy diets through fat supplementation

Modern dairy rations are often supplemented with rumen protected fat to increase the

energy intake early post partum (Beam and Butler, 1997). There are several possibilities

through which this could influence reproductive performance (reviewed by Staples et al.,

1998) but here again the reports about the final effects on fertility are contradictory (Staples,

1998; McNamara et al., 2003). In an attempt to improve the energy balance (DeFrain et al.,

Chapter 3: Reduced Fertility in Dairy Cows – A Review

35

2005), added fat stimulates milk production and thus energy loss, ultimately resulting in an

almost unchanged energy balance (McNamara et al., 2003). Supplemental fat increases the

size and the estradiol production of the preovulatory follicle (Lucy, 1991; Beam and Butler,

1997). This increased follicle size may have beneficial effects on both oocyte quality and

hence on corpus luteum function as has been explained above (Vasconcelos et al., 2001).

Moreover, the resulting higher high-density lipoprotein cholesterol concentrations in FF and

plasma may enhance progesterone secretion, supporting early embryo viablity (Ryan et al.,

1992; Lammoglia et al., 1996; McNamara et al., 2003). The more, depending on the type of

fatty acids, addition of fatty acids can reduce the secretion of prostaglandin metabolites and

hence may support the lifespan of the CL (Staples et al., 1998). Surprisingly, adding fat may

depress insulin concentrations and can even trigger an increased peripheral lypolysis (Staples

et al., 1998). The latter is however in contrast with the recent findings of DeFrain et al.

(2005).

Focusing on possible direct effects on oocyte and embryo, it has recently been

demonstrated that addition of 6% protected fat in the diet can alter the fatty acid profile both

in serum and in the FF (Adamiak et al., 2004a). Whether this is also true for the tubal or

uterine environment is not known. Furthermore, the change in FF fatty acid composition is

even reflected in the fatty acid content and profile of the COC proper (Adamiak et al., 2005).

When serum of these heifers was added to an in vitro embryo culture system, the resulting

embryos showed a higher total fatty acid content, an altered energy metabolism and a higher

incidence of apoptosis (Adamiak et al., 2004b). During prematuration and maturation in vivo,

there is a physiological lipid accumulation in the oocyte (Fair, 2003). Sata et al. (1999) and

Kim et al. (2001) demonstrated that oocytes and embryos in vitro are able to accumulate fatty

acids from their environment. This lipid accumulation is known to impair the quality of the

embryos by increasing their sensitivity to oxidative stress, chilling and cryopreservation (Abe

et al., 1999; Reis et al., 2003). The increased lipid accumulation has been associated with

suboptimal mitochondrial function and a deviation in the relative abundance of

developmentally important gene transcripts, all hampering the quality and hence the viability

of the embryo (Abe et al., 2002; Rizos et al., 2003). Whether excessive lipid accumulation is

present in oocytes and/or embryos from high yielding dairy cows due to fat inclusion in the

diet is not known. Further research should concentrate on this.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

36

From the above, it can be concluded that the plane of nutrition (energy content) does

have the potential to influence the oocyte developmental competence and the embryo viability

in a direct and/or indirect way. Frequently reported mechanisms are changes in progesterone,

oestrogen, IGF and insulin concentrations and alterations in the follicular and uterine

microenvironment. Concerning the specific reproductive physiology of high producing dairy

cows, our main assumption is that high energy diets turn out to be beneficial for oocyte

quality early post partum, most likely by reducing the depth and duration of the NEB

(Kendrick et al., 1999; Gwazdauskas et al., 2000). Later post partum however, when dairy

cows regain a positive energy balance, a (too) high intake of nutritional energy probably

results in an overstimulated and thus inferior oocyte (Armstrong et al., 2001) and in a

significant reduction of embryo quality. This may ultimately lead to reduced conception rates

and to a higher incidence of embryo mortality.

Dietary protein content

One of the strategies to support and stimulate milk production in early lactation is

increasing dietary crude protein levels (up to 19% or higher on a dry matter basis) (Butler

1998). A lot of attention has been paid to the protein part that is degradable by the rumen

bacteria and protozoa. An excessive intake of such degradable protein and a relative shortage

of energy (carbohydrates) to synthesize bacterial proteins will result in an accumulation of

excessive ammonia in the rumen (Sinclair et al., 2000b). This is absorbed through the ruminal

wall and will be converted into urea in the liver. This detoxification process costs energy and

thus may exacerbate the NEB early post partum, thereby reducing fertility (Butler, 1998). A

second source of urea produced by the liver is the deamination and metabolism of amino

acids.

In spite of the milk stimulating features, high dietary protein levels have been

associated with a hampered reproductive performance in most, but not all, studies (reviewed

by Butler, 2003 and Melendez et al., 2003). Futhermore, the possibility of confounding

between effects of protein intake and of the lactation induced NEB on reproductive

performance can make the correct interpretation of some study results difficult (Butler, 1998;

Gath et al., 1999; Kenny et al., 2001, Kenny et al., 2002a). High crude protein levels in the

diet do not appear to have deleterious impacts on the reinitiation of ovarian cyclicity in the

postpartum dairy cow. However, reduced conception rates (up to 30% in lactating cows and

20% in heifers) in animals with serum urea nitrogen concentrations exceeding 20 mg/dl (or

Chapter 3: Reduced Fertility in Dairy Cows – A Review

37

milk urea nitrogen concentrations > 19 mg/dl) have frequently been reported (Butler et al.,

1996; Westwood et al., 1998; Sinclair et al., 2000a; Melendez et al., 2003).

The major pathogenesis suggested for this conception failure (or early embryonic

mortality) is the potentially direct toxicity of the by-products of protein catabolism (ammonia

and urea) for oocyte and embryo. Murine embryos for example, cultured in presence of high

NH4+ concentrations displayed morphological, metabolic and genetic abnormalities (Gardner

and Lane, 1993; Lane and Gardner, 2003). However, it has also been documented that the

lactating dairy cow can metabolically adapt to prolonged high intakes of quickly degradable

nitrogen, probably leading to a neutralization of possible adverse effects of long-term high

urea concentrations on embryo growth (Dawuda et al., 2002; Laven et al., 2004). This could

not be confirmed in ewes (McEvoy et al., 1997).

High systemic urea concentrations have been associated with a reduction in uterine pH

(7.1 to 6.8) and an alteration in the ionic composition of uterine fluid both of which create a

hostile environment for the developing embryo (Jordan et al., 1983; Elrod and Butler, 1993).

This has recently been confirmed in vitro by Ocon and Hansen (2003). Furthermore,

endometrial cultures incubated with high amounts of urea secreted significantly higher

amounts of PGF2α compared to controls (Butler, 1998). Finally, it has been suggested that

such uterine environments are also hostile for the viability and motility of spermatozoa

(Westwood et al., 1998).

Fahey et al. (2001) did a very interesting observation. They saw a reduced embryo

quality in donor ewes fed high protein diets but diet type of embryo recipients had no effect

on survival of the transplanted embryos. Hence, they suggested that the effects of urea on

embryo quality are likely to be due to deleterious alterations in the environment of the follicle

and/or oviduct, rather than due to a changed uterine environment (Fahey et al., 2001;

Papadopoulos et al., 2001). Oocytes recovered from beef heifers that experienced elevated

ammonia concentrations both in serum and in FF, showed indeed a compromised

developmental competence in vitro (Sinclair et al., 2000a). Hammon et al. (2000a)

demonstrated that effects of ammonia on bovine oocytes in vitro depend on timing and

duration of exposure (Hammon et al., 2000b). Furthermore, since ammonia is also toxic for

granulosa cells in vitro, the cells lose their ability to support oocyte maturation in vitro

(Rooke et al., 2004). In contrast with ammonia, very little is known about the actual urea

Chapter 3: Reduced Fertility in Dairy Cows – A Review

38

concentrations in FF of high yielding dairy cows. Only Hammon et al. (2005) found a good

correlation for urea concentrations between plasma and follicular or uterine fluid in high

producing dairy cows early post partum. De Wit et al. (2001) reported a retarded nuclear

maturation and reduced fertilization and cleavage rates in bovine oocytes matured in the

presence of 6 mM urea probably through inhibition of the polymerization of tubulin into

microtubules. Similar toxic effects on oocyte maturation have been documented by Ocon and

Hansen (2003).

In conclusion, it can be said that notwithstanding all these sometimes conflicting

studies, there is evidence to assume that diets inducing high urea and ammonia concentrations

in blood can have detrimental effects on oocyte and embryo quality. This adverse effect can

act both at the level of the embryo (especially through ammonia) and the oocyte (particularly

through urea). The duration of such high protein diets is, however, also important because

cows usually are able to compensate for negative effects when such diets are fed for weeks.

Other possible clues affecting oocyte and embryo quality

Since decades, dairy cows have been strictly selected for high milk yield. Some

studies suggest that this genetic selection as such could also have an adverse effect on oocyte

quality. Snijders et al. (2000) collected oocytes from high and low genetic merit cows and

described a significantly lower developmental competence in vitro for oocytes originating

from high merit cows, irrespective of milk yield. Furthermore, a greater number of high than

of medium genetic merit cows were not pregnant at the end of the breeding season.

Surprisingly, there were no obvious differences in NEFA concentrations between both

groups, indicating no differences in EB (Snijders et al., 2001). In the latter study, no

differences were found in postpartum follicular development, suggesting that especially the

oocyte quality is impaired in high genetic merit cows, resulting in lower conception rates. In

contrast with the findings of Snijders et al. (2001), Veerkamp et al. (2003) and Horan et al.

(2005) suggested that high genetic merit for milk production is also associated with a more

severe NEB. This higher metabolic stress may explain poorer oocyte quality and

disappointing reproductive performance. Silke et al. (2002) did not find any significant

relationship between extent or pattern of late embryonic loss and genetic merit. Possible

Chapter 3: Reduced Fertility in Dairy Cows – A Review

39

influences of genetic selection are therefore more likely to operate on the oocyte or on the

early embryo (within two weeks of fertilization).

Along with selection towards higher milk production, modern dairy cows became

more sensitive to heat stress as their internal heat production significantly increased (reviewed

by Kadzere et al., 2002). In other words, the temperature at which dairy cows currently start

experiencing heat stress has shifted to a lower point. It has been proven that heat stress is

pernicious for reproduction (reviewed by De Rensis and Scaramuzzi, 2003). In addition to the

detrimental effects on energy balance, follicular dynamics and hypothalamus–pituitary–ovary

axis, it has also been suggested that high body temperatures can directly be toxic for the

oocyte and the embryo proper (Rocha et al., 1998).

It is generally accepted that high yielding dairy cows are more vulnerable for

metabolic and infectious diseases. Postpartum diseases are even suggested to be a more

important risk factor for reproductive failure compared to the NEB (Loeffler et al., 1999).

Especially the incidence of mastitis has increased probably due to a depressed immune system

early post partum (Ingvartsen et al., 2003). Mastitis early post partum but also intramammary

infections around the moment of AI are strongly associated with reduced conception rates

(Loeffler et al., 1999) and more specifically with higher risks of abortion within the next 90

days (Risco et al., 1999). The possible mechanisms involved in the link between infectious

diseases and embryonic mortality have been extensively reviewed by Hansen et al. (2004) and

are beyond the scope of this review.

Conclusions

Fertility in high yielding dairy cows is declining and there is increasing evidence to

assume that oocyte and embryo quality are two important factors in the complex pathogenesis

of reproductive failure. The oocyte and the embryo are vulnerable to all kinds of endocrine

and metabolic changes in their microenvironment. The knowledge about the specific

composition of that microenvironment in the follicle, the oviduct or the uterus is however

surprisingly scarce.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

40

Several mechanisms through which the oocyte and/or the embryo quality can be

affected in high yielding dairy cows have been proposed. Especially the NEB and the typical

milk stimulating diets can be associated with severe endocrine and metabolic alterations

which probably have the capacity to endanger the quality of the female gamete or embryo in a

direct or indirect way. Further research is of capital importance to gather scientific evidence

for the different clues which have been proposed in this review.

Chapter 3: Reduced Fertility in Dairy Cows – A Review

41

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Chapter 4

The Intrafollicular Environment in High Yielding Dairy Cows

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Chapter 4A

Metabolite and Ionic Composition of Follicular Fluid from different-sized Follicles and their Relationship

to Serum Concentrations in Dairy Cows

J.L.M.R. Leroy1, T. Vanholder1, J.R. Delanghe2, G. Opsomer1, A. Van Soom1, P.E.J. Bols3, A. de Kruif1

1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

2 Department of Clinical Chemistry, Faculty of Medicine, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium;

3 Laboratory for Veterinary Physiology, Departement of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk

Animal Reproduction Science 2004, 80: 201-211.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

58

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

59

Abstract

Metabolic changes in blood serum may be reflected in the biochemical composition of

follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to

examine the biochemical composition of follicular fluid harvested from different sized

follicles and its relationship with that of blood serum in dairy cattle.

Following slaughter, blood samples were collected from dairy cows (n = 30) and

follicular fluid aspirated from three size classes of non-atretic follicles (< 4 mm, 6 to 8 mm

and > 10 mm diameter). Samples remained independent between cows and between size

classes within cows. Serum and follicular fluid samples were assayed using commercial

clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and

metabolites (glucose, β-hydroxybutyrate, lactate, urea, total protein, triglycerides, non-

esterified fatty acids and total cholesterol).

Results showed that follicular fluid concentrations of glucose, β-hydroxybutyrate and

total cholesterol increased from small to large follicles and decreased for potassium, chloride,

lactate, urea and triglycerides. There was a significant concentration gradient for all variables

between their levels in serum and follicular fluid (P< 0.05). Significant correlations were

observed for chloride (r = 0.40), glucose (r = 0.56), β-hydroxybutyrate (r = 0.85), urea (r =

0.95) and total protein (r = 0.60) for all three follicle size classes and for triglycerides (r =

0.43), non-esterified fatty acids (r = 0.50) and total cholesterol (r = 0.42) for large follicles

(P< 0.05).

The results from the present study suggest that the oocyte and the granulosa cells of

dairy cows grow and mature in a biochemical environment that changes from small to large

follicles. Furthermore, the significant correlation between the composition of serum and

follicular fluid for the above mentioned metabolites suggests that metabolic changes in serum

levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the

oocyte and the granulosa cells.

Key Words Cattle, Fertility, Follicular fluid composition, Metabolites, Ovary, Serum concentrations

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

60

Introduction

Metabolic changes in serum concentrations, caused by a negative energy balance and a

high energy and high protein diet, occur in some high yielding dairy cows shortly after

parturition. These changes can induce pathological conditions such as hypoglycemia,

ketonemia, uremia, hyperlipidemia, hypercholesterolemia, and increased levels of non-

esterified fatty acids (NEFA) which may have a deleterious effect on fertility in dairy cows

(Butler and Smith, 1989; Harrison et al., 1990; Butler, 1998; Opsomer, 1999; Bertoni et al.,

2002). O’Callaghan and Boland (1999) suggested that the decline in fertility in high yielding

dairy cattle is mainly a problem of inferior oocyte and embryo quality, rather than being the

result of a disruption in gonadotropin secretion. Since it has already been shown that changes

in concentrations of gonadotropins, steroids and growth factors in follicular fluid of dairy

cows were linked with alterations in oocyte quality (Wehrman et al., 1993; Izadyar et al.,

1997; Driancourt and Thuel, 1998), it is not unlikely that metabolites which are present in the

follicular fluid can influence oocyte quality. Moreover, several in vitro studies showed that

metabolites, such as glucose, urea and β-hydroxybutyrate may influence the competence of

bovine oocytes to mature and, after fertilization, to grow to the blastocyst stage (Gomez,

1997; Hashimoto et al., 2000; Armstrong et al., 2001; De Wit et al., 2001).

The follicular fluid forms the biochemical environment of the oocyte before ovulation

(Edwards, 1974; Chang et al., 1976; Gosden et al., 1988; Józwik et al., 2001). It is an

avascular compartment within the mammalian ovary, separated from the perifollicular stroma

by the follicular wall, that constitutes a ‘blood-follicle barrier’ (Okuda et al., 1982;

Bagavandoss et al., 1983). Follicular fluid is in part an exsudate of serum and is in addition

partially composed of locally produced substances, which are related to the metabolic activity

of follicular cells (Gérard et al., 2002). This metabolic activity, together with the “barrier”

properties of the follicular wall, is changing significantly during the growth phase of the

follicle (Edwards, 1974; Zamboni, 1974; Bagavandoss et al., 1983; Wise, 1987; Gosden et al.,

1988). Therefore, a different biochemical composition of the follicular fluid in different sized

follicles can be expected.

Before focusing on possible effects of metabolic changes on follicle and oocyte

quality, it is necessary to determine physiological concentrations of the most common

metabolites in follicular fluid from differently sized follicles and to investigate to what extent

the serum and follicular fluid levels are correlated. Therefore, the aims of this study were 1) to

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

61

determine the chemical composition of follicular fluid in three different follicle sizes; 2) to

compare and correlate the biochemical composition of serum and of follicular fluid. Because

of their importance in the metabolism of dairy cows, concentrations of glucose, β-

hydroxybutyrate (β-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids

and total cholesterol were examined. In addition, the basic ionic composition (sodium,

potassium and chloride) of the follicular fluid and the serum was investigated.

Materials and Methods

Animals, ovary collection and sample preparation

Thirty adult dairy cows (Holstein Friesian) in good health and with normal

reproductive tracts upon macroscopical examination after slaughter were used for this study.

No pre-slaughter information was available for these animals. Collection of all samples was

performed on two different days of the same week. Ovaries were collected immediately after

slaughter and a blood sample was taken into capped disposable plastic tubes (unheparinized)

during exsanguination. Both ovaries and the blood sample were identified by using the eartag

number of the cow. Blood was allowed to coagulate for 20 minutes at 15° C and then cooled

at 4°C after which the ovaries and blood samples were transported on ice (4°C) to the

laboratory.

Ovaries were washed twice in cooled NaCl 0.9 % (4°C) and blotted dry. Three

different follicle classes, based on follicle diameter were considered for puncture: small

follicles (< 4 mm), medium follicles (6 to 8 mm) and large follicles (> 10 mm). Follicular

fluid was collected by aspiration with a 26 G needle and a 1ml syringe and pooled per follicle

class within cow. For each cow and follicle class, a different needle and syringe were used.

Hemorrhagic and morphologically atretic follicles, identified macroscopically according to

the method of Kruip and Dieleman (1982), were not sampled. Follicular fluid (at least 0.3 ml

per sample) was centrifuged (10,000 × g, 7 min) and the supernatant was collected for

analysis. The coagulated blood samples were centrifuged (1,400 × g, 30 min) within 1.5 hours

after collection and the serum was separated. Sample preparation was completed within 3

hours after slaughter. Samples were snap-frozen in CO2 ice (-65°C) and stored at –20°C until

biochemical assay, which took place within 3 days of ovary collection.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

62

Biochemical analyses

In each sample, the concentrations of sodium, potassium, chloride, glucose, lactate, β-

hydroxybutyrate (β-OHB), urea, total protein, triglycerides, non-esterified fatty acids (NEFA)

and total cholesterol were measured.

All analyses were performed at the Department of Clinical Chemistry, University

Hospital, Ghent, Belgium. The determination of metabolite levels in follicular fluid and blood

serum was done using wet chemistry techniques on two clinical chemistry autoanalysers

(Modular P and Hitachi 911, Roche Diagnostics). Sodium, potassium and chloride were

measured using indirect potentiometry. Measurements of glucose, lactate, urea, total protein,

triglycerides, and total cholesterol were performed using commercial photometric assays

(Roche Diagnostics GmbH, Mannheim, Germany). Commercial kits were also used for the

measurement of β-OHB (Sigma Diagnostics Inc., St. Louis, USA) and NEFA (Wako

Chemicals GmbH, Neuss, Germany). All measurements were carried out according to the

manufacturers’ instructions. The intra-assay and inter-assay coefficients of variation for all

analyses were below 5%.

Statistical analyses

Results are expressed as means ± SEM. The overall mean concentration ± SEM of

each metabolite and ion was calculated for follicular fluid and for blood serum in all cows.

The concentrations of each factor in the follicular fluid were compared between the three

follicle classes. A comparison was made for the levels in the follicular fluid of each follicle

class and those of serum. Concentrations in the three different follicle classes, were compared

using a Linear Mixed Effects Model (S-PLUS 2000, Cambridge, USA) in which the cow is

considered as a random effect. Correlation coefficients between follicular fluid and serum

levels of the same parameter were calculated and a paired samples t-test was performed to

compare concentrations found in the blood serum and the follicular fluid (SPSS 10.0 for

Windows, Chicago, Illinois, USA). A value of P < 0.05 was considered statistically

significant.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

63

Results

An average (± SEM) of 16.5 ± 1.3 small, 2.73 ± 0.3 medium and 1.33 ± 0.2 large

follicles were punctured. A larger number of small follicles was needed to obtain a sufficient

amount of follicular fluid for analysis. The concentration of ions and metabolites in the

follicular fluid from small, medium and large follicles is shown in Tables 1 and 2. Potassium,

chloride, lactate, urea and triglyceride concentrations decreased significantly as follicle size

increased (P< 0.05). The proportionate decrease was 61% for lactate and 43% for

triglycerides. Conversely, the concentrations of glucose β-OHB and total cholesterol

increased as follicle size increased and the values for glucose and β-OHB rose by 46% and

33%, respectively from small to large follicles. The increase in total cholesterol was smaller

but still significant (P< 0.05). The concentrations of glucose, lactate, β-OHB, urea, NEFA and

total cholesterol in all follicle classes varied considerably between animals.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

64

Table 1. Average concentrations (± SEM) of ions (sodium, potassium and chloride), glucose and lactate in follicular fluid of each follicle class and in serum of 30 dairy cows.

Na (mM) K (mM) Cl (mM) glucose (mM) lactate (mM) Small follicles (< 4 mm) 142.5 ± 0.34a 10.1 ± 0.21a *** 105.0 ± 0.50a ** 2.01 ± 0.10a *** 14.4 ± 0.35a ***

Medium follicles (6-8 mm) 142.4 ± 0.63a 7.9 ± 0.28b *** 104.0 ± 0.60b ** 2.85 ± 0.16b*** 9.4 ± 0.35b ***

Large follicles (>10 mm) 141.0 ± 1.14a 6.0 ± 0.23c *** 102.9 ± 0.76c ** 3.75 ± 0.18c *** 5.6 ± 0.37c ***

Blood serum 145.0 ± 0.641,2,3 * 5.0 ± 0.101,2,3 * 102.1 ± 0.641,2 * 4.77 ± 0.111,2,3 * 5.0 ± 0.321,2 *

a, b, c Data with different superscripts within a column differ significantly between follicle classes. 1, 2, 3 Concentrations in serum marked with 1, 2, 3

differ significantly from the concentrations found in small, medium and large follicles respectively. *, **, *** Statistical levels of significance are indicated with * (P<0.05), ** (P<0.01) and *** (P<0.001).

Table 2. Average concentrations (± SEM) of β- hydroxybutyrate, urea, total protein, triglycerides, non-esterified fatty acid and total cholesterol in follicular fluid of each follicle class and in serum of 30 dairy cows.

β-OHB (mM) urea (mM) total protein (g/dl)

triglycerides (mg/dl) NEFA (mM) total cholesterol

(mg/dl) Small follicles (< 4 mm) 0.29 ± 0.02a * 4.65 ± 0.35a *** 6.59 ± 0.10a 21.8 ± 0.60a *** 0.47 ± 0.04a 55.9 ± 3.39a *

Medium follicles (6-8 mm) 0.39 ± 0.03b * 4.30 ± 0.34b *** 6.36 ± 0.11a 16.6 ± 0.55b *** 0.50 ± 0.04a 62.7 ± 2.91b *

Large follicles (>10 mm) 0.43 ± 0.03c * 4.13 ± 0.34c *** 6.50 ± 0.10a 12.4 ± 0.45c *** 0.44 ± 0.08a 63.7 ± 3.23c *

Blood Serum 0.33 ± 0.022,3 * 4.00 ± 0.291,2 * 8.19 ± 0.111,2,3 * 17.0 ± 0.911,3 * 0.58 ± 0.083 * 147.9 ± 9.361,2,3 *

a, b, c Data with different superscripts within a column differ significantly between follicle classes. 1, 2, 3 Concentrations in serum marked with 1, 2, 3

differ significantly from the concentrations found in small, medium and large follicles respectively. *, **, *** Statistical levels of significance are indicated with * (P<0.05), ** (P<0.01) and *** (P<0.001).

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

65

The ion and metabolite serum levels of the thirty cows are presented in Tables 1 and 2.

The concentration of lactate, β-OHB, urea, triglyceride, NEFA and total cholesterol varied

considerably between animals (coefficient of variance = 35.5%, 39.4%, 41.5%, 29.1%, 75.9%

and 34.6% respectively). The serum concentrations of sodium, glucose, total protein and total

cholesterol were significantly higher than in small, medium and large follicles (P< 0.05). The

average total protein and cholesterol concentrations found in all follicular classes were 80%

and 41%, respectively, of the concentration found in serum. The concentration of glucose in

follicular fluid of small follicles was less than half of the level found in serum but only 21%

lower in large follicles. Potassium concentrations in serum were significantly lower than the

levels in all follicle classes and only half of the concentration found in small follicles (P<

0.05). Chloride, lactate and urea levels in small and medium sized follicles were significantly

higher than the serum levels but the values for large follicles were similar to those in serum

(P< 0.05). β-hydroxybutyrate was the only factor with a significantly lower concentration in

blood serum compared to the levels in medium and large follicles (P< 0.05). The serum

concentration of triglycerides was significantly lower than the level measured in small

follicles, (P< 0.05), similar in value to that in medium follicles but higher than in large

follicles (P< 0.05). NEFA concentrations were lower in all follicle sizes than in serum but the

difference was significant only for large follicles (P< 0.05).

The correlation coefficients between serum and follicular fluid of each follicle class

were calculated and significant (P< 0.05) correlation coefficients (r) are presented in Table 3.

High correlations between serum levels and levels in follicular fluid of all three follicular

classes were found for chloride, glucose, β-OHB, urea and total protein. The highest

correlation observed was for urea (Figure 1). The coefficient for β-OHB was also high in

medium and large follicles. There was no significant correlation between serum and follicular

fluid potassium levels for any follicle class. A significant correlation was observed between

concentrations in the follicular fluid of large follicles and in serum for all variates except

sodium, potassium and lactate.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

66

Table 3. Correlation coefficients (r) between ion and metabolite concentrations in follicular fluid and serum for each size of follicle. Values are presented for significant correlations (P<0.05; NS = not significant). Correlation bloodserum – foll. Fluid (r)

Na K Cl Glucose Lactate β-OHB Urea Total Protein Triglyceride NEFA Cholesterol

Serum - small foll. 0.47 NS 0.65 0.62 0.48 0.56 0.90 0.71 NS NS NS

Serum - medium foll. NS NS 0.74 0.48 NS 0.86 0.92 0.63 NS NS 0.66

Serum - large foll. NS NS 0.40 0.56 NS 0.85 0.95 0.60 0.43 0.50 0.42

urea concentration in serum (mM)

7654321

urea

con

cent

ratio

n in

larg

e fo

llicle

s (m

M)

8

7

6

5

4

3

2

1

0

Figure 1. Relationship between the urea concentration in serum and in follicular fluid of large follicles in 30 dairy cows (r = 0.953, P < 0.05).

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

67

Discussion

In the present studie, follicular fluid was sampled separately for each cow unlike

previous studies (Chang et al., 1976; Cabrera et al., 1985; Wise, 1987; Hammon et al., 2000),

where follicular fluid samples from different cows were pooled for technical reasons.

Obviously atretic follicles were excluded from aspiration (Chang et al., 1976; Homa and

Brown, 1992) but because up to 85% of all bovine antral follicles show signs of atresia, it is

possible that a proportion of follicles sampled in all follicle classes were atretic (Kruip and

Dieleman, 1982).

The sodium, chloride and potassium concentration in the follicular fluid were similar

to those given in other studies (Chang et al., 1976; Wise, 1987; Collins et al., 1997). The

concentration gradient for potassium between serum and follicular fluid suggests an active

inward transport of the cation (Gosden et al., 1988). Moreover, no correlation with serum was

found, indicating that potassium levels in follicular fluid may also be the result of local

metabolism.

Glucose plays an important role in ovarian metabolism since it is the major energy

source for the bovine, mouse and human ovary, possibly metabolized by the ovary through

anaerobic pathways leading to lactate formation (Leese and Lenton, 1990; Boland et al.,

1994; Rabiee et al., 1997b; Rabiee et al., 1999). We found that glucose and lactate

concentrations in follicular fluid were lower, respectively higher than those measured in

serum. Our data also show that the glucose concentration increases and lactate levels decrease

when the follicle diameter increases, which confirms the results of Landau et al. (2000) in

dairy cows and Chang et al. (1976) in sows. This could indicate that glucose metabolism is

less intensive in large follicles compared to small ones, resulting in a lower consumption of

glucose from follicular fluid and in a reduced secretion of lactate into the follicular fluid. An

increasing amount of follicular fluid is a second explanation for the increase in glucose and

the decrease in lactate, since in large follicles a relatively smaller number of granulosa cells

consumes glucose from and secretes lactate into a relatively larger amount of follicular fluid

(McNatty et al., 1978; Gosden et al., 1988). A further reason for this observation could be the

increased permeability of the blood-follicle barrier during follicular growth (Edwards, 1974;

Zamboni, 1974; Okuda et al., 1982; Bagavandoss et al., 1983). Consequently, an equilibrium

between the vascular compartment and follicular fluid can be achieved more easily in large

follicles. Leese and Lenton (1990) concluded that the glucose and lactate concentrations in

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

68

follicular fluid in women is a result of both glycolysis taking place in the mural granulosa

cells and influx of the same molecules from the plasma into the fluid. This is supported by our

data which show good correlations between blood serum and follicular fluid for glucose at

each follicular size. Hence, these correlation coefficients suggest that hypoglycemia may

reduce the glucose content in follicular fluid but this needs to be confirmed by further

investigation in hypoglycemic dairy cows. When interpreting these kind of data for

potassium, glucose and lactate, it is important to consider postmortem changes which can

induce increased potassium concentrations by leakage from damaged cells and turnover of

glucose to lactate by anaerobic glycolysis (Chang et al., 1976; Gosden et al., 1988). In a

preliminary study, no other metabolite concentrations changed significantly during transport

to the laboratory (Leroy et al., unpublished).

The strong correlation between β-OHB levels in follicular fluid of all three classes of

follicles and serum suggest that elevated levels in the serum of dairy cows (ketonemia) may

cause similar changes in the follicular fluid. Further studies with ketonemic cows are required

to confirm this conclusion. The significant increase of β-OHB from small to large follicles is

possibly caused by a local secretion of this ketone body by the follicle cells. This needs,

however, further investigation. Rabiee et al. (1999) showed that β-OHB can be used or

converted by the bovine and ovine ovary (Rabiee et al., 1997a; Rabiee et al., 1997b; Rabiee et

al., 1999).

The observation that follicular levels of urea were different between all follicle classes

was not expected. In small and medium sized follicles, the concentration of urea was

significantly higher than the serum concentrations, possibly caused by an active inward

transport or a local urea production by the follicle cells. Like Collins et al. (1997) in mares,

we found a very high correlation for urea between follicular fluid and blood serum. Reports

about the effect of elevated urea levels on fertility are contradictory, although all authors

agree that the possible adverse effect of diet induced elevated urea levels must act at the level

of the oocyte (Sinclair et al., 2000; Dawuda et al., 2002). The high correlation between

follicular fluid and serum suggests that elevated serum urea levels of dairy cows may be

reflected in the follicular fluid and hence, may influence oocyte quality. This requires further

investigation.

The total protein content of the follicular fluid did not differ between follicle classes

and was about 75% of that present in serum. The high correlation between total protein

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

69

content in follicular fluid and in serum suggests that a substantial part of the protein content in

follicular fluid originates from serum (Edwards, 1974; Wise, 1987).

The amount of triglycerides decreased from small to large follicles. Triglyceride levels

in small follicles were significantly higher than in serum and significantly lower in large

follicles. There was a significant correlation between the follicular fluid and the serum levels

of triglycerides only in large follicles. These data favour the idea that follicular triglyceride

levels are mainly a result of local metabolic processes. A relatively stable concentration of

triglycerides is maintained in the bovine ovarian follicle, regardless of increases in serum due

to physiological status or diet (Wehrman et al., 1991). Triglycerides probably do not pass

through the follicular membrane since they are transported primarily by the very low-density

lipoprotein fraction (VLDL) which is too large to pass through this barrier (Grummer and

Carroll, 1988). In follicular fluid, triglycerides may serve as an alternative energy source since

in vitro cultured cells can absorb and consume triglycerides out of the medium. Also oocytes

and embryos show lipid accumulation when cultured in triglyceride containing media (Kim et

al., 2001, Abe et al., 2002).

This also applies for non-esterified fatty acids (Abe et al., 2002). Non-esterified fatty

acids are transported in the blood by means of albumin and this complex can easily penetrate

the follicular wall. NEFA concentrations did not differ between the different follicle classes

and tended to be higher in serum. A significant correlation with serum levels of NEFA was

observed only for large follicles.

Total cholesterol in follicular fluid was about 42 % of the concentration found in

blood serum and there was a significant increase of the total cholesterol content from small to

large follicles. Cholesterol, present in follicular fluid, is bound to the high density lipoprotein

fraction (HDL) because the only other cholesterol-containing lipoprotein fraction, the low

density lipoprotein fraction (LDL), is too large to pass the blood-follicle barrier (Puppione,

1977; Grummer and Carrol, 1988; Wehrman et al., 1991; Bauchart, 1993). The higher total

cholesterol concentration in large follicles can be explained by the increased permeability of

the follicular wall in that follicle class, permitting the entrance of the larger HDL fraction

(Bagavandoss et al., 1983; Wehrman et al., 1991). A significant correlation between follicular

fluid and serum levels of cholesterol was noticed in medium and large follicles.

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

70

Conclusions

In conclusion, we observed an increase in the concentrations of glucose, β-OHB and

total cholesterol and a decrease in the concentrations of potassium, chloride, lactate, urea and

triglycerides in the follicular fluid from small to large follicles. Although we have not

evaluated changes in the biochemical composition of follicular fluid from one follicle during

its growth, our data, however, suggest that what we found in the different sized follicles

reflects what happens during the follicular growth. Our findings suggest that the oocyte and

the granulosa cells of dairy cows grow and mature in a changing biochemical environment

from small to large follicles and that this environment is correlated with serum levels of the

ions and metabolites studied here. Further research should concentrate on changes in these

metabolites in the follicular fluid of high producing dairy cows in vivo during the first weeks

of lactation and their effect on oocyte quality.

Acknowledgments

The authors thank Dr. M Berth for his excellent scientific and technical support, Dr. K

Moerloose for the critical reading of the manuscript, and Dr. J Dewulf and Dr. S De Vliegher

for statistical analyses. This research was funded by the Institute for the Promotion of

Innovation by Science and Technology in Flanders (Grant no° 1236).

Chapter 4A: Metabolite and Ionic Composition of Follicular Fluid

71

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Armstrong, D.G., McEvoy, T.G., Baxter, G., Robinson, J.J., Hogg, C.O., Woad, K.J., Webb, R., Sinclair, K.D.,2001. Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insuline-like growth factor system. Biol. Reprod. 64, 1624-1632.

Bagavandoss, P., Midgley, A.R., Wicha, M., 1983. Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy. J. Histochem. Cytochem., 633-640.

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Cabrera, V.O., Andino, N.V., Mateo De Acosta, O., 1985. Protein electrophoresis patterns of bovine and porcine ovarian follicular fluid. J. Endocrinol. Invest. 8, 489-493.

Chang, S.C.S., Jones, J.D., Ellefson, R.D., Ryan, R.J., 1976. The porcine ovarian follicle: I. Selected chemical analysis of follicular fluid at different developmental stages. Biol. Reprod. 15, 321-328.

Collins, A., Palmer, E., Bezard, J., Burke, J., Duchamp, G., Buckley, J. A., 1997. A comparison of the biochemical composition of equine follicular fluid and serum at four different stages of the follicular cycle. Equine Vet. J. Suppl. 25, 12-16.

Dawuda, P.M., Scaramuzzi, R.J., Leese, H.J., Hall, C.J., Peters, A.R., Drew, S.B., Wathes, D.C., 2002. Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows. Theriogenology 58, 1443-1455.

De Wit, A.A.C., Cesar, M.L.F., Kruip, T.A.M., 2001. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes. J. Dairy Sci. 84, 1800-1804.

Driancourt, M.A., Thuel, B., 1998. Control of oocyte growth and maturation by follicular cells and molecules present in follicular fluid. A review. Reprod. Nutr. Dev. 345-362.

Edwards, R.G., 1974. Follicular fluid. J. Reprod. Fertil. 37, 189-219. Gérard, N., Loiseau, S., Duchamp, G., Seguin, F., 2002. Analysis of the variations of follicular fluid

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Gomez, E., 1997. Acetoacetate and β-D-hydroxybutyrate as energy substrates during early bovine embryo development in vitro. Theriogenology 48, 63-74.

Gosden, R.G., Hunter, R.H.F., Telfer, E., Torrance, C., Brown, N., 1988. Physiological factors underlying the formation of ovarian follicular fluid. J. Reprod. Fertil. 82, 813-825.

Grummer, R.R., Carroll, D.J., 1988. A review of lipoprotein cholesterol metabolism: importance to ovarian function. J. Anim. Sci. 66, 3160-3173.

Hammon, D.S., Wang, S., Holyoak, G.R., 2000. Ammonia concentration in bovine follicular fluid and its effect during in vitro maturation on subsequent embryo development. Anim. Reprod. Sci. 58, 1-8.

Harrison, R.O., Ford, S.P., Young, J.W., Conley, J.W., Freeman, A.E., 1990. Increased milk production versus reproductive and energy status of high producing dairy cows. J. Dairy Sci. 73, 2749-2758.

Hashimoto, S., Minami, N., Yamada, M., Imai, H., 2000. Excessive concentration of glucose during in vitro maturation impairs the developmental competence of bovine oocytes after in vitro fertilisation: relevance to intracellular reactive oxygen species and glutathione contents. Mol. Reprod. Dev. 56, 520-526.

Homa, S.T., Brown, C.A., 1992. Changes in linoleic acid during follicular development and inhibition of spontaneous breakdown of germinal vesicles in cumulus-free bovine oocytes. J. Reprod. Fert. 94, 153-160.

Izadyar, F., Van Tol, H.T., Colenbrander, B., Bevers, M.M., 1997. Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through cumulus cells and not mediated by IGF-I. Mol. Reprod. Dev. 47, 175-180.

Józwik, M., Józwik, M., Wolczynski, S., Józwik, M., Szamatowicz, M., 2001. Ammonia concentration in human preovulatoy ovarian follicles. Eur. J. Obstet. Gynecol. and Reprod. Biol. 94, 256-260.

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McNatty, K.P., Smith, D.M., Makris, A., Osathanondh, R., Ryan, K.J., 1978. The microenvironment of the human antral follicle: interrelationships among the steroid levels in the antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro. J. Clin. Endocrinol. Metab. 49, 851-860.

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Chapter 4B

Metabolic Changes in Follicular Fluid of the Dominant Follicle in High Yielding Dairy Cows

Early Post Partum

J.L.M.R. Leroy1, T. Vanholder1, J.R. Delanghe2, G. Opsomer1, A. Van Soom1, P.E.J. Bols3, J Dewulf1, A. de Kruif1

1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

2 Department of Clinical Chemistry, Faculty of Medicine, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium;

3 Laboratory for Veterinary Physiology, Departement of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk

Theriogenology 2004, 62: 1131-1143

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

76

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

77

Abstract

Characteristics of the intrafollicular environment to which the preovulatory oocyte is

exposed may be one of the major factors determining subsequent fertility. The aim of our

study was to examine to what extent metabolic changes that occur in early postpartum high

yielding dairy cows are reflected in the follicular fluid (FF) of the dominant follicle (> 8mm).

Nine blood samples were taken per cow from nine high yielding dairy cows between 7

days before and 46 days after parturition. From day 14 post partum on and together with

blood sampling, FF samples of the largest follicle were collected from the same cows by

means of transvaginal follicle aspiration. Serum and FF samples were analysed using

commercial clinical and photometric chemistry assays for glucose, β-hydroxybutyrate (β-

OHB), urea, total protein (TP), triglycerides (TG), non-esterified fatty acids (NEFA) and total

cholesterol (TC).

All cows lost body condition during the experimental period (0.94 ± 0.09 points)

illustrating a negative energy balance during the experimental period. In FF, glucose

concentrations were significantly higher and the TP, TG, NEFA and TC concentrations were

significantly lower than in serum (P < 0.05). The concentrations of glucose, β-OHB, urea and

TC in serum and in FF changed significantly over time (P < 0.05). Throughout the study,

changes of all metabolites in serum were reflected by similar changes in FF. Especially for

glucose, β-OHB and urea the correlations were remarkably high.

The results from the present study confirm that the typical metabolic adaptations

which can be found in serum of high yielding dairy cows shortly post partum, are reflected in

follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa

cells.

Key Words

Dairy Cow, Fertility, Follicular fluid, High producing, Metabolites.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

78

Introduction

Over the past decades there has been a substantial decline in the reproductive

performance of high producing dairy cows (Butler, 2003; López-Gatius, 2003). The negative

energy balance (NEB) early post partum and the intake of a high-protein and high-energy diet,

are known to cause hormonal and biochemical changes in these cows (O’Callaghan and

Boland, 1999; Boland et al., 2001; Butler, 2000; Walters et al., 2002a; Walters et al., 2002b).

Physiological adaptations during the onset of lactation such as hypoglycemia, ketonemia,

uremia, increased levels of non-esterified fatty acids and subsequent lipid accumulation in the

liver can become pathological and hence may interfere with reproductive performance (Butler

and Smith, 1989; Harrison et al., 1990; Butler, 1998; Opsomer, 1999; Bertoni et al., 2002).

However, it is not completely clear how these biochemical changes are able to influence

reproductive outcome. O’Callaghan and Boland (1999) suggested that the decline in fertility

is mainly a problem of inferior oocyte and embryo quality. Altered concentrations of

gonadotropins, steroids and growth factors in follicular fluid (FF) have already been linked

with changes in oocyte quality (Wehrman et al., 1993; Izadyar et al., 1997; Driancourt and

Thuel, 1998). Furthermore, diets high in energy and protein, which are typically supplied to

high yielding cows, are known to alter oocyte and subsequent embryo quality probably

through a changed composition of the follicular and/or fallopian tubal fluid (O’Callaghan and

Boland, 1999; Armstrong et al., 2001; Boland et al., 2001; Dawuda et al., 2002; Kenny et al.,

2002).

A new approach to investigate the contribution of these metabolic adaptations to the

pathogenesis of reduced fertility is to mimic biochemical changes in an in vitro model to

study the possible effects on in vitro granulosa cell function (Vanholder et al., 2003) or on

oocyte maturation, fertilisation and subsequent embryo yield (Leroy et al., 2003). Such

studies already showed that parameters, such as glucose, urea and β-hydroxybutyrate (β-

OHB) may influence the competence of bovine oocytes to mature and, following fertilization,

to develop to the blastocyst stage (Gomez, 1997; Hashimoto et al., 2000;Armstrong et al.,

2001; De Wit et al., 2001).

However, despite all these interesting data one important step has not been

investigated yet. Little is known about the implications of postpartum biochemical serum

changes in the composition of the FF, embedding the granulosa cells and supporting the

oocyte to undergo the fine tuned processes of growth, pre- and final maturation. In a previous

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

79

study on dairy cows post mortem, we demonstrated that the oocyte and the granulosa cells

grow and mature in a changing biochemical environment from small to large follicles. The

biochemical composition of the FF was well correlated with the serum composition (Leroy et

al., 2004). However, these findings needed to be confirmed in living, high yielding dairy

cows which were subjected to repeated sampling of serum and FF during the first weeks after

calving.

Therefore, the objectives of the present study were 1) to investigate if the metabolite

concentrations in serum and FF are significantly different and 2) to assess to what extent

metabolic changes that occur in high yielding dairy cows early post partum are reflected in the

FF. Concentrations of glucose, β-OHB, urea, total protein (TP), triglycerides (TG), non-

esterified fatty acids (NEFA) and total cholesterol (TC) were examined because of their

importance in the metabolism of dairy cows.

Materials and Methods

Animals

Nine healthy multiparous Holstein-Friesian cows were used in this study. All

experimental work was performed at the research dairy farm of the University of Ghent

(Biocentrum Agri-Vet, Melle, Belgium) following protocol approval by the Ethical

Committee of the Faculty of Veterinary Medicine (Ghent University). Cows were milked on

average 2.2 times a day by means of an automated voluntary milking system. On the farm, the

average milk yield per cow was 9200 kg milk (3.90 % fat and 3.45 % protein) during 305

days of lactation.

After an average dry period of 55 ± 12 days in which the animals were fed corn silage,

straw ad libitum, dry cow minerals, magnesium and soybean meal, all cows calved normally

between September 2002 and April 2003. During the experimental period (first 50 days of

lactation), all cows were housed in a loose stable with cubicles and were fed according to their

requirements for maintenance and milk production. The ration consisted of high quality

roughages (corn silage and grass silage, sugar beet pulp), soybean meal and concentrates.

Propylene glycol (500 ml daily) was routinely given as an oral drench to all cows during the

first 3 days of lactation. One animal, that suffered from retained plancenta, was treated once

iu with tetracycline (4 g) at 24 hours post partum. The fetal membranes have been removed

the day after. One other animal suffered from a mild mastitis in one quarter. After an

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

80

intramammary treatment with antibiotics, the animal was cured within 3 days, well before the

first ovarian puncture. During the experimental period, daily milk yield (± SEM) of the

selected cows averaged 38.2 ± 2.7 kg per cow, ranging from 30.1 kg to 53.5 kg. Body

condition scores (BCS), based on the notation of Edmondson et al. (1989), were recorded by

the same experienced person using a score on a scale of 1-5 (with 0.25 increments).

Blood sampling

Blood samples were collected from each animal 7 days prior to the expected calving

date and at day 0, 11, 14, 20, 26, 33, 40 and 46 post partum. Blood was sampled from the

jugular vein into two unheparinized, silicone coated tubes (Venoject®, Autosep®, Gel + Clot.

Act.; Terumo Europe N.V., Leuven, Belgium) and in one tube with sodium fluoride (NaF)

(Venoject®, Terumo Europe N.V., Leuven, Belgium). Samples were taken between 1h00 pm

and 3h00 pm, two hours after automated milking at the latest and before any rectal

examination or ultrasound transvaginal aspiration. The coagulated blood samples and the

blood samples on NaF were centrifuged (1,400 × g, 30 min) within 1.5 hours after collection

and the serum or plasma was collected.

Ultrasound examination and follicular fluid sampling

On day 11 post partum all animals showed normal uterine involution and follicular

growth on one or both ovaries upon ultrasound examination. On day 14, 20, 26, 33, 40 and 46

post partum (experimental sessions), dominant follicles with a diameter greater than 0.8 cm

were subjected to ultrasound guided transvaginal aspiration as described by Bols et al. (1995).

Briefly, the rectum was emptied and the perineum and external genitalia were cleansed

carefully. Cows received epidural anaesthesia (5 cc Procain HCl 4% with adrenalin, Eurovet

N.V., Heusden-Zolder, Belgium) to prevent them from straining. An OPU device, equipped

with a 5.0 MHz mechanical multi angle probe transducer (Esaote / Pie Medical NV,

Maastricht, The Netherlands) and a needle guidance system (Pie Medical) was inserted

vaginally and both ovaries were visualized through rectal manipulation. Before aspiration, the

number of different sized follicles (< 4 mm, 4-8 mm, > 8 mm) was recorded per ovary.

Subsequently, follicles were punctured and the FF was aspirated by a second operator,

following positioning along the biopsy line. Hereto, the needle (TERUMO NEOLUS 21GX2”

0.8X50, Leuven, Belgium) was attached by means of a stainless steal connector to an extra

thin silicon tube (inner diameter: 0.034”; Silclear TM Tubing, Multi Purpose Medical Grade

Silicone Tubing, Degania Silicone/Israel) and a 5 ml syringe (PlastipakTM, Madrid, Spain)

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

81

was used to aspirate the FF from the punctured follicle. The largest and the second largest

follicle (if present) with a diameter greater than 8 mm were aspirated. Attention was paid to

prevent blood contamination. Follicular fluid samples with obvious blood contamination were

omitted from further processing. The collected FF was cooled immediately (4°C).

Subsequently, FF samples were centrifuged (10,000 × g, 10 min) and the supernatant was

collected for analysis. Sample preparation was completed within 3 hours after each session.

Blood and FF samples were snap-frozen in CO2 ice (-65°C) and stored at –22°C until

biochemical assay.

Hormone analyses

To identify possible atresia of the punctured follicles, a progesterone (P4) and

estradiol-17β (E) analysis was carried out on each FF sample. Follicular fluid with a ratio

E/P4 < 1 was considered to originate from an obviously atretic follicle and was omitted for

biochemical analysis (Wise, 1987; Badinga et al., 1992; Landau et al., 2000). Progesterone

was extracted with petroleum ether from 20 μl of FF that was diluted 3 times. Estradiol was

extracted with diethyl ether from 20 μl of FF that was diluted 100 times. Estradiol-17β and

progesterone concentrations were assessed through a radio immuno assay (RIA), as described

earlier (Henry et al., 1987). The detection limit for E was 5 pg and the intra- and inter-assay

coefficients of variation were 5.75% and 8.30% respectively. The RIA for P4 had a detection

limit of 5 pg and intra- and inter-assay coefficients of variation of 7.05 % and 8.75 %

respectively.

Biochemical analyses

In each sample, the concentrations of glucose, β-OHB, urea, TP, TG, NEFA and TC

were measured. All analyses were performed at the Department of Clinical Chemistry,

University Hospital, Ghent, Belgium. The determination of metabolite levels in FF and blood

serum was done using wet chemistry techniques on two clinical chemistry automated

analysers (Modular P and Hitachi 911, Roche Diagnostics). Measurements of glucose, urea,

TP, TG, and TC were performed using commercial photometric assays (Roche Diagnostics

GmbH, Mannheim, Germany). Commercial kits were also used for the measurement of β-

OHB (Sigma Diagnostics Inc., St. Louis, USA) and NEFA (Wako Chemicals GmbH, Neuss,

Germany). All measurements were carried out according to the manufacturer’s instructions.

The intra-assay and inter-assay coefficients of variation for all analyses were below 5%.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

82

Statistical analyses

All data are presented as means ± S.E.M. Only the data collected from day 14 post

partum onwards (availability of both serum and FF samples) were used in the statistical

model. However, to have an overall view on serological changes pre- and postpartum, the

serum concentrations before day 14 post partum are presented in the figures. Since data were

correlated (repeated measurements in the same animal) a linear mixed effects model with cow

as random factor (s-Plus 2000, Cambridge, USA) was used 1) to investigate if the metabolite

concentrations were significantly different in the serum compared to FF (effect of

compartment), 2) to evaluate if the metabolite concentrations changed significantly over time

(effect of days postpartum) and 3) to estimate to what extent the changes in serum and FF

metabolite concentrations were parallel during the test period (interactions compartment X

time postpartum). A non significant compartment X time interaction indicates that the

concentrations of the metabolite of interest changes similarly over time in both compartments.

The data for NEFA and TC concentrations were log-transformed for normality reasons.

Normal correlations (Pearson) were calculated between serum and FF levels at each moment

post partum (SPSS 11.0 for Windows, Chicago, IL, USA). A paired samples t-test was used to

compare milk yield and BCS at the onset and at the end of the experimental period (SPSS

11.0 for Windows, Chicago, IL, USA). Values of P < 0.05 were considered statistically

significant.

Results

From 7 days prior to the expected parturition (varying between 11 to 3 days prior to

the real day of parturition) up to 46 days post partum, all cows showed a significant loss in

BCS (an average BC loss of 0.94 ± 0.09 points) (P < 0.05) (Figure 1). From day 11 up to day

46 post partum, the average daily milk yield increased with 7.2 kg, from 35.7 ± 2.3 kg to 42.9

± 3.5 kg. An average of 1.2 ± 0.1 follicles were punctured per session per cow and a total

amount of 1.62 ± 0.14 ml FF was aspirated. Due to atresia, based on the E/P4 ratio in the FF,

or because of blood contamination, 5 FF samples (5.8% of all FF samples) were excluded

from any further analysis. In all analysed FF samples, the average (± SEM) E/P4 ratio was

15.6 ± 2.7.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

83

2

2.5

3

3.5

-7 0 11 14 20 26 33 40 46

days to parturition

Bod

y C

ondi

tion

Scor

e

Figure 1. Average (± SEM) body condition scores of 9 high yielding dairy cows during the experimental period

The profiles of the concentrations of glucose, β-OHB, urea, TP, TG, NEFA and TC in

serum and in the FF throughout the experimental period are shown in Figures 2 to 8. The P-

values for the effect of compartment, time and interactions compartment X time are reported

in Table 1. Concentrations of TP and TC in serum transiently decreased at parturition.

Glucose concentrations in serum decreased during the first two weeks after parturition and

increased during the period thereafter. The TP concentrations stayed relatively stable after two

weeks post partum. After a significant decrease at parturition, the serum concentration of TG

remained low and TC concentrations gradually rose. Urea concentrations in serum doubled

around parturition and remained relatively stable for the rest of the experimental period.

Serum β-OHB concentrations gradually increased after parturition and peaked at day 33 (1.62

± 0.34 mM). Besides a marked increase up to 0.50 ± 0.08 mM in the serum concentration of

NEFA around parturition, there was no significant change in the profile during the period

thereafter (P = 0.05).

Throughout the study the concentration of glucose in the FF was circa 0.34 mM higher than in

serum. The opposite relation was found for TP, TG, NEFA and TC (Table 1, P-values of

compartment effect).

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

84

Table 1. Results of the linear mixed effects model (repeated measurement). Significances of the effect of compartment, time and of the interaction compartment X time on the concentrations of the measured parameters (S-Plus, P-values).

P-values Compartment effect (serum or follicular fluid)

Time effect (days post partum)

Compartment X time interaction

Glucose < 0.01 < 0.01 0.40 β-OHB 0.48 0.02 0.91 Urea 0.25 0.06 0.56 Total Protein < 0.01 0.60 0.73 Triglycerides < 0.01 0.80 0.15 Log(NEFA) 0.02 0.05 0.86 Log(Cholesterol) < 0.01 < 0.01 0.02

Bold values indicate significant effects (P < 0.05).

Because none of the calculated interactions (compartment X time) were significant,

changes in the FF for glucose, β-OHB, urea, TP, TG and NEFA during the study period were

similar as changes of the same metabolite in serum (same slopes of profiles) (Table 1). For

TC however, there was a significant compartment X time interaction (different slopes of

profiles) (Table 1, Figure 8).

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

85

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

-7 0 11 14 20 26 33 40 46

days to parturition

Glu

cose

(mM

)

.

0

0.5

1

1.5

2

2.5

-7 0 11 14 20 26 33 40 46

days to parturition

B-h

ydro

xybu

tyra

te (m

M)

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

-7 0 11 14 20 26 33 40 46

days to parturition

Ure

a (m

M)

Figure 2. Average (± SEM) glucose concentrations (mM) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period

Figure 3. Average (± SEM) β-hydroxybutyrate concentrations (mM) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Figure 4. Average (± SEM) urea concentrations (mM) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

86

5.5

6

6.5

7

7.5

8

-7 0 11 14 20 26 33 40 46

days to parturition

tota

l pro

tein

(g/d

l)

0

5

10

15

20

25

30

-7 0 11 14 20 26 33 40 46

days to parturition

trig

lyce

rides

(mg/

dl)

0

0.1

0.2

0.3

0.4

0.5

0.6

-7 0 11 14 20 26 33 40 46

days to parturition

NEF

A (m

M)

Figure 5. Average (± SEM) total protein concentrations (g/dl) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Figure 6. Average (± SEM) triglyceride concentrations (mg/dl) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Figure 7. Average (± SEM) NEFA concentrations (mM) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

87

40

60

80

100

120

140

160

180

200

-7 0 11 14 20 26 33 40 46days to parturition

Cho

lest

erol

(mg/

dl)

Correlations between serum and FF levels were also calculated per experimental

session post partum (without taking any time effect into consideration) and correlation

coefficients are shown in Table 2. A good correlation existed at almost all experimental

sessions for glucose, β-OHB, urea and TC.

Table 2. Correlation coefficients (r’s) between metabolite concentrations in follicular fluid and serum per experimental session in nine dairy cows. Correlations

(r) Glucose β-OHB Urea Total Protein Triglycerides NEFA Total

Cholesterol14 days pp 0.834* 0.996** NS NS 0.892** NS NS 20 days pp 0.788* 0.972** 0.929** NS NS NS 0.787* 26 days pp 0.733* 0.992** 0.987** NS 0.872** NS NS 33 days pp 0.925** 0.976** 0.990** NS 0.710* 0.845** 0.918** 40 days pp 0.916** 0.971** 0.973** 0.860** NS NS 0.862** 46 days pp 0.901* 1.00** 0.782* NS NS 0.908* 0.948*

Values are presented for significant correlations (* P < 0.05; ** P < 0.01; NS: not significant).

Figure 8. Average (± SEM) total cholesterol concentrations (mg/dl) in serum and follicular fluid of 9 high yielding dairy cows during the experimental period.

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

88

Discussion

Characteristics of the intrafollicular environment in which the preovulatory oocyte

grows and matures, may be one of the major factors determining subsequent fertility. To our

knowledge, this is the first time that biochemical serum changes, are compared to changes in

the FF in high yielding dairy cows early post partum. However, FF was only sampled from

day 14 post partum onwards because of reduced approachability of the ovaries in the

puerperium.

The concentration of glucose in serum showed a marked decrease during the first two

weeks of lactation followed by a steady increase in the period thereafter. Butler (2000) and

others described a decrease in serum glucose concentration during the period of negative

energy balance but other studies could not confirm this finding (Rukkwamsuk et al., 1999; Ill-

Hwa and Gook-Hyun, 2003). Landau et al. (2000) showed that a low intrafollicular glucose

concentration coincides with a low insulin concentration in the FF and that the levels of both

parameters are influenced by the diet. We found that the FF glucose concentration was closely

correlated with the serum levels and that it was consistently higher than in serum, possibly

due to an active inward transport. This finding strongly suggest that postpartum changes in

glycaemia are well reflected in the FF of dominant follicles but that the oocyte is more or less

protected from low glucose concentrations.

Glucose and β-OHB concentrations were negatively correlated, both in serum and in

FF (r = -0.56 and -0.83, respectively) suggesting that β-OHB is a good indicator for

hypoglycemia. The average serum β-OHB concentration peaked at 33 days post partum (1.62

mM). This concentration has been associated with signs of subclinical ketosis (Busato et al.,

2002). The β-OHB concentrations in serum and in the FF were similar and both slopes of

profile were exactly the same. Based on these strong correlations between serum and FF

concentrations throughout this study, it can be stated that elevated β-OHB levels in serum

(ketonemia) will appear in the FF as well. These findings confirm what has been assumed in

earlier work (Leroy et al., 2004).

Urea concentrations showed an important increase during the first week post partum

and remained high in the weeks thereafter. Collins et al. (1997) as well as our own group

(Leroy et al., 2004) found in respectively mares and cows post mortem a very high correlation

for urea between FF and blood serum during the first weeks of lactation. Although reports

about the effect of elevated urea levels on fertility are contradictory, most authors agree that

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

89

the possible adverse effect of diet induced elevated urea levels must act at the level of the

oocyte (Sinclair et al., 2000; De Wit et al., 2001; Dawuda et al., 2002). Based on our results,

it can be stated that elevated serum urea levels are reflected in the FF and hence, may affect

oocyte quality.

The TP content in the FF remained stable during the experimental period and was

about 80% of that present in serum. The similar slopes of the TP profiles in serum and FF

during the study indicate that a substantial part of the protein content in FF originates from

serum (Wise, 1987; Edwards, 1974).

During a period of NEB, lipolysis causes an increase of NEFA concentrations in

serum during the first weeks postpartum. The serum NEFA levels in our study remained

relatively high during the experimental period. The repeated measurement analysis of our data

revealed that the NEFA concentrations in FF parallelled those in serum. This finding has been

confirmed by previous studies on cows subjected to an acute dietary restriction to mimic a

period of NEB (Comin et al., 2002; Jorritsma et al., 2003). However, the FF concentrations

remained consistently lower than the levels in serum. Furthermore, there was a much higher

variation in serum NEFA concentrations between animals compared to FF concentrations (an

average coefficient of variation of 58% and 30%, respectively). Both findings suggest that

there might be a mechanism to protect the oocyte and the granulosa cells from high NEFA

concentrations, which are shown to be toxic in vitro (Mason et al., 1999; Yanase et al., 2001;

Vanholder et al., 2003).

Similar profiles of TG and TC in serum around parturition and during the first weeks

post partum were described earlier and are characteristic for dairy cows (Varman and Schultz,

1968; Puppione, 1977; Guédon et al., 1999). However, the changes of TG and TC in FF

during this period have never been investigated before. Following parturition, the TG

concentration remained relatively low in serum and FF while the TC concentration doubled.

This observation is partially caused by the mammary conversion of TG-rich lipoproteins (β-

lipoproteins or very low and low density lipoproteins) to TC-rich lipoproteins (α-lipoproteins

or high density lipoproteins) (Puppione, 1977; Bauchart, 1993; Guédon et al., 1999).

Wehrman et al. (1991) demonstrated that the TG concentration in the FF is relatively stable,

regardless of an increase in the serum level due to physiological status or diet. However,

dietary fat supplementation is known to increase serum and FF cholesterol concentrations

(Wehrman et al., 1991; Lammoglia et al., 1996). We also found that FF total cholesterol

levels rise when serum concentrations increase but both increases of TC were at different

rates (75.5% in serum versus 57.3% in FF) (Figure 8). This is confirmed by the significant

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

90

compartment X time interaction we found for log(TC) (Table 1). We also found that the FF

cholesterol concentration is only 43 – 48 % (at day 14 and day 46 post partum, respectively)

of the serum concentration. This finding suggests that the relative importance of the smallest

high density lipoprotein complexes (small HDL), decreased during ongoing lactation. This

small HDL is the only lipoprotein fraction that can pass the blood-follicle barrier and hence is

the only lipoprotein present in the FF (Brantmeier et al., 1987; Grummer and Carroll, 1988;

Wehrman et al., 1991). However, the relative importance of the complete HDL cholesterol

fraction (large, medium and small HDL complexes) in the amount of TC in serum remained

stable during the lactation (85%) (results not shown).

Conclusions

In conclusion, we found that the typical postpartum biochemical changes in the serum

concentration of glucose, β-OHB, urea, TP, TG, NEFA and TC are well reflected in the

follicular fluid of the dominant follicle. However, the oocyte and the granulosa cells seem to

be protected from low glucose levels and from high NEFA concentrations.

These findings may be crucial in the understanding of the pathogenesis of subfertility in high

yielding dairy cattle, by affecting the quality of both the oocyte and the granulosa cells. This

knowledge should be taken into account in planning further in vivo and in vitro research

concerning fertility problems in high-producing dairy cattle.

Acknowledgments

The authors thank Dr. J Penders and Dr. M Coryn for their excellent scientific and

technical support, Dr. K Moerloose and Ir. JLJP Leroy for the critical reading of the

manuscript, and J Mestach and G Spaepen for the indispensable help in the IVF lab. This

research was funded by the Institute for the Promotion of Innovation by Science and

Technology in Flanders (Grant no° 13236).

Chapter 4B: Metabolic Changes in Follicular Fluid in High Yielding Dairy Cows Early Post Partum

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Izadyar F, Van Tol HT, Colenbrander B, Bevers MM. Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through cumulus cells and not mediated by IGF-I. Mol Reprod Dev 1997;47:175-180.

Jorritsma R, Groot MW, Vos PL, Kruip TA, Wensing T, Noordhuizen JP. Acute fasting in heifers as a model for assessing the relationship between plasma and follicular fluid NEFA concentrations. Theriogenology 2003;60:151-61.

Kenny DA, Humpherson PG, Leese HJ, Morris DG, Tomos AD, Diskin MG, Sreenan JM. Effect of elevated systemic concentrations of ammonia and urea on the metabolite and ionic composition of oviductal fluid in cattle. Biol Reprod 2002;66:1797-1804.

Lammoglia MA, Willard ST, Oldham JR, Randel RD. Effects of dietary fat and season on steroid hormonal profiles before parturition and on hormonal, cholesterol, triglycerides, follicular patterns, and postpartum reproduction in Brahman cows. J Anim Sci 1996;74:2253-2262.

Landau S, Braw-Tal R, Kaim M, Bor A, Bruckental I. Preovulatory follicular status and diet affect the insulin and glucose content of follicles in high yielding dairy cows. Anim Reprod Sci 2000;64:181-197.

Leroy JLMR, Vanholder T, Delanghe JR, Opsomer G, Van Soom A, Bols PEJ, de Kruif A. Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows. Anim Reprod Sci 2003b. In Press.

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Leroy JLMR, Vanholder T, Van Soom A, Opsomer G, Bols P, de Kruif A. Effect of oleïc acid during in vitro maturation on fertilisation, first cleavage and embryo development of bovine cumulus-oocyte-complexes. Reprod Dom Anim 2003a;38:328 (abstr.).

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Mason TM, Goh T, Tchipashvili V, Sandhu H, Gupta N, Lewis GF, Giacca A. Prolonged elevation of plasma free fatty acids desensitizes the insuline secretory response to glucose in vivo in rats. Diabetes 1999;48:524-530.

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Chapter 5

Negative Energy Balance in High Yielding Dairy Cows and the Consequences

for Oocyte Quality

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Chapter 5A

Non-esterified Fatty Acids in Follicular Fluid of the Dominant Follicle in High Yielding Dairy Cows

and their Effect on Developmental Capacity of Bovine Oocytes in vitro

J.L.M.R. Leroy1, T. Vanholder1, B. Mateusen1, A. Christophe2, G. Opsomer1, A. de Kruif1, G. Genicot3, A. Van Soom1

1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

2 Department of Internal Medicine, Division of Nutrition, Faculty of Medicine, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium;

3 Institut des Sciences de la Vie, Unité des Sciences Vétérinaires, Catholic University of Louvain, Place Croix du Sud 5 box 10, B-1348 Louvain-la-Neuve, Belgium.

Reproduction 2005, 130: 485-495

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

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Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

99

Abstract

In this study concentration and composition of non-esterified fatty acids (NEFA) in

follicular fluid (FF) of high yielding dairy cows were determined during the period of

negative energy balance (NEB) early post partum. NEFA were then added during in vitro

maturation at concentrations measured previously in FF to evaluate their effect on the

oocyte’s developmental competence.

At 16 and 44 days post partum, FF of the dominant follicle and blood were collected

from nine high yielding dairy cows. Samples were analysed for NEFA concentration and

composition. NEFA concentrations in FF (0.2 - 0.6 mmol/l) during NEB remained ± 40%

lower compared to serum (0.4 – 1.2 mmol/l). The NEFA composition differed significantly

between serum and FF with oleic acid (OA), palmitic acid (PA) and stearic acid (SA) being

the predominant fatty acids in FF. Based on these results, 5115 oocytes were matured for 24

hours in serum-free media with or without (negative control) the addition of 0.200 mmol/l

OA, 0.133 mmol/l PA or 0.067 mmol/l SA solved in ethanol or ethanol alone (positive

control). Matured oocytes were fertilized and cultured for 7 days in SOF medium.

Addition of PA or SA during oocyte maturation had negative effects on maturation,

fertilization and cleavage rate and blastocyst yield. More (late) apoptotic cumulus cells were

observed in cumulus oocyte complexes matured in presence of SA or PA. Ethanol or OA had

no effect. These in vitro results suggest that NEB may hamper fertility of high yielding dairy

cows through increased NEFA concentrations in FF affecting oocyte quality.

Key Words

Fertility decline, Follicular fluid, High yielding dairy cow, Non-esterified fatty acid, Oocyte

quality

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

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Introduction

Reduced fertility in high yielding dairy cows has been reported world-wide during the

last decades (Lucy 2001). Ovarian dysfunction early post partum (pp), leading to delayed

resumption of cyclicity and prolonged calving intervals, is one of the major and thoroughly

studied drawbacks of this high productivity (Opsomer et al. 1998; Shrestha et al. 2004a). It is

only recently that an important role has been attributed to the oocyte and embryo quality in

determining the final fertility outcome. Some studies already suggested that the decline in

fertility is mainly caused by an inferior oocyte and embryo quality rather than being related to

an ovarian/endocrine dysfunction (Harrison et al., 1990; O’Callaghan & Boland, 1999, Horan

et al., 2005). A remarkable decline in first-service conception rates from around 65% in the

fifties to well below 40% in 2001 has been reported by Butler (2003). A significant reduction

in oocyte quality has been seen in high yielding dairy cows (Kruip 1995; Gwazdauskas et al.

2000; Sartori et al. 2002; Snijders et al., 2002; Walters et al. 2002) and can result in reduced

conception rates or in a higher prevalence of early embryonic mortality (Boland et al. 2001;

Lucy et al. 2001; Silke et al. 2002). Britt (1994) hypothesized that follicles grown during the

period of NEB early pp could be affected by unfavourable metabolic changes and may

contain a developmentally incompetent oocyte. It has recently been shown that the

composition of follicular fluid (FF) is subjected to these metabolic adaptations early pp

(Leroy et al. 2004). Subsequently, after a growing and maturation phase of several weeks, this

inferior oocyte will be ovulated at the moment of first insemination (Britt, 1994). One of the

major metabolic changes during the period of NEB is the increased non-esterified fatty acid

(NEFA) concentrations in serum which are strongly correlated with the depth of NEB.

Recently, it has been demonstrated that elevated NEFA levels are toxic for bovine

(Vanholder et al. 2005) and human (Mu et al. 2001) granulosa cell growth and function in

vitro. Similar cytotoxic effects were described in pancreatic β-cells (Cnop et al. 2001;

Maedler et al. 2001), Leydig cells (Lu et al. 2003) and blood mononuclear cells (Lacetera et

al. 2002).

Until now, knowledge about the influence of elevated NEFA levels as encountered

during NEB in vivo on oocyte developmental capacity in vitro is very scarce or even absent.

Furthermore, very little is known about the NEFA concentration and NEFA composition in

the intrafollicular environment in relation to the serum composition. This knowledge is

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indispensable to investigate the effect of in vivo intrafollicular NEFA concentrations during a

period of NEB in an in vitro maturation (IVM) model.

In the present study we wanted to clarify possible interactions between high NEFA

concentrations and oocyte quality, being a potential contributing factor in the pathogenesis of

subfertility in modern high yielding dairy cows. Therefore, the aims of the present study were

(1) to investigate the concentration and composition of NEFA in serum and in FF of the

dominant follicle in high yielding dairy cows during and shortly after the period of NEB; and

(2) to imitate these NEB associated FF NEFA concentrations in an IVM model to test their

effect on oocyte developmental competence.

Material and Methods

NEFA concentration and composition in serum and FF of the dominant follicle

a. Animals

Nine healthy multiparous Holstein-Friesian cows were used in this study. All

experimental work was performed at the research dairy farm of the Ghent University

(Biocentrum Agri-Vet, Melle, Belgium) following protocol approval by the Ethical

Committee of the Faculty of Veterinary Medicine (Ghent University). Cows were milked on

average 2.2 times a day by means of an automated voluntary milking system. The average

milk yield per cow in the herd was 10,200 kg milk (4.1 % fat and 3.4 % protein) during 305

days of lactation. After an average dry period of 55 days, all cows calved normally between

October 2003 and March 2004. During the experimental period (first 50 days of lactation), all

cows were housed in a loose stable with cubicles and were fed according to their requirements

for maintenance and milk production. The ration consisted of high quality roughages (corn

silage and grass silage, sugar beet pulp), soybean meal and concentrates. All animals showed

a normal puerperium and uterine involution. One animal suffered from a mild mastitis in one

quarter. After an intramammary treatment with antibiotics, the animal was cured within 3

days, well before the first ovarian puncture. Body condition scores (BCS) based on the

notation of Edmondson et al. (1989), were recorded by the same experienced operator using a

score on a scale of 1-5 (with 0.25 increments).

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b. Blood and FF sampling

Blood samples were collected from each animal 7 days prior to the expected calving

date, at the day of parturition and at days 16 (severe NEB) and 44 (improving NEB) pp.

Blood was sampled from the jugular vein into two unheparinized, silicone coated tubes

(Venoject®, Autosep®, Gel + Clot. Act.; Terumo Europe N.V., Leuven, Belgium). Any stress

prior to blood sampling was avoided. Samples were taken between 1.00 pm and 3.00 pm, two

hours after automated milking at the latest and before any other handling of the animals was

performed. The coagulated blood samples were centrifuged (1,400 × g, 30 min) within 1.5

hours after collection and the collected serum was stored under N2 atmosphere at -80°C until

analysis.

On day 11 pp an ultrasound examination of the genital tract was performed in all cows

to monitor uterine involution and follicular growth. On day 16 and 44 pp only dominant

follicles with a diameter greater than 0.8 cm were subjected to ultrasound guided transvaginal

aspiration as described previously (Leroy et al. 2004). Attention was paid to prevent blood

contamination. Follicular fluid samples with obvious blood contamination were omitted from

further processing. The collected FF was cooled immediately (4°C). Subsequently, FF

samples were centrifuged (10,000 × g, 10 min) and the supernatant was collected for analysis.

Within 2 hours after each session, the FF samples were frozen under N2 atmosphere at –80°C

until analysis.

c. Analyses

To identify possible atresia of the punctured follicles, a progesterone (P4) and

estradiol-17β (E2) analysis was carried out on each FF sample as previously described (Leroy

et al. 2004). Follicular fluid with a ratio E2/P4 < 1 was considered to originate from an atretic

follicle and was omitted from biochemical analysis (Badinga et al. 1992; Landau et al. 2000).

The analyses for total NEFA concentration were done using wet chemistry techniques

on a clinical automated analyser (Hitachi 911, Roche Diagnostics, Mannheim, Germany). A

commercial kit was used (Wako Chemicals GmbH, Neuss, Germany) according to the

manufacturer’s instructions. The intra- and inter-assay coefficients of variation were below

5%.

The composition of the NEFA fraction in serum and FF samples was determined as

follows. The total lipid fraction was extracted with methanol/chloroform according to a

modified method of Folch et al. (1957). In brief, 100 μl of 1N HCl, 1 ml of methanol and 2 ml

of chloroform were added to 1 ml of serum or FF. After centrifugation at 4°C, the upper phase

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and the interface were removed by aspiration and filtration, respectively. The filtrate was

evaporated to dryness under a N2 flow and the residue was dissolved in chloroform. To avoid

any fatty acid oxidation, the samples were kept under N2 atmosphere. Non-esterified fatty

acids were isolated by thin layer chromatography on rhodamine-impregnated silica gel plates

using petroleum ether (bp 60-80°C; Merck Belgolab, Overijse, Belgium) and acetone (85:15

by volume) as mobile phase. The free fatty acid band was scraped off and the fatty acids were

converted into methyl esters by esterification using 2 ml of a mixture of

methanol/chloroform/HCl (fuming 37%) (80:20:4 by volume) as methylating agent for 4 h at

95°C. After cooling and addition of 2 ml of distilled water, the methyl esters were extracted

with petroleum ether (bp 40-60°C) and evaporated to dryness under a N2 flow. The fatty acids

were analysed by temperature-programmed capillary gas chromatography (Varian model

3500 gas chromatograph, Walnut Creek, CA, USA) on a 60 m x 250 μm (L x ID) x 0.2 μm

film thickness 10% cyanopropylphenyl - 90% biscyanopropyl polysiloxane column (Rtx®-

2330, Restek, USA). The injection and detection temperatures were set at 285°C. The starting

temperature of the column was 165°C, which, after 1 min, was increased to 230°C at a rate of

2°C/min. The carrier gas was nitrogen with a linear velocity of 18.1cm/s. Peak identification

was done based on the retention times using authentic standards. Peak integration and

calculation of the fatty acid compositions were automatically performed using appropriate

software (Varian Star 5.52, 1998). The results for individual fatty acids were expressed as

weight % of the amount of total fatty acids.

Addition of oleic acid, palmitic acid or stearic acid during IVM of bovine oocytes

a. Materials and media

Chemicals and media were obtained from Sigma (Bornem, Belgium) and from

Gibco/InvitrogenTM life technologies (Merelbeke, Belgium). A modified HEPES-buffered

Tyrode’s balanced salt solution, termed HEPES-TALP, consisted of 114 mmol/l NaCl, 3.1

mmol/l KCl, 2 mmol/l NaHCO3, 0.3 mmol/l NaH2PO4, 10 mmol/l HEPES, 2.1 mmol/l CaCl2,

0.4 mmol/l MgCl2, 10 mmol/l sodium lactate, 0.2 mmol/l sodium pyruvate, 3 mg/ml fatty acid

free bovine serum albumin (BSA) and 10 μg/ml gentamycine sulphate. Oleic acid (OA, cis

C18:1), palmitic acid (PA, C16:0) and stearic acid (SA, C18:0) were dissolved in pure ethanol

(Vel/Merck Eurolab, Zaventem, Belgium) at a concentration of 50, 25 and 12.5 mg/mL,

respectively. Murine epidermal growth factor (EGF) was solved at a concentration of 1μg/ml

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in bicarbonate buffered Medium 199 with Earle’s and glutamine (TCM 199) and with 0.1%

w/v fatty acid-free BSA.

The serum-free maturation media (pH = 7.2) contained TCM199, one fatty acid

dissolved in ethanol (cfr. Infra) and EGF (20 ng/ml). Fertilization medium consisted of

Tyrode’s balanced salt solution supplemented with 25 mmol/l NaHCO3, 10 mmol/l sodium

lactate, 0.2 mmol/l sodium pyruvate, 6 mg/ml fatty acid-free BSA, 10 μg/ml gentamycin

sulphate and 10 μg/ml heparin. The embryo culture medium consisted of Synthetic oviduct

fluid (SOF) (Minitüb, Tiefenbach, Germany) supplemented with 40 μl/ml Basal medium

eagle (BME), 10 μl/ml Minimum essential medium (MEM), 0.2 mmol/l sodium pyruvate and

50 μl/ml Fetal calf serum (FCS) (N.V. HyClone, Europe S.A., Erembodegem, Belgium).

Percoll™ was purchased from Amersham Biosciences (Uppsala, Sweden), heparin

from Leo Pharma (Zaventem, Belgium), ethanol from Vel/Merck Eurolab (Zaventem,

Belgium), and Hoechst 33342 from Molecular Probes (Leiden, The Netherlands).

b. In vitro production of embryos

Ovaries and oocytes were collected as described by Tanghe et al. (2003). After

collection, ovaries were rinsed in physiological saline (0.9% NaCl) with 0.5% kanamycin.

The IVM was performed as follows. Immature cumulus oocyte complexes (COCs) were

aspirated from follicles 2-6 mm in diameter. Only grade I COCs were used for further culture

following selection under a stereo microscope. After several washings in HEPES-TALP, the

COCs were cultured in groups of 50-60 for 24 h at 38.5 °C in 500 μl of serum-free maturation

medium in a humidified 5% CO2 incubator.

After IVM, fertilization was performed as described by Tanghe et al. (2003). Briefly,

all groups of COCs were coincubated per 100-120 with spermatozoa at a final concentration

of 106 sperm cells/ml for 20 h at 38.5 °C in fertilization medium in a humidified 5% CO2

incubator. For all experiments, frozen bull semen from the same ejaculate was thawed and

live spermatozoa were selected by centrifugation on a discontinuous Percoll® gradient (90 and

45%). The final sperm-egg ratio was adjusted to 5000:1.

After coincubation with spermatozoa, the presumptive zygotes were vortexed for 4

minutes to remove excess sperm and cumulus cells. After several washings with HEPES-

TALP and modified SOF medium, presumptive zygotes were cultured per 25 in 50 μl droplets

of modified SOF medium with 5% FCS, under mineral oil (modular incubator: 39 °C, 5%

CO2, 5% O2 and 90% N2) until 8 days after fertilization. For each replicate, four drops of

embryos were prepared per treatment.

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c. Analyses

Maturation and fertilization rate

After IVM or fertilization, COCs or presumptive zygotes were vortexed for 4 or 2 min,

respectively. The denuded matured oocytes/presumptive zygotes were fixed in 2%

paraformaldehyde and 2% glutaraldehyde in PBS for at least 24 h (4°C), and stained for 10

min with 10 µg/mL Hoechst 33342 (Molecular Probes, Leiden, The Netherlands). The

matured oocytes/presumed zygotes were mounted in 100% glycerol and evaluated by means

of a Leica DMR fluorescence microscope (Van Hopplynus N.V., Brussels, Belgium) (400 X

magnification). To evaluate the maturation rate of the oocytes, the nuclear stage was recorded

as being in first metaphase (MI), anaphase or telophase (AT) and second metaphase with

extruded polar body (MII, successful nuclear maturation). To investigate the fertilization rate,

following stages were distinguished: MII, the presence of 2 pronuclei (2PN, successful

fertilization) and the presence of more than 2 pronuclei (>2PN, polyspermy).

Lipid content

To investigate whether IVM in the presence of a fatty acid (PA or SA) influenced the

lipid content in the matured and denuded oocytes, the selected oocytes were fixed, stained

with 10 μg/ml Nile Red (Molecular Probes, Inc., Eugene, Oregon, USA) for 3 h and analysed

as described before (Genicot et al., 2005). The emitted fluorescent light was evaluated at a

wavelength of 582 ± 6 nm with an inverted fluorescence microscope (Excitation: 400-500nm

and Emission: 515LP) using a 10 X objective. The fluorescence was amplified with a

photomultiplier, quantified with a photometer attached to the microscope (MPV-SP, Leitz,

Wetzlar, Germany) and calculated by the MPF Bio Software (Leitz). The results were

expressed in arbitrary units of fluorescence.

Morphology of COCs after IVM

After IVM, COCs were evaluated morphologically for cumulus expansion by means

of a binocular microscope (40 X magnification). The presence of apoptosis in cumulus cells

of COCs matured in the control group (with ethanol) and in the test group (SA or PA) was

evaluated by means of propidium iodide (PI) and annexin V staining (VybrantTM Apoptosis

Assay kit #3, Molecular Probes, Eugene, Oregon, USA). Positive control COCs were

incubated during the last 12 h of IVM with 1 µM staurosporine to induce apoptosis. After 24

h of IVM, COCs were first washed for 20 seconds in annexin binding buffer at 37°C and

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incubated for 15 minutes in the presence of FITC conjugate of annexin V (25µl/ml) and PI

solution (3µg/ml) according to the manufacturer’s recommendations for the VybrantTM

Apoptosis Assay kit #3. Then COCs were washed for 20 seconds in annexin binding buffer

and transferred per 3 to a drop of pre-warmed PBS (37°C) on a microscopic slide. The stained

samples were examined with a Leica TCS SP2 laser scanning spectral confocal system (Leica

Microsystems GmbH, Heidelberg, Germany) linked to a Leica DM IRB inverted microscope

(Leica Microsystems GmbH, Wetzlar, Germany). An Argon laser was used to excite FITC

(488 nm) and PI (586 nm) fluorochromes. Positive labelling for annexin V on the outer

surface membrane was observed as bright yellow to green staining. Late apoptotic and

necrotic cells displayed a PI positive nucleus (red). The total COC was evaluated by multiple

cross sections set at 3µm intervals. Analysis of the images was performed with Leica confocal

software.

d. Experimental design

Each fatty acid in the IVM medium was tested for its effect on cleavage rate (48h after

fertilization) and blastocyst yield (8 days after fertilization). To explain possible observed

effects on the developmental competence, fertilization and maturation rates were investigated

in separate replicates. Per experiment one fatty acid was tested and a negative and positive

control group were included. The negative control group consisted of TCM199 and EGF (20

ng/ml). The sole difference in the positive control group was the addition of an equal volume

of ethanol as used in the fatty acid group. In the fatty acid group, OA, PA or SA dissolved in

ethanol were added to reach a final concentration of 200 μM, 133 μM or 67 μM, respectively.

The fatty acid concentrations tested in this IVM model were based on the results of the in vivo

experiment where the highest NEFA concentration observed in the FF during the NEB was

0.6 mmol/l and the average relative importance of OA, PA and SA at that time was 33%, 23%

and 13%, respectively. In total 5115 oocytes were cultured. The number of oocytes and

replicates per experiment are shown in table 1.

Table 1. Number of bovine oocytes (and number of replicates) per experiment (one fatty acid tested per experiment including a negative and positive control group). Experiment Maturation rate Fertilization rate Cleavage and blastocyst yield Oleic acid (C18:1) 338 (2) 437 (2) 752 (3) Palmitic acid (C16:0) 450 (2) 487 (2) 845 (3) Stearic acid (C18:0) 478 (2) 476 (2) 852 (3)

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

107

To evaluate the effect of maturation in the presence of one fatty acid on lipid content,

144 oocytes were evaluated (two replicates, 9 to 20 oocytes per group). Per replicate, 4 groups

were compared: immature oocytes, oocytes matured in the presence of PA or SA and oocytes

matured in positive control medium.

To detect the presence of apoptosis/necrosis, 10 COCs from each group (positive

control, negative control and fatty acid group) were stained as described earlier (two

replicates).

As an extra control of the described IVM model, also the effect of basal NEFA

concentrations during IVM was investigated: 66.7 μM OA, 44.3 μM PA and 22.3 μM SA.

These concentrations are based on the basal concentrations observed in the FF at day 44 pp,

well after the period of NEB (total NEFA concentration of 0.2 mmol/l, see below).

Statistics

Data are expressed as means ± SEM. All statistical procedures were carried out with

SPSS 11.0 for Windows, (Chicago, IL, USA). Values of P < 0.05 were considered statistically

significant.

a. NEFA concentration and composition in serum and FF of the dominant follicle

The absolute NEFA concentrations in serum and in FF early and late pp were

compared with a paired sample t-test (paired samples within the same animal in a different

compartment (serum vs. FF) or in a different time frame (early vs. late postpartum)). There

were no departures from normality. The different fatty acids, expressed as percentages in the

NEFA fraction, were compared between serum and FF by a non parametric Wilcoxon Signed

Ranks test.

b. Addition of oleic acid, palmitic acid or stearic acid during IVM of bovine oocytes

The proportion of oocytes that cleaved at 48h after fertilization and the proportion of

oocytes and cleaved zygotes that developed up to the blastocyst stage at Day 8 after

fertilization were calculated for each culture droplet (experimental unit). Four droplets were

used per replicate and per treatment. No data transformations were necessary for inequality of

variance between groups or for normality reasons. Data were analysed using a two-way

ANOVA and a post-hoc Scheffé test. Treatment was inserted as fixed factor and replicate as

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

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random factor together with the interaction term (treatment X replicate) (mixed model). In the

absence of a significant interaction term, the term was left out from the final model.

The proportion of oocytes that had reached the MI, AT or MII stage and the proportion

of oocytes/zygotes that were in the MII, 2PN or >2PN stage, were calculated per treatment

group and per replicate. Data were analysed using a binary logistic regression model in which

treatment, replicate and the interaction of these two factors were included. In the absence of a

significant interaction term, the term was left out from the final model.

The data of the lipid determination (arbitrary units of emitted fluorescence) were

normally distributed and were analysed using a two-way ANOVA with treatment as fixed

factor and replicate as random factor.

Results

NEFA concentration and composition in serum and FF of the dominant follicle

From 7 days prior to the expected parturition date (varying between 18 and 3 days

prior to the real day of parturition) up to 44 days pp, all cows displayed a loss in BCS (on

average 0.83 ± 0.15 points) (P < 0.05). From day 16 up to day 44 pp, the average daily milk

yield increased by 5.6 kg, from 35.9 ± 1.8 kg to 41.5 ± 2.0 kg.

On average, 1.54 ± 0.2 ml FF was aspirated from 1.14 ± 0.15 follicles per cow and per

session. Nine percent of all FF samples were excluded from further analysis due to atresia,

based on an E2/P4 ratio < 1, or because of blood contamination. In the FF samples which were

analysed, the average E2/P4 ratio was 13.15 ± 2.17.

In serum the NEFA concentration increased significantly around parturition and was

still high at 16 days pp (0.4 – 1.2 mmol/l). At 44 days pp, the serum NEFA concentrations

were again at the basal level (0.1 – 0.3 mmol/l). Similarly, a significant decrease was also

found in the FF from day 16 to day 44 pp. The FF NEFA concentrations early pp (day 16)

ranged from 0.2 to 0.6 mmol/l and were on average 47 ± 6.4 % lower than those in serum.

Later pp (day 44) there was no significant difference in NEFA concentrations between serum

(0.1 – 0.3 mmol/l) and FF (0.1 – 0.3 mmol/l) (Figure 1).

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

109

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

7 d prepartum parturition 16 d postpartum

44 d postpartum

NEFA

(+/-

SEM

) mm

ol/l

Figure 1. Mean non-esterified fatty acid (NEFA) concentrations (± SEM) in bovine serum (black line) and in follicular fluid (dotted line) at different time points relative to parturition. Serum NEFA concentrations, marked with a, b, differ significantly between different time points. Follicular fluid NEFA concentrations marked with 1, 2 differ significantly between different time points. Non-esterified fatty acid concentrations at one time point marked with *, differ significantly between serum and follicular fluid (P < 0.05).

Both in serum and in FF OA, PA and SA were the three predominant free fatty acids

(Figure 2). The NEFA composition differed significantly between the two compartments.

Early pp the relative concentration of SA in FF was significantly lower compared to serum.

Linoleic acid (LA, C18:2), as a percentage of the NEFA, on the other hand was higher in FF

than in serum. At 44 days pp, almost all investigated fatty acids differed in relative

concentration in serum compared to FF. Parallel with the decrease of the NEFA concentration

from early to later pp, there was a change in the composition of the NEFA fraction both in

serum and in FF. In serum, the relative concentrations of SA and LA increased and the

concentrations of PA and OA decreased significantly. In FF similar significant changes for

OA and LA were observed as in serum.

a

b* b

a

1*

2

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

110

A.

0

5

10

15

20

25

30

35

40

45

C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2

free fatty acid

%

B.

0

5

10

15

20

25

30

35

40

45

C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2

free fatty acid

%

Figure 2. Mean percentage (± SEM) of the predominant fatty acids in the non-esterified fatty acid lipid fraction in serum (dark bars) and in FF (pale bars) early (day 16) (A) and late (day 44) (B) post partum: myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), margaric acid (C17:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). Fatty acids marked with * have a significantly different relative concentration in serum compared to follicular fluid (P < 0.05).

Addition of oleic acid, palmitic acid or stearic acid during IVM of bovine oocytes

Maturation in the presence of OA had no significant effect on the oocyte

developmental capacity in terms of cleavage or blastocyst yield (data not shown). However,

addition of SA resulted in a significantly lower cleavage rate and subsequent blastocyst yield

(Table 2) (P < 0.05). Similarly, there was a strong tendency for a reduced cleavage rate (P =

0.07) and blastocyst yield relative to the number of cultured oocytes (P = 0.06) or to the

number of cleaved zygotes (P = 0.12) after maturation in the presence of PA (Table 3). The

fertilization rate was significantly reduced for the oocytes matured in the presence of PA or

* *

*

*

*

* *

*

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

111

SA (P < 0.05). Moreover, the presence of PA or SA during the IVM delayed the progression

through meiosis, expressed as a significantly higher number of oocytes still in MI and a

concomitant lower relative number of oocytes in MII (Table 2 and 3) (P < 0.05).

Table 2. Effect of stearic acid (C18:0) added to the maturation medium on maturation and fertilization rate, cleavage rate (± SEM) at 48h after fertilization (pi) and number of blastocysts (± SEM) at 8 days pi relative to the number of bovine oocytes put in culture or relative to the cleaved zygotes. Negative control Positive control Stearic acid (C18:0) Maturation rate (%)

Metaphase I 9.2a 18.6b* 26.0b*

Ana-/Telophase1 16.1a 11.6a 18.4a

Metaphase II 74.8a 67.8a 54.0b

Fertilization rate (%) Metaphase II 10.7a 8.8a 23.4b

2 Pronuclei 69.7a 72.2a 55.6b

> 2 Pronuclei 12.5a 12.1a 12.5a

Cleavage rate at 48h pi (%) 76.9 ± 3.2a 77.4 ± 2.7a 57.9 ± 3.6b

% blastocysts from oocytes 33.3 ± 3.6a 34.4 ± 2.1a 21.3 ± 3.5b

% blastocysts from cleaved 43.1 ± 4.3a 44.4 ± 2.1a 39.6 ± 7.0a

a,b Data within a row marked with different superscripts, differ significantly (P < 0.05). * P = 0.1 1 Significant interaction term “treatment X replicate”.

Table 3. Effect of palmitic acid (C16:0) added to the maturation medium on maturation and fertilization rate, cleavage rate (± SEM) at 48h after fertilization (pi) and number of blastocysts (± SEM) at 8 days pi relative to the number of bovine oocytes put in culture or relative to the cleaved zygotes. Negative control Positive control Palmitic acid (C16:0) Maturation rate (%)

Metaphase I 9.1a 12.5a 24.1b

Ana-/Telophase 15.9a,b 10.5a 19.9b

Metaphase II 75.0a 77.1a 63.2b

Fertilization rate (%) Metaphase II 21.6a 20.2a 33.5b

2 Pronuclei 64.0a 59.2a 43.4b

> 2 Pronuclei1 7.0a 5.8a 11.6a

Cleavage rate at 48h pi (%) 76.6 ± 2.3a 74.5 ± 2.6a,b* 66.6 ± 3.2b*

% blastocysts from oocytes 22.4 ± 2.0a 24.6 ± 1.5a$ 17.2 ± 3.0a$

% blastocysts from cleaved 29.1 ± 2.4ab§ 33.2 ± 1.8a 22.7 ± 4.1b§

a,b Data within a row marked with different superscripts, differ significantly (P < 0.05). 1 Significant interaction term “treatment X replicate”. * P = 0.07 $ P = 0.06 § P = 0.12

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

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Maturation of oocytes in the presence of PA or SA had no effect on the lipid content

of single bovine oocytes. The arbitrary units of emitted fluorescent light were similar in the

four groups (data not shown).

After IVM in PA or SA, COC morphology was evaluated and compared with control

COCs. Poor expansion of the COCs cultured in the presence of PA or SA was obvious

(Figure 3). After staining and evaluation with laser scanning confocal microscopy all COCs in

the SA or PA group displayed a high proportion of apoptotic or late apoptotic/necrotic cells (>

40% of the cells were positive). In the positive control group only few cells of the COCs (<

10% of the cells) were apoptotic (Figure 4).

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

113

A. B.

Figure 3. Cumulus oocyte complexes after 24h of maturation in positive control medium (well expanded) (A.) and in medium with added stearic acid (poor expansion) (B.) (40 X magnification).

A. B.

Figure 4. Cumulus oocyte complexes from the positive control group (A.) and the stearic acid group (B.) after staining with Annexin V and propidium iodide for detection of apoptotic (green cell membranes) or late apoptotic/necrotic cells (green cell membranes and red nucleus) (100 X magnification). The white circle represents the position of the oocyte. A relative higher abundance of annexin V and PI positive cells can be appreciated in the stearic acid group.

No effect of ethanol during the IVM could be observed on all evaluated outcome

variables. Similarly, IVM of oocytes in the presence of positive energy balance associated

concentrations of the three tested fatty acids, had no effect on any of the tested variables.

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

114

Discussion

In the present study it was hypothesized that possible toxic effects of NEFA on oocyte

quality may be a partial explanation for the fertility decline in modern high yielding dairy

cows. Therefore, we aimed first to determine the NEFA concentration and composition in FF

of high yielding dairy cows in relation to serum early and later pp. Secondly, the three

predominant NEFA in the FF of the dominant follicle, were added in an in vitro maturation

model at concentrations observed in vivo, to investigate their effect on the developmental

capacity of the oocyte.

The results of the in vivo study show a significant increase in serum NEFA

concentrations around parturition and elevated levels are maintained up to two weeks pp. At

44 days pp the NEFA concentrations had returned to prepartum levels. This change in NEFA

concentration with time pp is in accordance with other studies and is a major characteristic of

the NEB early pp. The NEB together with low insulin concentrations and the release of stress

associated catecholamines increases the degree of lipolysis and decreases the rate of re-

esterification of free fatty acids in the adipose tissue (Chilliard et al. 1998; Vernon 2002).

Moreover, all animals displayed a significant loss in body condition early pp, confirming the

presence of NEB. Several studies have associated the NEB with delayed resumption of

ovarian activity and reduced conception rates, finally leading to suboptimal fertility (Zurek et

al. 1995; Beam & Butler 1999; de Vries & Veerkamp 2000).

Focussing on the FF early pp, the NEFA concentrations were elevated but still

significantly lower than in serum. This remarkable concentration gradient confirms what has

been suggested in earlier work (Leroy et al. 2004). Later on pp, both serum and FF NEFA

concentrations were basal again and no concentration differences were present. These

findings suggest that, at least to some extent, the vulnerable oocyte and granulosa cells are

protected from too high and possibly toxic NEFA concentrations during the NEB in high

yielding dairy cows. Elevated NEFA concentrations in serum and in FF have also been

described in heifers and lactating cows that were subjected to an acute dietary restriction

(Comin et al. 2002; Jorritsma et al. 2003). Our results also demonstrate that OA, PA and SA

are the three predominant free fatty acids both in serum and in FF. This was also shown by

Yao et al. (1980) in pigs. Moallem et al. (1999) however, found that LA dominated in the

NEFA fraction of bovine FF. Furthermore, we observed that the NEFA composition in serum

early pp differs from that later on pp.

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

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Differences in serum or FF albumin concentration, on which NEFA are bound and

transported, has been suggested to account for the observed NEFA gradient (Yao et al. 1980).

We only found a 7% lower albumin concentration in FF compared to serum early and later pp

(data not shown). Therefore, it is unlikely that this small albumin gradient is the only factor

responsible for the observed differences in NEFA concentrations. Literature about the

properties of the follicle-blood barrier and their effects on albumin and thus NEFA

concentrations is contradictory (Zamboni 1974; Wise 1987).

In the presence of high NEFA levels, a substantial portion of the NEFA can be

partitioned to low density lipoproteins (LDL) (Chung et al. 1995). Especially the saturated

fatty acids are bound on LDL, while the unsaturated ones are preferably bound on albumin

(Chung et al. 1995). The fact that LDL are absent in FF (Brantmeier et al. 1987; Wehrman et

al. 1991), may explain the observed differences early pp in the concentration and composition

of NEFA in FF compared to serum in our study. Indeed, the results show a lower fraction of

SA (saturated) and a higher fraction of LA (unsaturated) in the NEFA present in FF compared

to serum. Also active transport, desaturating enzymes and selective uptake or metabolisation

by intrafollicular cells (Yao et al. 1980) could be responsible for the observed differences in

NEFA concentration and composition in the two compartments early and later pp.

Conclusively, it can be stated that mimicking NEB associated NEFA concentrations in IVM

models should be based on the intrafollicular rather than on the serum concentrations.

After investigating the NEFA fraction in the FF of high yielding dairy cows during

NEB we were able to test the effect of elevated concentrations of the three major unbound

NEFA on in vitro oocyte maturation. Although NEFA in FF are mainly bound to albumin,

especially the unbound fraction is directly involved in the fatty acid uptake by cells (Berk &

Stump 1999). The importance of the albumin bound fatty acids in this process remains a

matter of discussion. It does seem though that both forms of fatty acids are taken up by the

cells suggesting the physiological significance of the total NEFA concentration (McArthur et

al. 1999; Synak et al. 2003). In preliminary experiments with fatty acids free albumin and

with albumin bound OA, albumin itself exerted a negative effect on the oocyte’s

developmental competence (Leroy et al. 2003). To avoid such effects, we used unbound fatty

acids dissolved in ethanol, as has been done by others (Hinckley et al. 1996; Hirabara et al.

2003; Vanholder et al., 2005).

Supplementation of the medium with elevated concentrations of PA or SA resulted in

a negative effect on the progression of meiosis. The subsequent fertilization and cleavage rate

and blastocyst formation were significantly reduced. Oleic acid had no effect on any of these

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

116

outcome variables which confirms that maturation and fertilization proceeded normally

(Rizos et al. 2002). Two other studies which have investigated the effect of fatty acids on

oocyte maturation differ from ours in the fact that they added fetal calf serum and applied

albumin bound fatty acids in supraphysiological concentrations (Homa & Brown 1992;

Jorritsma et al. 2004).

The reduced fertilization rate and hampered in vitro development are most likely

carry-over effects of the delayed or blocked maturation. Therefore, based on the present

study, it is impossible to give evidence on how the maturation in the presence of PA or SA

directly influenced the oocyte’s developmental capacity after maturation. Only IVM in the

presence of PA tended to have a negative effect on the rate of blastocyst formation relative to

the cleaved zygotes. It is clear, however, that the major impact of PA and SA is on the oocyte

maturation itself. A combination of the three fatty acids in one IVM set up also negatively

affected oocyte quality. Unfortunately, because there was a tendency for subtle aggregation

and precipitation of the added fatty acids, data were not fully reliable and hence are not

shown.

Parallel with the results of the present study, it has been shown earlier in our lab that

PA and SA and not OA exert a toxic effect on bovine granulosa cell growth and function in

vitro (Vanholder et al., 2005). Similar results were observed in human granulosa cells (Mu et

al. 2001) and in rat Leydig cells in vitro (Lu et al. 2003). These studies demonstrated the

induction of apoptosis by PA and SA, probably through ceramide production or through a

down-regulation of the apoptosis inhibitor Bcl-2 and the up-regulation of an apoptosis

mediator such as Bax. Our observations of the poorly expanded COCs after maturation in the

presence of PA or SA seem to be due to the induction of apoptosis as well, since a massive

degree of late apoptotic and even necrotic cumulus cells were detected. Iseki et al. (1995)

documented the presence of fatty acid binding proteins in rat granulosa cells, illustrating the

possibility of fatty acid uptake. The existence of such receptors in the cell membrane of

bovine cumulus cells, however, has never been described. Others found that especially

saturated fatty acids can induce peripheral insulin resistance and thus blocking of glucose

uptake in muscle cells (Hirabara et al. 2003). The more, insulin depletion in pancreatic β-cells

can also be triggered by an increased prevalence of apoptosis and necrosis after incubation

with saturated fatty acids (Mason et al. 1999; Cnop et al. 2001; Maedler et al. 2001).

Jorritsma et al. (2004) suggested that changes in membrane properties of the oocyte could be

responsible for the observed negative effects of albumin bound OA in the IVM medium.

Whatever the mechanisms, our results clearly indicate that exposure of COC to PA or SA

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

117

during 24h has a deleterious effect on cumulus cell health and survival. Because a healthy

cumulus investment is indispensable for correct oocyte maturation (Tanghe et al. 2002), the

oocyte is most likely indirectly affected by these fatty acids.

Oocytes are said to be able to accumulate fatty acids from their environment,

potentially changing their lipid content and composition (Kim et al. 2001; Adamiak et al.

2005). Lipid accumulation in oocytes and embryos can reduce their quality and cryotolerance

(Abe et al. 2002). But, in contrast with Xenopus oocytes (Zhou et al. 1994), a fatty acid

binding protein on the oolemma of bovine oocytes has never been described. Shimabukuro et

al. (1998) attributed the lipotoxicity of added NEFA in β-cell cultures to the accumulation of

intracellular lipids, inducing ceramide and NO production, finally resulting in apoptosis. To

test the possibility of such lipid accumulation in the oocyte, we analysed the lipid content of

mature oocytes after IVM in presence of PA or SA. No lipid accumulation, however, could be

detected. This suggests that lipid accumulation in oocytes is probably not involved in the

observed negative effects of the free fatty acids in this study.

The findings of the present study support the hypothesis of Britt (1994), confirming

that metabolic changes during a period of NEB (in casu: high NEFA concentrations) may

have detrimental effects on the developmental capacity of the oocyte. It is however important

to mention that the combined in vitro and in vivo model used in this study was not entirely

appropriate in investigating the described carry-over effect on oocyte quality. Our results only

document on the FF composition in the dominant follicle during the NEB which was

mimicked in vitro. Quiescent follicles, which embed the oocytes of interest, however, provide

a much poorer isolation of the oocyte from the extrafollicular environment and blood serum,

probably exposing the growing oocyte to even higher NEFA concentrations (Zamboni 1974;

Fair 2003). The more, in this study the COCs were exposed to elevated NEFA levels for only

24 h, whereas in vivo the oocytes are exposed to such levels for weeks. The ideal model

should cultivate primordial follicles in high NEFA conditions for several days or even weeks.

Moreover, extrapolating in vitro results from this well defined IVM model to the real in vivo

situation should always be done with caution. Being the only practical approach, the model

used in the present study revealed for the first time possible toxic effects of high

intrafollicular NEFA concentrations on the developmental competence of bovine oocytes in

vitro. Acute fatty acid mobilization caused by food restriction or reduced appetite (illness or

lameness) later pp also involves a fast NEFA rise both in serum as well as in FF (Comin et al.

2002; Jorritsma et al. 2003). The present study demonstrates that even a very short (24 h)

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

118

exposure to elevated NEFA levels just prior to ovulation can be detrimental to the

developmental capacity of the pre-ovulatory oocyte.

Conclusions

It can be concluded that even though FF NEFA levels are high during the period of

NEB early pp, the concentration remains remarkably lower than in serum. Furthermore, the

NEFA composition in FF differs from that of serum. In vitro oocyte maturation in the

presence of NEB associated concentrations of PA and SA is hampered, leading to reduced

fertilization rate and developmental competence. The data of the present study suggest that

toxic effects of elevated FF NEFA concentrations on oocyte quality may be one of the factors

through which NEB exerts its negative effects on fertility in high yielding dairy cows.

Future research should concentrate on the cellular mechanisms through which fatty

acids can exert a toxic effect on COCs.

Acknowledgments

The authors thank J. De Clercq, J. Mestach and G. Spaepen for their excellent

technical support, and K. Moerloose, M. Coryn and P.E.J. Bols for the critical reading of the

manuscript. This research was funded by the Institute for the Promotion of Innovation by

Science and Technology in Flanders (Grant no° 13236).

Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality

119

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Chapter 5B

The in vitro Development of Bovine Oocytes after Maturation in Glucose and β-Hydroxybutyrate

Concentrations associated with Negative Energy Balance in Dairy Cows

JLMR Leroy, T Vanholder, G Opsomer, A Van Soom, A de Kruif

Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

Reproduction in Domestic Animals, In Press.

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

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Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

127

Abstract

Negative energy balance (NEB) in high yielding dairy cows early post partum may

affect oocyte quality. Therefore, we tested the effect of two different β-hydroxybutyrate

(BHB) and glucose concentrations, which are associated with subclinical or clinical ketosis,

during IVM on the developmental competence of bovine oocytes.

In experiment 1, subclinical ketosis conditions were imitated. Oocytes were matured

in 4 different serum-free media with 2 glucose concentrations (g1 = 2.75 mmol/l or G1 = 5.5

mmol/l glucose) and with or without BHB (BHB1 = 1.8 mmol/l BHB). Following maturation

groups were used: g1, G1, g1:BHB1 and G1:BHB1. In experiment 2, clinical ketosis

conditions were mimicked by using the concentrations: g2 = 1.375 mmol/l or G2 = 3.1

mmol/l glucose and BHB2 = 4.0 mmol/l BHB. The combinations used were: g2, G2,

g2:BHB2 and G2:BHB2. After IVM and IVF, presumptive zygotes were routinely cultured

for 7 days in SOF (5% FCS). At respectively 48h and 8 days pi, cleavage rate and number of

blastocysts were recorded.

The results demonstrated that the maturation conditions mimicking subclinical

(g1:BHB1) and clinical ketosis (g2:BHB2) resulted in an impaired developmental competence

of the oocyte after maturation. Especially the moderately low (g1) or extremely low glucose

(g2) concentrations were responsible for this detrimental effect which was associated with a

blocked cumulus expansion. Only in moderately low glucose conditions (g1:BHB1), BHB

exerted an additive toxic effect during oocyte maturation resulting in a reduced blastocyst

rate. Conclusively, our results may suggest that subclinical and clinical ketosis can affect the

oocyte’s developmental competence most likely through a directly adverse effect of the low

glucose concentrations on oocyte maturation. Only in subclinical conditions this harmful

effect may be aggravated by BHB.

Key Words

Glucose, β-Hydroxybutyrate, Ketosis, Negative energy balance, Oocyte quality

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

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Introduction

In the 21st century a disappointing reproductive performance has become a major issue

for the modern dairy industry (Lucy, 2001; Bousquet et al., 2004). Numerous studies on high

yielding dairy cows have reported on retarded onset of ovarian activity post partum,

disordered ovarian cyclicity (cystic ovarian disease or prolonged luteal phases) and reduced

oestrus symptoms (Beam and Butler, 1997; Opsomer et al., 1998; Lopez et al., 2004). This

reproductive failure has frequently been linked to the negative energy balance (NEB) early

post partum (Ducker et al., 1985; Butler, 2003). During the early postpartum period, the high

yielding dairy cow faces with an energy deficit due to the imbalance between energy intake

through feed and energy expenditure through milk yield. This imbalance associated with the

so called energy prioritization towards milk production is accompanied by a massive degree

of lipolysis and is typically featured by high non-esterified fatty acid (NEFA) and β-

hydroxybutyrate (BHB) in combination with low glucose concentrations in serum (Baird,

1982; Chilliard et al., 1998; Herdt, 2000).

Apart from the disturbed ovarian function, also reduced conception rates and a high

incidence of early embryonic mortality have been put forward as major factors of

reproductive failure in high producing dairy cows (Dunne et al., 1999; Bilodeau-Goeseels and

Kastelic, 2003; Bousquet et al., 2004). These observations strongly suggest that not only the

endocrine signalling is disturbed but that also the quality of the oocyte and/or embryo proper

can be adversely affected by the NEB (O’Callaghan and Boland, 1999). Very recently it has

been shown that lactating dairy cows produce embryos of inferior quality compared to non-

lactating dairy heifers (Sartori et al., 2002; Leroy et al., 2005a) or beef cows (Leroy et al.,

2005a). Even oocyte quality is said to be reduced in cows that suffer from a severe NEB

(Kruip et al., 1995; Wiltbank et al., 2001; Walters et al., 2002). This could partly be

explained by the high NEFA concentrations in serum which are paralleled in follicular fluid

(Leroy et al., 2004) thereby exerting toxic effects on oocyte maturation (Leroy et al., 2005b)

and on granulosa cell growth and function (Vanholder et al., 2005). The more, it has also been

documented that changes in glucose and BHB concentrations in serum are well reflected in

follicular fluid of the dominant follicle (Leroy et al., 2004). As a consequence, bovine oocytes

are exposed to elevated BHB and low glucose concentrations in case of subclinical or clinical

ketosis. Investigating the effect of such an exposure on the developmental competence of

bovine oocytes could be an interesting next step in unravelling the pathways to subfertility.

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

129

Therefore, the purpose of the present study was to imitate glucose and BHB

concentrations, which are typically associated with subclinical and clinical ketosis, in an in

vitro maturation model to test their effect on oocyte developmental competence.

Material and Methods

Materials and media

Chemicals and media were obtained from Sigma (Bornem, Belgium) and from

Gibco/InvitrogenTM life technologies (Merelbeke, Belgium). A modified HEPES-buffered

Tyrode’s balanced salt solution, termed HEPES-TALP, consisted of 114 mmol/l NaCl, 3.1

mmol/l KCl, 2 mmol/l NaHCO3, 0.3 mmol/l NaH2PO4, 10 mmol/l HEPES, 2.1 mmol/l CaCl2,

0.4 mmol/l MgCl2, 10 mmol/l sodium lactate, 0.2 mmol/l sodium pyruvate, 3 mg/ml fatty acid

free bovine serum albumin (BSA) and 10 μg/ml gentamycine sulphate. Murine epidermal

growth factor (EGF) was solved at a concentration of 1μg/ml in bicarbonate buffered Medium

199 with Earle’s and glutamine (TCM 199) and with 0.1% w/v fatty acid-free BSA.

The serum-free maturation media (pH = 7.2) contained TCM199 (5.5 mmol/l glucose),

DMEM (without glucose), D-glucose and a sodium salt of DL-β-hydroxybutyrate and EGF

(20 ng/ml). In the first experiment the ratio of TCM199:DMEM was 1:1 in order to obtain a

glucose concentration of 2.75 mmol/l (glucose concentration in follicular fluid during

subcinical ketosis). For the second experiment a TCM199:DMEM ratio of 1:3 was used to

obtain a glucose concentration of 1.375 mmol/l (corresponding to glucose concentration in

follicular fluid during clinical ketosis). Higher glucose concentrations were achieved by

adding appropriate amounts of D-glucose (see below). Fertilization medium consisted of

Tyrode’s balanced salt solution supplemented with 25 mmol/l NaHCO3, 10 mmol/l sodium

lactate, 0.2 mmol/l sodium pyruvate, 6 mg/ml fatty acid-free BSA, 10 μg/ml gentamycin

sulphate and 10 μg/ml heparin. The embryo culture medium consisted of Synthetic oviduct

fluid (SOF) (Minitüb, Tiefenbach, Germany) supplemented with 40 μl/ml Basal medium

eagle (BME), 10 μl/ml Minimum essential medium (MEM), 0.2 mmol/l sodium pyruvate and

50 μl/ml Fetal calf serum (FCS) (N.V. HyClone, Europe S.A., Erembodegem, Belgium).

Percoll™ was purchased from Amersham Biosciences (Uppsala, Sweden), heparin

from Leo Pharma (Zaventem, Belgium), ethanol from Vel/Merck Eurolab (Zaventem,

Belgium), and Hoechst 33342 from Molecular Probes (Leiden, The Netherlands).

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

130

In vitro production of embryos

Ovaries and oocytes were collected as described by Tanghe et al. (2003). After

collection, ovaries were rinsed in physiological saline (0.9% NaCl) with 0.5% kanamycin.

The IVM was performed as follows. Immature cumulus oocyte complexes (COCs) were

aspirated from follicles of 2-6 mm in diameter. Only grade I COCs were used for further

culture following selection under a stereomicroscope. After several washings in HEPES-

TALP, the COCs were cultured in groups of 50-60 for 24 h at 38.5 °C in 500 μl of serum-free

maturation medium in a humidified 5% CO2 incubator.

After IVM, fertilization was performed as described by Tanghe et al. (2003). Briefly,

all groups of COCs were coincubated per 100-120 with spermatozoa at a final concentration

of 106 sperm cells/ml for 20 h at 38.5 °C in fertilization medium in a humidified 5% CO2

incubator. For all experiments, frozen bull semen from the same ejaculate was thawed and

live spermatozoa were selected by centrifugation on a discontinuous Percoll® gradient (90 and

45%). The final sperm-egg ratio was adjusted to 5000:1.

After coincubation with spermatozoa, the presumptive zygotes were vortexed for 4

minutes to remove excess sperm and cumulus cells. After several washings with HEPES-

TALP and modified SOF medium, presumptive zygotes were cultured per 25 in 50 μl droplets

of modified SOF medium with 5% FCS, under mineral oil (modular incubator: 39 °C, 5%

CO2, 5% O2 and 90% N2) until 8 days after fertilization. For each replicate, four drops of

embryos were prepared per treatment.

Experimental design

In a first experiment, in which subclinical ketosis conditions were mimicked, oocytes

were matured either in standard in vitro glucose concentrations (G1 = 5.5 mmol/l glucose

corresponding to the routinely used glucose concentration in TCM based maturation media)

or in a moderately hypoglycaemic environment (g1 = 2.75 mmol/l glucose) with or without

elevated BHB concentrations (BHB1 = 1.8 mmol/l BHB). Both these moderately low glucose

and elevated BHB concentrations were within the ranges which had been measured in

follicular fluid of the dominant follicle in high yielding dairy cows early post partum in

association with subclinical ketosis (Leroy et al., 2004). Following 4 different maturation

groups were used: g1, G1, g1:BHB1 and G1:BHB1. In total 1034 oocytes were cultured in

three different replicates.

In a second experiment, in which clinical ketosis conditions were mimicked, oocytes

were matured in normal in vivo glucose concentrations (G2 = 3.1 mmol/l glucose as a control)

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

131

or in a severely hypoglycaemic environment (g2 = 1.375 mmol/l glucose) with or without

very high BHB concentrations (BHB2 = 4.0 mmol/l BHB) which had been measured less

frequently in follicular fluid of the dominant follicle in high yielding dairy cows (Leroy et al.,

2004). These very low glucose and high BHB concentrations are typically associated with

clinical ketosis (Zdzisinska et al., 2000). Following 4 different maturation groups were used:

g2, G2, g2:BHB2, G2:BHB2. In total 1421 oocytes were cultured in four different replicates.

The developmental competence of the oocytes was investigated by evaluating

cleavage rate and number of blastocysts at 48 hours and at day 8 after fertilisation,

respectively.

Statistics

The proportion of oocytes that cleaved at 48h after fertilization and the proportion of

oocytes and cleaved zygotes that developed up to the blastocyst stage at Day 8 after

fertilization were calculated and are expressed as means. All statistical procedures were

carried out with SPSS 11.0 for Windows, (Chicago, IL, USA). Values of P < 0.05 were

considered statistically significant.

In preparation of the final statistical analysis, the importance of randomness of

‘replicate’ was investigated by means of a logistic regression in which replicate was inserted

as random factor and therapy as fixed factor (MLwiN). Since the randomness of replicate

turned out to be not important, data were analysed using a binary logistic regression model

(experimental unit is one oocyte) in which treatment (4), replicate (3 or 4) and the interaction

of these two factors were included (SPSS 11.0). In the absence of a significant interaction

term, this term was left out from the final model. Because 4 different treatment groups were

analysed, 3 pairwise comparisons (which were not independent) had to be made. Therefore,

the P-values were corrected (Bonferroni correction).

Results

Maturation of bovine oocytes at a glucose (g1) concentration, which is associated with

subclinical ketosis in dairy cows, tended to have an adverse effect on cleavage rate at 48h pi

(P = 0.08) compared to G1 or G1:BHB1 (Table 1). This tendency of toxicity became

significant when subclinical BHB concentrations were added to the low glucose maturation

medium (g1:BHB1) which resulted in a lower cleavage rate compared to G1 and G1:BHB1.

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

132

A significantly additive (negative) effect of simultaneous exposure to low glucose and BHB

during IVM could be observed for blastocyst yield compared to maturation in low glucose

alone (g1) or G1:BHB1 (P < 0.05) and compared to G1 (P = 0.07). In other words, only in

moderately hypoglycaemic conditions BHB turned out to exert an extra toxic effect during

maturation on the oocyte’s developmental competence. Addition of BHB in control glucose

concentrations had no effect. Once the zygotes have been cleaved, a similar additive toxic

carry over effect of BHB could be observed on embryo development after maturation in

moderately low glucose conditions.

Table 1. Cleavage rate at 48h PI, number of formed blastocysts relative to the number of cultured oocytes or to the number of cleaved zygotes at day 8 PI. Oocytes were matured in 2.75 mM (g1) or in 5.5 mM (G1) glucose with or without the presence of 1.8 mM β-hydroxybutyrate (BHB1) (subclinical ketosis). g1 G1 g1BHB1 G1BHB1 Cleavage rate at 48h pi (%) 57.13 ± 3.16ab 66.18 ± 4.16b 55.54 ± 2.78a 66.56 ± 3.34b

% blastocysts from oocytes 25.33 ± 2.33a 23.48 ± 3.32ab 17.41 ± 3.62b 27.46 ± 3.06a

% blastocysts from cleaved zygotes

45.30 ± 4.46a 33.50 ± 4.31ab 31.21 ± 5.40b 40.75 ± 4.15ab

ab Data marked with different superscripts per row differ significantly between groups.

For the second experiment, it was decided to use the physiological glycaemia level

(3.1 mmol/l) as normal control medium (G2) in stead of the routine IVM glucose

concentration (5.5 mmol/l) as in experiment 1. Exposure to the metabolic environment

associated with severe clinical ketosis during the period of IVM (g2:BHB2) but also exposure

to very low glucose concentrations alone (g2), showed to be harmful for the oocyte’s

developmental competence (Table 2). However, in contrast with experiment 1, no significant

additive effect of high BHB concentrations in low glucose conditions could be observed.

Moreover, after maturation in a very low glucose environment (g2 or g2:BHB2), COC’s

showed almost no cumulus expansion (Figure 1). Thus, especially the very low glucose

concentrations during oocyte maturation resulted in a hampered cleavage and blastocyst

formation, irrespective of the BHB concentration (g2 or g2:BHB2). Concerning the blastocyst

formation from zygotes, a negative ‘carry over effect’ of clinical ketosis maturation

conditions (g2:BHB2) could be observed on embryo development.

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

133

Table 2. Cleavage rate at 48h PI, number of formed blastocysts relative to the number of cultured oocytes or to the number of cleaved zygotes at day 8 PI. Oocytes were matured in 1.375 mM (g2) or in 3.1 mM (G2) glucose with or without the presence of 4.0 mM β-hydroxybutyrate (BHB2) (clinical ketosis). g2 G2 g2BHB2 G2BHB2 Cleavage rate at 48h pi (%) 53.89 ± 2.34a 64.47 ± 2.20b 46.05 ± 3.35a 64.93 ± 2.81b

% blastocysts from oocytes 16.88 ± 3.66a 23.72 ± 2.23b 11.37 ± 1.58a 22.76 ± 3.10b

% blastocysts from cleaved zygotes

30.12 ± 6.52ab 36.56 ± 3.14a 29.30 ± 5.84b 33.77 ± 4.28a

abData marked with different superscripts per row differ significantly between groups.

A. B. Figure 1. Cumulus oocyte complexes after 24h of maturation in control medium (glucose 3.1 mM: well expanded cumulus investment) (A.) and in medium with low glucose concentration (1.375 mM: poor expansion of the cumulus investment) (B.) (40 X magnification).

Discussion

In the present study the effect of elevated BHB and low glucose levels during in vitro

maturation on the oocyte’s developmental capacity was investigated. We showed that the

maturation conditions mimicking (sub)clinical ketosis resulted in an impaired developmental

competence of the oocyte after maturation. Only in moderately low glucose conditions, BHB

exerted an additive toxic effect during oocyte maturation. In experiment 2, especially the very

low glucose concentrations rather than the high BHB concentrations as such were toxic for

oocytes during their maturation, blocking cumulus expansion and leading to a reduced

developmental competence. The effects of subclinical or clinical ketosis associated glucose

and BHB concentrations on oocyte maturation in vitro have, to our knowledge, never been

studied before.

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

134

Ketone bodies represent an integral part of ruminant intermediary metabolism and

they provide a major form of energy to peripheral tissue during negative energy balance,

when glucose concentrations are low due to for example an increasing milk production early

post partum (Duffield, 2000). Literature about the effect of such elevated ketone

concentrations on several cell types is not unequivocal. Lacetera et al. (2002) did not find any

effect of 3.6 mM BHB on mononuclear cell function in vitro which confirmed earlier findings

(Franklin et al., 1991; Nonnecke et al., 1992). Others described a toxic effect of BHB on

neutrophil function, concluding that high BHB concentrations are a causal link between NEB

and the immune depression which has been frequently reported in early postpartum cows

(Hoeben et al., 1997; Suriyasathaporn et al., 2000). The reason why BHB only exerted an

additive toxic effect at concentrations seen in mild ketosis (in moderately low glucose

conditions) is not known. Sartorelli et al. (2000) described similar findings in ovine

neutrophils which only displayed a decreased bactericidal activity when incubated in 2.4

mmol/l BHB and not after incubation in 4.8 mmol/l BHB. No explanation is however given.

In contrast with our findings, Zdzisinska et al. (2000) did not find any effect of a combination

of low glucose and high BHB levels on endothelial cell function.

It is known that BHB may be utilized as energy source for several cell types under

aerobic conditions in the citric acid cycle (Veech, 2004). Gomez et al. (2002) demonstrated

that at least early bovine embryos are capable of consuming BHB as an alternative energy

source. Whether this is also the case in the COC, is not known. Since addition of BHB in the

maturation medium with low glucose concentration did not improve the reduced cleavage and

blastocyst formation in the present study, it is most likely that COCs are not able to use BHB

as an alternative source of energy. To the contrary, addition of BHB aggravated the harmful

effects of moderately low glucose concentrations as discussed above. In the cumulus cells

glucose is predominantly metabolized via the glycolytic pathway for the production of

pyruvate and lactate, which are the oocyte’s preferred substrates for ATP production (Cetica

et al., 2002). These pyruvate and lactate molecules can not be produced from BHB though

since BHB is inserted as acetyl-CoA in the Krebs cycle (Stryer, 1995). In the oocyte proper,

glucose is predominantly metabolized in the pentose phosphate pathway (PPP) for DNA or

NADPH synthesis (reviewed by Sutton et al., 2003). Also in this particular pathway BHB can

not serve as an alternative substrate (Nehlig, 2004). Glucose is thus an indispensable molecule

during oocyte maturation both for energy supply and for the meiotic progression (DNA

synthesis) which ultimately determines the oocyte’s developmental capacity (Downs and

Utecht, 1999; Cetica et al., 2002; Sutton et al., 2003). This is clearly illustrated by our results

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

135

which demonstrate that reducing the glucose concentration in the maturation medium to 2.75

mmol/l or even to 1.375 mmol/l, significantly hampers the subsequent oocyte’s cleavage (for

g1 and g2) and blastocyst formation (for g2). Hashimoto et al. (2000) confirmed this by

demonstrating that addition of glucose (1.5 or 5.5 mmol/l compared to absence of glucose)

during IVM improves the nuclear maturation, embryo cleavage and blastocyst development.

Also Iwata et al. (2004) described a beneficial effect of 5.56 mmol/l glucose compared to 1.5

mmol/l glucose, on nuclear maturation and developmental rate, which is in line with our

results. The nuclear maturation stage was not investigated in the present study, but we did

observe obvious reduction in cumulus expansion when COCs were matured in hypoglycaemic

conditions, irrespective of BHB. This observation confirms that glucose is necessary to

sustain an adequate cumulus expansion as it acts as a substrate for the formation of

extracellular matrix (hyaluronic acid) which cannot be synthesized from BHB (Sutton-

McDowall et al., 2004).

Conclusions

Based on our results, it can be concluded that cows experiencing clinical ketosis create

an adverse biochemical environment for optimal oocyte maturation in follicular fluid,

resulting in a hampered developmental competence. Not the very high BHB concentrations

but the concomitant hypoglycaemic conditions seem to be responsible for this adverse effect

on oocyte quality. Only in moderately low glucose conditions, BHB aggravate the toxic effect

of the glucose concentrations associated with subclinical ketosis. These findings may reveal

one of the pathways in the complex interaction between NEB and subfertility in general or

oocyte quality more specifically in the modern high producing dairy cow.

Acknowledgments

The authors thank J. Mestach and G. Spaepen for their excellent technical support, and

K. Moerloose for the critical reading of the manuscript. This research was funded by the

Institute for the Promotion of Innovation by Science and Technology in Flanders (Grant no°

13236).

Chapter 5B: Effect of (sub)clinical ketosis on oocyte quality

136

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Chapter 6

A New Technique to Evaluate the Lipid Content of Single Oocytes and Embryos

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Chapter 6A

The Use of Fluorescent Dye, Nile Red, to Evaluate the Lipid Content of Single Mammalian Oocytes

J.L.M.R. Leroy1, G Genicot 2, A Van Soom 1, I Donnay 2

1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

2 Catholic University of Louvain, Institut des Sciences de la Vie, Unité des Sciences vétérinaires, Place Croix du Sud 5 box 10, B-1348 Louvain-la-Neuve, Belgium.

Adapted from Theriogenology 2005, 63: 1181-1194.

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Chapter 6A: Lipid Evaluation of Single Oocytes

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Abstract

This study aimed to investigate the use of Nile red, a fluorescent dye specific for

intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was

hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result

in a higher amount of emitted fluorescent light.

Following fixation and subsequent staining of denuded oocytes, the fluorescence of

the whole oocyte was visualized by fluorescence microscopy and quantified with a

photometer and photomultiplier connected to the microscope. The peak of fluorescence was

observed in the yellow spectrum (590nm) and the fluorescence was restricted to the lipid

droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 μg/ml

yielded similar results. After fixation, a minimum of 2h staining was necessary to reach

maximal fluorescence which remained stable for several hours. The position of the

microscopic focus within the oocyte had no influence on the amount of measured

fluorescence. Successive measurements of the same oocyte yielded very similar results

indicating the repeatability of the method. Finally, the technique was validated by comparing

the lipid content of bovine, porcine and murine immature oocytes, which are known to

contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8

fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less

fluorescent than porcine oocytes.

Based on this study, it can be said that this rather fast and easy technique allows for

the relative quantification of the lipid content (present in the lipid droplets) of one single

oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine

oocytes correlated with the known lipid contents in these three species. This technique could

be used to compare the lipid content of oocytes originating from different donors, from

different sized follicles or cultured in various conditions.

Key Words

Fluorescence, Lipid content, Mammalian oocyte, Nile red.

Chapter 6A: Lipid Evaluation of Single Oocytes

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Introduction

It is known for quite some time that culture environment can influence oocyte and

embryo morphology and metabolism (Lonergan et al., 2003; Rizos et al., 2003). Kim and co-

workers (2001) suggested the uptake of serum lipids during the in vitro maturation of bovine

oocytes. The dark appearance of bovine embryos cultured in the presence of serum has been

attributed to an increased lipid content (Abe et al., 1999; Ferguson et al., 1999; Abe et al.,

2002). Three mechanisms have been suggested by which serum can interfere with lipid

metabolism in embryos: (a) serum may increase lipid neosynthesis (especially triglycerides)

in the embryo (Abd El Razek et al., 2000), (b) lipoproteins present in the serum could be

internalised by the cells, increasing the intracellular lipid content (Ferguson et al., 1999; Sata

et al., 1999) or (c) the presence of serum can alter lipid metabolism of mitochondria, leading

to an increased storage of intracellular lipids (Abe et al., 2002; Abe et al., 2003). A

combination of these mechanisms is also possible. Reis and co-workers (2003) demonstrated

that especially neutral lipids accounted for the embryonic fatty acid accumulation in the

presence of serum. Whatever the mechanism(s), an increase in intracellular lipids impairs the

quality of the embryos by increasing their sensitivity to oxidative stress and cryopreservation

(Reis et al., 2003). Moreover, the sensitivity to chilling and cryotolerance of mammalian

oocytes and embryos seems directly correlated to their lipid content (Kim et al., 2001; Abe et

al., 2002).

Several methods have already been used to evaluate the lipid content in oocytes and

embryos. Fatty acid composition can be analysed by thin-layer or gas chromatography.

However, these techniques require from 4 up to 1000 oocytes or embryos to be analysed

together (McEvoy et al. 2000; Kim et al., 2001; Sinclair et al., 2002; Reis et al., 2003). Kit-

based assays can be used to measure the different classes of lipids in pools of 100 oocytes or

embryos (Kim et al., 2001). It is also possible to measure the triglyceride content of 1 to 3

embryos using an enzymatic assay coupled to microfluorescence detection (Ferguson et al.,

1999; Sturmey and Leese, 2003). This technique is more sensitive but is difficult to carry out.

Neosynthesis of lipids can be quantified by the incorporation of labelled oleic acid, followed

by HPLC (Abd El Razek et al., 2000). Crosier et al. (2000; 2001), Abe et al. (2002), Rizos et

al.(2003) and Kikuchi et al. (2002) used electron or light microscopy after staining with

Sudan Black B for the evaluation of the number, density or size of the intracellular lipid

droplets in embryos grown in different culture conditions. This technique does not permit to

Chapter 6A: Lipid Evaluation of Single Oocytes

147

evaluate the whole embryo because only a few slices are analysed. When analysing a limited

number of oocytes (following ovum pick-up) or flushed embryos in an in vivo study, all

abovementioned techniques are labour-intensive and/or impossible to perform on a single

oocyte or embryo.

Therefore, a lipid specific fluorescent dye, Nile red, was used for the first time to

visualize the lipid droplets and to evaluate the lipid content of single oocytes. The more lipid

droplets present in an oocyte, the higher the amount of emitted fluorescent light will be after

staining. In order to allow the evaluation of the lipid content in single oocytes or embryos,

fluorescence was quantified using a photometer connected to a microscope. The fluorescence

of Nile red is quenched in an aqueous environment. In a hydrophobic lipid environment

however, Nile red fluoresces yellow to orange. After staining with Nile red, neutral lipids, like

triglycerides (lipid droplets), fluoresce yellow (580–596 nm, 590-nm peak fluorescence)

while polar lipids (phospholipid bilayers) fluoresce in the orange spectrum (597–620 nm,

600-nm peak fluorescence) (Greenspan and Fowler, 1985). Nile Red is commonly used to

visualize intracellular lipid droplets in all kinds of cells (Henault and Killian, 1993;

Greenspan et al., 1985; Than et al., 2003). The use of Nile red to evaluate the lipid content in

macrophages has been reported by Koren et al. (1990).

The aim of the present study was to test the sensitivity, the specificity and the

repeatability of this new technique for the visualisation of the lipid droplets and the evaluation

of the lipid content in lipid droplets in single mammalian oocytes. The following parameters

were tested: specificity of the dye for the lipid droplets, spectrum of emission, dye

concentration, duration of equilibration, importance of the plane of focus during the

measurement and stability and repeatability of the measurements. Finally, this study aimed to

validate the technique by analysing oocytes of different species (cattle, pig and mouse) known

to have different lipid contents.

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Materials and Methods

All experimental procedures were approved and conducted in accordance with the UK

Animals (Scientific procedures) Acts, 1986 and with the guidelines of the animal ethics

committee of the catholic University of Louvain, Belgium. The chemicals used in this study

were purchased from Sigma-Aldrich (Steinheim, Germany) unless otherwise indicated.

Oocyte recovery

Bovine cumulus oocyte complexes (COCs) were collected by puncturing follicles (2 -

6 mm) from abattoir cow ovaries. Pig COCs were collected by puncturing 3 to 5 mm follicles

from ovaries of slaughtered 6 month old pigs. Mouse COCs were collected by slicing ovaries

of ten week old mice (n=4) after cervical dislocation. Only COCs with an intact, non-

expanded cumulus investment (Grade I) were used for further analysis (22). COCs were

selected in Hepes-buffered TCM-199.

Oocyte staining

Selected COCs were vortexed for 10 min in Hepes-199 and the denuded oocytes were

fixed in a 500µl 2% glutaraldehyde and 2% formaldehyde solution. Oocytes were then fixed

for at least 24h. They were transferred in individual wells of a 386-well microplate (cliniplate

384 labsystem, Helsinki, Finland) containing 25µL of a 10µg/ml Nile Red solution

(Molecular Probes, Inc., Eugene, OR, USA) dissolved in physiological saline (0.9% NaCl)

with 1mg/ml polyvinylpyrrolidone. Oocytes were stained overnight in the dark and at room

temperature unless otherwise indicated. The Nile Red stock solution (1mg/ml) was prepared

by dilution in DMSO and stored at room temperature in the dark. Final concentrations were

obtained by diluting the stock with the saline solution.

Photometer measurement of single oocytes

Lipid droplets were visualised using a fluorescence microscope and a 20x and 60x

objective. The amount of emitted fluorescent light of the whole oocyte was evaluated at 582 ±

6 nm with an inverted fluorescence microscope (Excitation: 400-500nm and Emission:

515LP) using a 10x objective. The fluorescence was amplified with a photomultiplier,

quantified with a photometer attached to the microscope (MPV-SP, Leitz, Wetzlar, Germany)

and calculated by the MPF Bio Software (Leitz). The results were expressed in arbitrary units

Chapter 6A: Lipid Evaluation of Single Oocytes

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of fluorescence. A UV light filter was necessary to avoid bleaching. The spectrum of emission

was evaluated using the MPV Spectra Software (Leitz). Unless otherwise indicated, one

measurement was performed per oocyte.

Experimental design

Experiment 1: Kinetics of saturation

To investigate the kinetics of saturation of Nile Red, the fluorescence of 30 bovine

oocytes was evaluated immediately after the transfer into the dye solution (time 0), then every

hour during 12 hours and at 24h, 48h and 72h. A group of 10 oocytes was stained one day

ahead to saturate the oocytes with the dye and was measured in parallel with the 30 oocytes as

a control group. Those 10 oocytes were measured simultaneously with the experimental

group. The 386 well plate was protected from light and covered after each measurement to

avoid evaporation. All manipulations were performed at room temperature.

Experiment 2: Importance of the accuracy of the focus

It was suspected that the position of the focal plane within the oocyte could influence

the results. Therefore, five stained bovine oocytes were analysed at different focal planes,

starting with the equatorial plane. Under and above this equatorial plane, five additional

measurements were performed. The distance between each measurement plane was 20 µm.

Experiment 3: Effect of the dye concentration

To test the effect of the dye concentration on the emitted fluorescence light, different

groups of 5 to 15 bovine oocytes were incubated in parallel in dye concentrations of 100, 10,

1, 0.1 or 0.01µg/mL of Nile red. The same staining protocol was used as described earlier.

Experiment 4: Repeatability of the measurement

Thirty one bovine oocytes were measured twice at a one-hour interval. Oocytes were

not selected for their quality in this experiment in order to obtain a broad spectrum in lipid

content. A correlation coefficient was calculated between the first and the second

measurement. Five oocytes were measured ten times and the smallest significant difference

was calculated using a regression model where the response is a deterministic function of the

repetition number (in order to take into account the bleaching effect).

Chapter 6A: Lipid Evaluation of Single Oocytes

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Experiment 5: Comparison between different species

To validate the technique, 75 bovine, 78 porcine and 29 murine immature oocytes

were stained after collection and their fluorescence measured as previously described.

Statistical analysis

One-way ANOVA was used to analyse data in experiments 1, 3 and 5 (JMP software;

SAS Institute Inc. SAS Campus Drive Cary, NC 27513). A two-way ANOVA (mixed model)

with oocyte as random factor was used to analyse experiment 2. Analysis of variance was

followed by the post hoc Scheffé test. A paired t-test in combination with a Spearman

correlation was used to compare the two measurements in Experiment 4 and the smallest

significant difference was calculated by means of a regression model. The results are

presented as mean ± SD. The threshold for significance was set at P < 0.05.

Results

After evaluation of the spectrum, the average maximum emission was found at 589 nm

(Figure 1), which corresponds to the emission spectrum of apolar lipids. The fluorescence

seemed to be restricted to the droplets corresponding to the dark spots observed with the

transmitted light and likely to be lipid droplets (Figure 2).

Figure 1. Spectrum of fluorescence emission of a bovine immature oocyte stained with Nile Red. The maximum emission was found at 589 nm.

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 2. Stained immature bovine oocytes (after Nile red staining) with a granulated (A and B) cytoplasm under visible light (A) and after UV excitation (B). Dark zones in the cytoplasm (with normal light) correspond with clusters of lipid droplets (white circles) (x 200 magnification). Fluorescence after Nile red staining is restricted to the lipid droplets (C) (arrow) (x 600 magnification).

Experiment 1: Kinetics of saturation

Maximum fluorescence was reached after 2 hours of staining and was then equal to the

control group stained the day before (Figure 3). The staining remained stable during the

following hours. The decrease observed after 24h might be related to variations in the light

intensity of the UV lamp, which was switched off between the last 3 measurements.

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 3. Kinetics of intensity of the fluorescence (mean ± SEM) in relation to the duration of staining (30 bovine oocytes – plain line). Control oocytes were stained one day before (10 oocytes – doted line). *Significantly different from control oocytes (P < 0.05).

Experiment 2: Importance of the accuracy of the focus

The results presented in Figure 4 show that the measurement of light emission by the

oocytes stained with Nile red is not affected by the variation of the focal plane within the

oocyte volume (intervals of 20 μm). As a consequence, there is no need to focus at the

equatorial plane of the oocytes when taking the measurements.

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 4. Influence of the focus plane on the measurement of fluorescence of 5 immature bovine oocytes. Units on the X-axis are related to the distance from the equatorial plane of the oocyte. (positive values: focus under the equatorial plane; negative values: focus above the equatorial plane). Each line represents 1 oocyte.

Experiment 3: Effect of the dye concentration

A concentration of 10 µg/ml is recommended by Molecular Probes for various types

of cells. Figure 5 shows that concentrations of 0.1 to 10 µg/ml gave similar results, while at

0.01 µg/ml, the fluorescence significantly decreased (P < 0.05). At 100 µg/ml, a significant

decrease in fluorescence intensity was also observed which might be related to the large

amount (20%) of DMSO in the staining medium (P < 0.05).

← Towards objective

Away from objective →

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 5. Effect of the dye concentration on the intensity of fluorescence (Mean ± SEM). a,b: values with different superscripts are significantly different (P < 0.05).

Experiment 4: Repeatability of the measurement

Repeated measurements within one oocyte at 1h intervals gave very reproducible

results while large variations were observed between oocytes (Figure 6). The paired sample t-

test revealed that the second measurement resulted in slightly higher light emission, probably

due to variation in the UV lamp intensity (P < 0.05). The correlation coefficient between the

first and the second measurement was very high (r2 = 0.98). The smallest difference in nile

red fluorescence intensity that can be detected within the same run between two oocytes

corresponds to roughly 1% (range: 0.8 to 1.2%).

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 6. Comparison of the fluorescence intensity of 31 different bovine oocytes measured two times with a one-hour interval.

Experiment 5: Comparison between different species

Figure 7 and 8 show the results of the mean fluorescence intensity of cattle, pig and

mouse oocytes after Nile red staining. A highly significant difference was observed between

the three species: the mean fluorescence of porcine oocytes was higher than that of bovine

oocytes which was in turn higher than that of murine oocytes (in arbitrary units of

fluorescence ± SD): 513±147, 233±97 and 75±32 respectively, (P < 0.001).

Oocyte ID

Chapter 6A: Lipid Evaluation of Single Oocytes

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Figure 7. Comparison of the lipid content of immature bovine, porcine and murine oocytes. (Mean ± SD). a,b: values with different superscripts are significantly different (P < 0.001).

Figure 8. Immature murine (A and B) bovine (C and D) and porcine (E and F) oocytes stained with Nile red under visible light (A, C and E) and after UV excitation (B, D and F). (x 200 magnification).

Chapter 6A: Lipid Evaluation of Single Oocytes

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Discussion

In this study the Nile red staining technique was tested for the first time to evaluate the

lipid content in single mammalian oocytes. The peak of fluorescence in stained bovine

oocytes was observed in the yellow spectrum (590nm) which corresponds to neutral lipids

(Greenspan and Fowler, 1985). Moreover, the fluorescence proved to be very specific for

lipid droplets and no fluorescence was observed in the cytosol or in the nuclear compartment.

These findings indicate that the fluorescing lipid droplets mainly contain triglycerides, which

confirms previous studies (Kim et al., 2001). Nile red has been used in previous work to

evaluate the apolar lipid content of cells (Koren et al., 1990). The fluorescence of single

macrophages incubated with acetylated low-density lipoproteins was measured by

microfluorimetry and correlated very well with cholesterol ester content measured by gas

chromatography. The shift in fluorescence in relation to the polarity of the lipid has been used

to detect oxidation of LDL (Greenspan and Lou, 1993) and to differentiate between neutral

lipid and phospholipid rich regions in cells and tissues (Bonilla and Prelle, 1987; Hjelle et al.,

1991; Brown et al., 1992; Smyth and Warton, 1992; Henault and Killian, 1993; Klinkner et

al., 1995).

The technique described in this article, is rapid and easy to perform and allows for the

evaluation of single oocytes. Hundreds of oocytes can be evaluated on the same day and

oocytes can be fixed several days before the staining. Oocytes from different experiments or

replicates can thus be analysed on the same day. The duration of staining can be reduced to 2h

or prolonged for a long period without a significant effect on the fluorescence intensity.

Repeated measurements on the same oocyte gave reproducible results but a filter on the UV

light was necessary to avoid excessive bleaching. A wide range of dye concentrations (0.1 to

10μg/ml) gave similar results. The decrease in fluorescence intensity observed at 100μg/ml

might be related to the increased concentration of DMSO in the medium (20%) which could

lead to the dissolving of the intracellular lipid droplets.

The use of a microfluorimeter connected to a microscope proved to be necessary as the

use of an automated multiplate spectrophotometer seemed to give unreliable results (data not

shown). Moreover it is important to mention that, in the absence of standards, only the

relative amount of lipids present in lipid droplets can be estimated. Furthermore, it does not

allow evaluation of the qualitative composition of lipids (lipid fractions or identification of

the fatty acids). However, the advantage of this technique is that we can compare the lipid

Chapter 6A: Lipid Evaluation of Single Oocytes

158

content between single oocytes originating from different donors or from different treatments

in vitro or in vivo.

In order to test and validate the technique, the lipid content of individual bovine,

murine and porcine oocytes was compared for the first time in the same experiment. Porcine

oocytes contained 2.4 fold more lipid in droplets than bovine oocytes. This is in agreement

with previous studies. A similar ratio in triglyceride content (2.3) is observed if we compare

the data obtained by Sturmey and Leese (2003) and Ferguson and Leese (1999). McEvoy et

al. (2000) observed a 2.6 fold ratio in the content of fatty acids between the two species using

gas chromatography on pools of 1000 oocytes (the ratio was up to 3.2 for fatty acid contained

in triglycerides). As expected, the murine oocytes contained less lipid compared to these of

pigs (pig:mouse = 6.8) and cattle (cattle:mouse = 2.8). The transparency of the oocytes under

the stereomicroscope was indicative for the lipid content: murine oocytes appeared more

transparent than bovine oocytes, and porcine oocytes had the darkest ooplasm (Figure 8). The

triglyceride content of immature bovine oocytes is 59 ng/oocyte in the study by Ferguson and

Leese (1999) and 58ng in the study by Kim et al. (2001) while that of pig oocytes is 135ng

(2003). The total lipid content of porcine oocytes reached 156 ng (McEvoy et al., 1997). Only

one study, performed in 1969 by Loewenstein and Cohen (Loewenstein and Cohen, 1964),

mentioned the lipid content in mouse oocytes. The value found in this study, 4ng per oocyte,

seems low by comparison with our relative results, but the difference might be related to the

indirect technique used. In their study, lipid content was estimated by the difference in oocyte

weight before and after lipid extraction.

It is also possible to apply the Nile red technique to embryos. Preliminary results

indicated that the technique is sensitive enough to detect differences in lipid content of day 6

morulae that were cultured either in the presence or the absence of serum (Leroy et al., 2003).

Lipid seems to be a source of energy for the oocyte and the early embryo.

Triglycerides represent the major component of intracellular lipids in immature oocytes and

may be metabolised during oocyte maturation, fertilization and first embryonic cleavage in

cattle (Ferguson and Leese, 1999; Kim et al., 2001). A close association between lipid

droplets and mitochondria was observed during maturation (Kruip et al., 1983) and during

fertilization (Fleming and Saacke, 1972) which confirms the role of oocyte lipid droplets as

energy stores. In porcine oocytes, a decrease in triglycerides is also observed after maturation

but no significant changes seem to occur during in vitro embryo development up to the

blastocyst stage where a significant increase is observed (Sturmey and Leese, 2003). In vivo

studies on the other hand demonstrated that the lipid content in bovine oocytes or embryos

Chapter 6A: Lipid Evaluation of Single Oocytes

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may be influenced by some physiological parameters such as feed, lactational stage and breed

(Adamiak et al., 2004; Leroy et al., 2004).

Conclusions

In conclusion, this study illustrates that Nile Red staining followed by the

quantification of the emitted fluorescent light with a photometer is suitable for the

visualization and comparison of the lipid contents of single mammalian oocytes. This easy

and rapid technique will be used to correlate lipid content and mitochondrial activity in

oocytes and early embryos of various origins (in vivo vs in vitro, from different donors…) or

cultured in various conditions (with or without serum).

Acknowledgments

The authors thank J. Mestach , G. Spaepen and P. Bombaerts for their excellent

technical support, and T. Vanholder, P.E.J. Bols and R. De Roover for the critical reading of

the manuscript. The authors also acknowledge B. Moreau for his statistical advice. This

research was partially funded by the Institute for the Promotion of Innovation by Science and

Technology in Flanders (Grant no° 13236), by Action de Recherche Concertée (Communauté

française de Belgique) and by the European Commission (grant. QLK3-CT1999-00104).

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Leroy JLMR, Genicot G, Opsomer G, Donnay I, Van Soom I. A comparison of the lipid content of immature and mature bovine oocytes and of morulae after staining with Nile Red. Proceedings of the 19th scientific meeting of the AETE, Rostock, 12-13th September 2003:180 (abstr.).

Leroy JLMR, Goossens L, Geldhof A, Vanholder T, Opsomer G, Van Soom A, de Kruif A. Embryo quality and colour in Holstein Friesian and Belgian Blue cattle in relation to donor blood cholesterol and triglycerides. Reprod Fert Dev 2004;16:211 (abstr.).

Loewenstein JE, Cohen AI. Dry mass, lipid content and protein content of the intact and zona-free mouse ovum J Embr Exp Morphol 1964;12:113–121.

Lonergan P, Rizos D, Gutierrez-Adan, Fair T, Boland MP. Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns. Reprod Domest Anim 2003;38:259-267.

McEvoy TG, Coull GD, Broadbent PJ, Hutchinson JS, Speake BK. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida. J Reprod Fertil 2000;118:163-70.

McEvoy TG, Coull GD, Speake BK, Staines ME, Broadbent PJ. Estimation of lipid and fatty acid composition of zona-intact pig oocytes. J Reprod Fert Abstract Series 1997;20:10.

Reis A, Rooke JA, McCallum GJ, Ewen M, Staines ME, Lomax MA, McEvoy TG. Fatty acid content of polar and neutral lipids from bovine blastocysts produced in vitro in the presence or absence of serum. Reproduction Abstract series 2003;30:57-58 (abstr).

Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP, Lonergan P. Bovine embryo culture in the presence or the absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod 2003;68: 236-243.

Sata R, Tsujii H, Abe H, Yamashita S, Hoshi H. Fatty acid composition of bovine embryos cultured in serum-free and serum-containing medium during early embryonic development. J Reprod Dev 1999;45:97-103.

Sinclair KD, Rooke JA, McEvoy TG. Regulation of nutrient uptake and metabolism in pre-elongation ruminant embryos. Reproduction 2002; Suppl 61:371-385.

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Smyth M, Wharton W. Differentiation of A31T6 proadipocytes: a flow cytometric analysis. Exp Cell Res 1992;199:29–38.

Sturmey RG, Leese HJ. Energy metabolism in pig oocytes and early embryos. Reproduction 2003;126:197-204.

Than NG, Sumegi B, Bellyei S, Berki T, Szekeres G, Janaky T, Szigeti A, Bohn H, Than GN. Lipid droplet and milk lipid globule membrane associated placental protein 17b (PP17b) is involved in apoptotic and differentiation processes of human epithelial cervical carcinoma cells. Eur J Biochem 2003;270:1176-1188.

Chapter 6B

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

J.L.M.R. Leroy1, G Genicot 2, I Donnay 2,A Van Soom 1

1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium;

2 Catholic University of Louvain, Institut des Sciences de la Vie, Unité des Sciences vétérinaires, Place Croix du Sud 5 box 10, B-1348 Louvain-la-Neuve, Belgium.

Reproduction in Domestic Animals 2005, 40: 76-78

.

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

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Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

167

Abstract

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid

content of immature bovine oocytes is correlated with the morphological appearance of the

ooplasm.

Oocytes with a uniform dark cytoplasm contained significantly more intracellular

lipids in lipid droplets compared to oocytes with a granulated or pale cytoplasm (P<0.05) .

Furthermore, this lipid analysing technique was applied for the first time on single bovine in

vitro embryos showing a significant increase of the lipid content in lipid droplets after culture

in the presence of serum (P<0.05).

Key Words

Embryo, Fluorescence, Lipid content, Nile red, Oocyte.

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

168

Introduction

The lipid content of oocytes and embryos is an important parameter linked to quality

and cryotolerance. It is known for quite some time that the lipid content can be influenced by

the culture environment of the oocyte and the embryo (Lonergan et al., 2003; Rizos et al.,

2003). Especially, the presence of serum is said to be responsible for excessive lipid

accumulation through increased lipid uptake from the medium and/or through disturbance of

the mitochondrial metabolism (Abe et al., 1999; Ferguson and Leese, 1999; Kim et al., 2001).

Also the origin of the oocyte or the embryo (in vitro or in vivo, species, breed, physiological

state and nutrition) proved to be a determining factor for the lipid content (Visintin et al.,

2002; Adamiak et al, 2004; Leroy et al., 2004).

Recently, a new and reliable technique was developed to evaluate the lipid content of

single bovine oocytes in a semi quantitative way (no absolute but relative evaluation of the

lipid content). This can be done by staining the oocytes with Nile Red which is a fluorescent

dye specific for intracellular lipid droplets (Genicot et al., 2004). The amount of emitted

fluorescent light proved to be correlated with the lipid content. The technique showed to be

highly sensitive and repeatable and in contrast with previously described techniques

(Ferguson and Leese, 1999; McEvoy et al., 2000; Kim et al., 2001), a single oocyte could be

analysed (Genicot et al., 2004).

The morphological appearance of the ooplasm of immature bovine oocytes, which is

commonly used as quality parameter (de Loos et al., 1989; Hawk and Wall, 1994), may be

influenced by the lipid content. Moreover, the known fact that bovine embryos accumulate

lipids when being cultured in the presence of serum, needs to be confirmed with this new

technique. Therefore, the aim of this study was (1) to compare the lipid content of immature

cumulus free bovine oocytes with a uniformly dark, a granulated or a very pale ooplasm and

(2) to apply the technique on day 6 morulae which were cultured either in the presence or the

absence of serum.

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

169

Materials and Methods

For the first experiment, bovine cumulus oocyte complexes (COCs) were collected by

puncturing follicles (2-6 mm) from abattoir cow ovaries. After vortexing (9 min), the cumulus

free oocytes were divided in three groups using a stereomicroscope (40x): Group I containing

oocytes with a uniformly dark ooplasm; Group II containing oocytes with gray and granulated

ooplasm and Group III with oocytes having a very pale appearance of the ooplasm. The

selected oocytes (N = 88, two replicates, 8 to 20 per group) were fixed, stained with 10 μg/ml

Nile Red (Molecular Probes, Inc., Eugene, OR, USA) and analysed as described before

(Genicot et al., 2004).

In the second experiment grade I COCs were matured per groups of 100 in TCM 199

(Invitrogen™ Life Technologies, Merelbeke, Belgium) supplemented with 20% FCS (N.V.

HyClone Europe S.A., Erembodegem, Belgium) for 24h (39°C, 5% CO2 in air atmosphere

with 100% humidity) (de Loos et al, 1989). After maturation a routine fertilisation was

performed (sperm oocyte coincubation for 20h, 1*106 sp/ml). The cumulus cells and

spermatozoa were mechanically removed from the presumptive zygotes. Subsequently they

were washed and placed per groups of 25 in 50 μl droplets of SOF (Minitüb, Tiefenbach,

Germany) either with 5% FCS or with 0.3% BSA (essentially fatty acid free) (Sigma-Aldrich,

Bornem, Belgium) and cultured for 5 days (38.5°C, 5% CO2, 5% O2 and 90% N2). Only

Grade I morulae (N = 55, two replicates, 12 tot 20 per group) were selected for further

analysis (Lindner and Wright, 1983). After fixation and staining, the embryos were analysed

as described before (Genicot et al., 2004).

A two-way ANOVA (mixed model) with replicate as random factor and a post hoc

Tukey test, was used to analyse the results from the immature oocytes. The data of the

morulae were analysed with the same test, leaving the post hoc test (only two groups) (SPSS

11.0 for Windows, Chicago, IL, USA). The results are presented as mean ± SD. The threshold

for significance was set at P < 0.05.

Results

Immature dark oocytes emitted a higher amount of fluorescence light compared to

granulated and pale oocytes (P<0.05). Pale oocytes had a signicantly lower fluorescence light

emission compared to the oocytes with a granulated ooplasm (P<0.05) (Figure 1).

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

170

Morulae that were cultured in the presence of serum contained more lipid droplets expressed

as a significant higher amount of emitted fluorescence light (± SD), compared to the morulae

that were cultured in serum free conditions (575 ± 117 versus 407 ± 123 arbitrary

fluorescence units, respectively) (P<0.05).

150

250

350

450

550

650

750

850

dark granulated pale

arbi

trar

y flu

ores

cenc

e un

its (+

/- SD

)

Figure 1. Amount of emitted fluorescence light of stained immature oocytes with a different appearance of the ooplasm. a, b, c Bars with a different superscript, differ significantly (P<0.05).

Discussion

In this study the Nile Red lipid analysis technique was used for the first time to

correlate the morphological appearance of the ooplasm of immature bovine oocytes with its

lipid content. Our results indicate that the lipid content contributes to the morphological

appearance of the ooplasm. It has been proven already that the dark clusters which can be

noticed in the ooplasm correspond to aggregates of lipid droplets (Genicot et al., 2004). Many

studies have already demonstrated that culture of immature oocytes with a coarsely granulated

or very pale ooplasm resulted in lower blastocyst yields (Hawk and Wall, 1994; Bilodeau-

Goeseels and Panich, 2002). Thus, this may suggest that a certain amount and an even

distribution of intracellular organelles such as lipid droplets are crucial for in vitro

development up to the blastocyst stage. After all, intracellular lipids are suggested to have a

significantly metabolic role as energy source for protein synthesis which supports the

cytoplasmic and nuclear maturation of the oocyte (Sturmey and Leese, 2003).

a

bc

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

171

We also demonstrated that culture in the presence of serum, causes a 30% increase of

the lipid content in lipid droplets of day 6 morulae. An increased lipid uptake from the

medium and/or an impaired mitochondrial metabolism are said to be responsible for the

observed lipid accumulation (Sata et al., 1999).This has been shown in other studies (Abe et

al., 1999; Abe and Hoshi, 2003; Reis et al., 2003) and has now been confirmed with this new

technique in our lab. The advantage of our technique however, is that it can be performed on

one single embryo. This is an interesting feature which makes it possible to investigate the

lipid content of in vivo embryos which are often in short supply.

Conclusions

By means of the newly developed lipid evaluation technique we were able to

demonstrate that the morphological appearance of the ooplasm of immature oocytes is

correlated with lipid content. The technique was also applied with success on single morulae

to demonstrate intracellular lipid accumulation due to culture in the presence of serum.

Acknowledgments

The authors thank J. Mestach , G. Spaepen for their excellent technical support, and T.

Vanholder and P.E.J. Bols for the critical reading of the manuscript. This research was

partially funded by the Institute for the Promotion of Innovation by Science and Technology

in Flanders (Grant no° 13236), by Action de Recherche Concertée (Communauté française de

Belgique) and by the European Commission (grant. QLK3-CT1999-00104).

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

172

References

Abe H, Hoshi H, 2003: Evaluation of bovine embryos produced in high performance serum-free media. J. Reprod. Dev.49 193-202.

Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H, 1999: Ultrastructure of bovine embryos developed from in vitro-matured and –fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Mol. Reprod. Dev. 53 325-335.

Adamiak SJ, Mackie K, Ewen M, Powell KA, Watt RG, Rooke JA, Webb R, Sinclair KD, 2004: Dietary carbohydrates and lipids affect in vitro embryo production following OPU in heifers. Reprod. Fert. Dev. 16 193-194 (abstr.).

Bilodeau-Goeseels S, Panich P, 2002: Effects of quality on development and transcriptional activity in early bovine embryos. Anim. Reprod. Sci. 71 143-155.

de Loos F, van Vliet C, van Maurik P, Kruip TA,1989: Morphology of immature bovine oocytes. Gamete Res. 24 197-204.

Ferguson EM, Leese HJ, 1999: Triglyceride content of bovine oocytes and early embryos. J. Reprod. Fert. 116 373–378.

Genicot G, Leroy JLMR, Van Soom A, Donnay I, 2004: The use of a fluorescent dye, Nile Red, to evaluate the lipid content of single mammalian oocytes. Theriogenology, in Press.

Hawk HW, Wall RJ,1994: Improved yields of bovine blastocysts from in vitro-produced oocytes. I. Selection of oocytes and zygotes. Theriogenology 41 1571-1583.

Kim JY, Kinoshita M, Ohnishi M, Fukui Y, 2001: Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes. Reproduction 122 131–138.

Leroy JLMR, Goossens L, Geldhof A, Vanholder T, Opsomer G, Van Soom A, de Kruif A, 2004: Embryo quality and colour in Holstein Friesian and Belgian Blue cattle in relation to donor blood cholesterol and triglycerides. Reprod. Fert. Dev. 16 211 (abstr.).

Lindner GM, Wright RW, 1983: Bovine embryo morphology and evaluation. Theriogenology 20 407-416.

Lonergan P, Rizos D, Gutierrez-Adan, Fair T, Boland MP, 2003: Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns. Reprod. Domest. Anim. 38 259-267.

McEvoy TG, Coull GD, Broadbent PJ, Hutchinson JS, Speake BK, 2000: Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida. J. Reprod. Fertil. 118 163-70.

Reis A, Rooke JA, McCallum GJ, Ewen M, Staines ME, Lomax MA, McEvoy TG, 2003: Fatty acid content of polar and neutral lipids from bovine blastocysts produced in vitro in the presence or absence of serum. Reproduction Abstr. Series 30 57-58 (abstr).

Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP, Lonergan P, 2003: Bovine embryo culture in the presence or the absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol. Reprod. 68 236-243.

Chapter 6B: Lipid Content in Oocytes and Embryos, a practical approach

173

Sata R, Tsujii H, Abe H, Yamashita S, Hoshi H, 1999: Fatty acid composition of bovine embryos cultured in serum-free and serum-containing medium during early embryonic development. J. Reprod. Dev. 45 97-103.

Sturmey RG, Leese HJ, 2003: Energy metabolism in pig oocytes and early embryos. Reproduction 126 197-204.

Visintin JA, Martins JF, Bevilacqua EM, Mello MR, Nicacio AC, Assumpcao ME, 2002: Cryopreservation of Bos taurus vs Bos indicus embryos: are they really different? Theriogenology 57 345-59.

Chapter 7

Comparison of Embryo Quality in High Yielding Dairy Cows, in Dairy Heifers

and in Beef Cows

J. L. M. R. Leroy1, G. Opsomer1, S. De Vliegher1, T. Vanholder1, L. Goossens2,

A. Geldhof2, P. E. J. Bols3, A. de Kruif1, A. Van Soom1

1Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, University

of Ghent, Salisburylaan 133, B-9820 Merelbeke, Belgium. 2Flemish Cattle Breeding Association, Van Thorenburghlaan 14, B-9860 Oosterzele, Belgium.

3Laboratory of Veterinary Physiology, Departement of Veterinary Sciences, University of Antwerp,

Universiteitsplein 1, B-2610 Wilrijk, Belgium.

Theriogenology, In Press

Chapter 7: Embryo Quality in Dairy Cows

176

Chapter 7: Embryo Quality in Dairy Cows

177

Abstract

The purpose of this study was to compare embryo quality of lactating Holstein

Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue

beef cows (BB) and to identify factors that are associated with embryo quality in LHFC and

NLHFH. After superovulation and embryo recovery at day 7, embryos (n = 727 from 47

LHFC, 27 NLHFH and 50 BB) were scored morphologically for quality, colour and

developmental stage. Blood samples and data concerning parity, age, milk production and

management were collected. Data were compared univariably between the three groups. A

multivariable regression model was built with quality and colour of the LHFC and NLHFH

embryos as dependent variables.

Only 13.1% of LHFC embryos were categorized as excellent compared to 62.5% and

55.0% of the embryos in NLHFH and BB, respectively. Almost none of the NLHFH or BB

embryos displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC

embryos. Only 4% of all LHFC embryos reached blastocyst stage compared to 23.2% and

17.3% in NLHFH and BB. Based on the multivariable regression analysis, “physiological

status” (lactating or not) together with the serum total protein concentration of LHFC and

NLHFH, was significantly associated with embryo quality and colour.

Thus, LHFC display an inferior embryo quality compared to NLHFH and BB.

Producing milk or not seems to be significantly associated with embryo quality. Therefore,

reduced embryo quality on day 7 following AI, could be an important factor in the subfertility

problem in modern high yielding dairy cows.

Key Words

Dairy cow, Embryo colour, Embryo quality, Fertility decline, High yielding

Chapter 7: Embryo Quality in Dairy Cows

178

Introduction

Dairy cow fertility has been declining for the last decades (Lucy, 2001). In addition to

cystic ovarian disease (Vanholder et al., 2002), the main problems in modern dairy cow

fertility are delayed oestrus and ovulation post partum (Opsomer et al., 1998), reduced

expression of estrus (Lopez et al., 2004), and lowered conception rates (Sartori et al., 2002).

Even though this subfertility problem seems to be associated with increased milk yield, most

authors agree that it rather reflects a multifactorial problem (Butler, 1998; Veerkamp et al.,

2003) in which only recently, attention has been paid to oocyte and embryo quality.

Firstly, high yielding dairy cows experience a major adaptation in their energy

metabolism post partum to sustain the level of milk production. This metabolic change is well

reflected in the follicular fluid (Leroy et al., 2004a; Leroy et al., 2004b) and has been

suggested to exert a negative effect on the growing and maturing oocyte (Britt, 1994).

Elevated non-esterified fatty acid levels, associated with negative energy balance, are indeed

detrimental to the oocyte developmental capacity (Leroy et al., 2004c) and granulosa cell

function in vitro (Vanholder et al., 2005). Secondly, modern rations typically fed to high

yielding dairy cows, are high in protein and energy and are said to influence the follicular,

tubal and uterine micro-environment, directly influencing the oocyte and embryo quality

(Elrod and Butler, 1993; McEvoy et al., 1997; Kenny et al., 2002). In vivo, these “metabolic

stressors” can lead to increased embryonic mortality, which is a major cause of reproductive

failure in dairy cows (Britt, 1994; Boland et al., 2001; Sartori et al., 2002; Walters et al.,

2002). Finally, genetic selection for high milk production, as such, may also be a cause of

reduced fertility as has been suggested by Snijders et al. (2000). Dairy cows with a high

genetic merit yielded oocytes with an inferior developmental capacity in vitro.

Until now, however, little was known about embryo quality of lactating Holstein

Friesian cow (LHFC) donors in comparison with beef cows or with non-lactating Holstein

Friesian heifers (NLHFH). Investigating this enables us to study the effect of differences in

“breed” or “physiological status” on embryo quality. Apart from these two parameters, other

factors can be associated with embryo quality in high yielding dairy cows but have not yet

been identified. So far, most studies on embryo quality in superovulated donors have included

only one or two factors potentially influencing the embryo quality at recovery (Wiebold,

1988; Sartori et al., 2002).

Chapter 7: Embryo Quality in Dairy Cows

179

One of the most commonly used non-invasive techniques to evaluate embryo quality

in commercial embryo transfer is based on morphological appearance (Lindner and Wright,

1983). Embryo colour or darkness is of increasing interest in this morphological evaluation.

Dark embryos show an excessive accumulation of lipid droplets, which in turn can be

influenced by the biochemical composition of the embryonic environment (Abe et al., 1999;

Abe et al., 2002). The large amounts of intracellular lipids can compromise embryo quality

through impaired mitochondrial function (Abe et al., 2002) and can reduce cryotolerance,

finally resulting in low pregnancy rates (Hill and Kuehner, 1998). Therefore, recording the

colour as an indicator of lipid content of flushed embryos upon collection might be an extra

valuable quality parameter.

Because it could be a potential contributing factor in the pathogenesis of subfertilty in

modern high yielding dairy cows we wanted to investigate whether embryo quality of LHFC

is reduced. Therefore, the aims of the present field study were: (1) to compare embryo quality

and colour among LHFC, NLHFH and Belgian Blue beef cows (BB) in relation to 4 serum

parameters, which have previously been linked with embryo quality in vivo and in vitro: urea,

total protein (TP), total cholesterol (TC) and triglycerides (TG); (2) to identify factors

associated with embryo quality and colour in NLHFH and LHFC.

Material and Methods

Animals, Embryo Recovery and Blood Sampling

The field trial was set up in close cooperation with the embryo transfer team of the

Flemish Cattle Breeding Association (Oosterzele, Belgium). All embryo recoveries (ER) were

performed between October 2002 and October 2003 by the same experienced veterinary

surgeon. Lactating Holstein Friesian cows (n = 47), NLHFH (n = 27) and BB (n = 50),

belonging to 72 privately owned herds, underwent 142 ER. None of the BB cows was

lactating at the time of ER. The procedure was identical for all sessions. In brief, animals

were injected i.m. during their mid-luteal phase with exogenous gonadotropin: pFSH – pLH

(5/1) (Stimufol®: Merial, Belgium), twice daily in decreasing doses during 4 consecutive

days. On the third day (approximately 60 hours after start of treatment), animals were injected

with a prostaglandin analogue (cloprostenol 750 μg i.m.) (Estrumate®: Schering-Plough,

Belgium) to induce regression of the corpus luteum. Approximately 60 and 72 hours after

prostaglandin injection, animals were inseminated with frozen-thawed semen from one of the

Chapter 7: Embryo Quality in Dairy Cows

180

67 different bulls with proven fertility, and which were of the same breed as the embryo

donor.

Embryos and ova were harvested non-surgically on day 7 after AI. Immediately before each

ER, donor blood was sampled from the coccygeal vein into two unheparinized, silicone-

coated tubes (Venoject®, Autosep®, Gel + Clot. Act.; Terumo Europe N.V., Belgium). The

coagulated blood samples were centrifuged (1400 X g, 20 min) within 30 min after collection

and the serum was frozen (-20°C) within hours until assay. In each serum sample, the

concentrations of urea, TP, TC and TG were measured using commercial photometric assays

(Roche diagnostics GmbH, Mannheim, Germany).

Embryo Evaluation and Data Collection

From each donor cow the following data per ER were recorded: breed, date of birth,

parity, date of last parturition and, if it occured, the number of days from previous ER. In

addition, for LHFC the average daily milk, milk fat and milk protein production during the

month preceding ER was recorded. A milk production score was calculated for each

individual LHFC to rank the performance of that specific cow within the herd. In addition, the

herd average milk production during the month preceding ER was recorded (expressed as

milk production per cow per day).

Immediately following ER, oocytes and embryos were counted and the transferable

embryos were morphologically scored for quality, colour and developmental stage, by the

same experienced operator throughout the whole study (stereomicroscope, 90 X

magnifications). The morphological quality of transferable embryos was graded in four

classes (excellent, good, fair and poor) based on the method described by (Lindner and

Wright, 1983). Pictures (figure 1), which were taken with the same stereomicroscope, were

used to estimate the colour of the transferable embryo as ‘pale’ or ‘dark’. Embryos with an

intermediate darkness were allocated to the ‘medium’ category (Hill and Keuhner, 1998).

Apart from the unfertilized oocytes, embryos in earlier developmental stages than morulae

were categorized as ‘degenerated’.

Chapter 7: Embryo Quality in Dairy Cows

181

A. B. Figure 1. Two embryos categorized as pale (A.) and dark (B.). Both embryos are morulas. (90 X magnification).

All embryos were recovered for commercial purposes. Therefore, experimental

freezing and thawing was not possible. Because a substantial number of embryos were still

stored in liquid nitrogen at the time of statistical analysis and several embryos have been

exported to foreign countries, it was impossible to record pregnancy rates after transfer.

Statistical procedure

The study consists of two parts. In the first part, embryo quality and colour together

with some other parameters were compared between NLHFH, LHFC and BB in a univariable

way. In the second part of the study, only the data of the Holstein Friesian group (NLHFH

and LHFC) were used because investigating the fertility decline in the dairy breed was a main

purpose of this study.

Data have been analyzed using logistic regression to identify factors associated with

embryo quality and colour. Data are expressed as mean ± SEM.

Comparison between NLHFH, LHFC and BB.

A non-parametric Kruskal-Wallis H test was used to compare the number of recovered

embryos and oocytes per ER among LHFC, NLHFH and BB, and to compare parity and the

number of days between parturition and ER among LHFC and BB. Differences in serum

concentrations (continuous variables) between the three groups were analyzed with a one way

ANOVA and a post hoc Scheffé test. The rates of ER with at least one commercially usable

embryo (recovery rates), embryo quality, colour and developmental stage were compared with

Chapter 7: Embryo Quality in Dairy Cows

182

a X2 –test. All these statistical analyses were done using SPSS 11.0 for Windows, Chicago, Il,

USA.

Factors associated with embryo quality and colour in NLHFH and LHFC.

In the second part of the study, only the data of the LHFC and NLHFH were used, as

explained before. Prior to statistical analysis, data were carefully explored and checked for

unlikely values. No data were excluded for this reason. Embryo recovery sessions with

missing values were not included in the final statistical model.

Embryo quality and colour were used as the two dependent variables. Both were

recoded to binary variables: excellent embryos (0) and other embryos (1) (good, fair and

poor) for embryo quality; pale embryos (0) and other embryos (1) (medium and dark) for

embryo colour. From the few donors that were flushed more than once, only one batch was

randomly selected for further analysis.

To deal with clustering of embryos within a donor and clustering of donors within

herd, multilevel logistic regression models with cow and herd as random factors were fitted

(MlwiN) (Rasbash et al., 2000). The regression model building involved several steps.

Initially, unconditional associations were tested per “physiological status group” (defined as

NLHFH or LHFC) between the two dependent variables separately (embryo colour and

quality) and the independent variables (Table 1). Statistical significance at this step was

assessed at P < 0.2.

Secondly, to prevent multicollinearity in the further analysis, Pearson correlations

coefficients were calculated among the significant variables per animal group. If 2 variables

had a correlation coefficient ≥ 0.6, only one was selected for further analysis, based on

biological reasoning.

In the third step, multivariable models were fitted for both embryo quality and colour

per physiological status group (LHFC vs. NLHFH) with the remaining variables.

Finally, the data sets for both physiological status groups were merged, because all of

the remaining significant variables were available in both groups. In addition, an extra

independent variable, i.e. physiological status (lactating or not: (0) for NLHFH and (1) for

LHFC), was created and added to all models. Using the resulting dataset, multivariable

models were fitted for both outcome variables with the remaining independent variables. All

possible two way interactions were tested. Non-significant variables were removed using

backward elimination at P < 0.05 (Wald’s test). Based on the final model, odds ratios were

Chapter 7: Embryo Quality in Dairy Cows

183

calculated as expβ (β being the estimate of the tested independent variable) with 95%

confidence intervals.

Table 1. Independent variables tested unconditionally for associations with embryo quality and colour in each physiological status group: (nulliparous) non-lactating Holstein Friesian heifers (NLHFH) and lactating Holstein Friesian cows (LHFC). NLHFH LHFC

Number of embryos per flushing Number of embryos per flushing Ratio transferable embryos/ total ovulation1 Ratio transferable embryos/ total ovulation1

Season2 Season2

Serum total cholesterol (mg/dl) Serum total cholesterol (mg/dl) Serum urea (mM) Serum urea (mM) Serum total protein (g/dl) Serum total protein (g/dl) Serum triglycerides (mg/dl) Serum triglycerides (mg/dl) Age (days) - - Kg dry matter in milk3

- Lactation score4 - Parity5

1ratio of the number of recovered transferable embryos and the sum of the flushed unfertilized ova, degenerated embryos and transferable embryos. 2categorical, coded as (1) spring, (2) summer, (3) autumn and (4) winter. 3average daily dry matter (fat and protein) production, during the month preceding embryo recovery. 4a score ranking the cows’ production of dry matter (fat and protein) relative to the herd average. 5categorical, coded as (0) for parity 1, (1) for parity 2 and (3) for parity > 2.

Results

Descriptive Analysis

The average daily milk production of the LHFC during the month preceding the ER

was 32.9 ± 1.1 kg and the average milk fat and protein content was 4.0 ± 0.1 % and 3.5 ± 0.05

%, respectively. The LHFC included in this study displayed on average an 11% better milk

production when compared with their respective herd average. The average daily milk

production during the last month prior to ER of the dairy herds involved was 28.7 ± 0.5 kg

milk with 4.0 ± 0.03 % fat and 3.4 ± 0.02 % protein.

Comparisons between NLHFH, LHFC and BB

In total 54, 33 and 55 ER were performed on 47 LHFC, 27 NLHFH and 50 BB,

respectively. This resulted in 328 LHFC, 168 NLHFH and 231 BB embryos (727 embryos in

total). The rates of ER with at least one commercially usable embryo (recovery rate) were

comparable: 79.6% for LHFC, 81.8% for the NLHFH and 87.3% for the BB (P > 0.05).

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Concerning the superovulatory response (the number of oocytes ovulated calculated as the

number of recovered transferable and degenerated embryos and unfertilized oocytes) there

was no significant difference between LHFC, NLHFH or BB: 10.2 ± 1.1, 8.1 ± 1.1 and 9.2 ±

0.8 in LHFC, NLHFH and BB, respectively (P > 0.05). Embryo rate (ratio of the number of

transferable embryos and the number of oocytes ovulated), cleavage rate (ratio of number of

degenerated plus transferable embryos and the number of oocytes ovulated) and the ratio of

the transferable embryos and cleaved embryos were similar between the three donor groups.

All other data concerning blood parameters and embryo batch or donor characteristics are

presented in Table 2. The results of embryo quality, colour and developmental stage are

shown in Figure 2. Lactating Holstein Friesian cows produced significantly more good and

fair quality embryos and significantly fewer excellent quality embryos compared to BB and

NLHFH (P < 0.05). The average embryo colour was significantly darker in LHFC with

almost no dark embryos in NLHFH and none in BB (P < 0.05). Embryo development also

seemed to be slower in LHFC with relatively more early morulae and fewer blastocysts

compared to the other two donor groups (P < 0.05).

Table 2. Results (mean ± SEM) of evaluated parameters for lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue cows (BB). LHFC NLHFH BB

N transferable embryos per ER 6.1 ± 0.7 5.1 ± 0.8 4.2 ± 0.4 N degenerated embryos per ER 1.3 ± 0.3 1.2 ± 0.3 1.1 ± 0.2 N unfertilized oocytes per ER 2.8 ± 0.6 1.8 ± 0.4 3.9 ± 0.7 Days between ER and preceding parturition 230.8 ± 24.9 - 366.5 ± 103.6 Parity 2.0 ± 0.5a - 2.5 ± 0.3b

Serum urea (mM) 4.5 ± 0.2a 2.8 ± 0.2b 3.8 ± 0.2c

Serum total protein (g/dl) 7.6 ± 0.1a 6.6 ± 0.1b 7.3 ± 0.1a

Serum total cholesterol (mg/dl) 183.2 ± 5.3a 104.8 ± 3.8b 105.9 ± 4.1b

Serum triglycerides (mg/dl) 17.2 ± 0.6a 23.8 ± 0.9a,b 28.3 ± 3.3b a, b, c Data with different superscripts differ significantly between each column (P < 0.05).

Factors associated with Embryo Quality and Colour of NLHFH and LHFC

The structure of the data used in this part of the study is shown in Table 3. In total 416

embryos were scored as having an excellent (0) (n = 126) or a minor quality (1) (n = 290), or

having a pale (0) (n = 261) or darker colour (1) (n = 155). In NLHFH, unconditional

associations were found between embryo quality on the one hand and the ratio transferable

embryos/total ovulation, serum TC and TP on the other hand. The more, association between

embryo colour and season, serum TP and age of the NLHFH donor turned out to be

Chapter 7: Embryo Quality in Dairy Cows

185

significant. In the LHFC group, there were significant interactions between embryo quality

and the number of embryos per flushing, serum TP and urea. The same was true for colour in

the LHFC embryos. After merging the data sets of both LHFC and NLHFH, the final model

was fitted. The results, expressed as the odds for having a darker or inferior embryo, are

presented in Table 4. High total protein levels in serum of the embryo donor were associated

with improved embryo quality, including a paler colour. Milk production was associated with

an inferior embryo quality and a darker appearance.

Table 3. Structure of the data as analysed in the second part of the study. Level N Range Herd 36 -

Animal1 59 1 to 6a

Embryo 416 1 to 17b

aThe range of cows and/or heifers per herd. bThe range of embryos per embryo batch. 1The animal level corresponds to the embryo batch level since only one batch per cow was used for the final analysis. Table 4. Independent variables significantly associated with embryo colour and quality in the lactating Holstein Friesian cows (LHFC) and non-lactating Holstein Friesian heifers (NLHFH) (P < 0.05).

EMBRYO QUALITY1 Independent variable Odds ratio 95% CI2 Serum total protein 0.21 0.06 – 0.71 Number of embryos per flushing 0.85 0.69 – 1.05 Physiological status

NLHFH Reference - LHFC 3.61 3.41 – 44.5

Physiological status x number of embryos3 NLHFH (0) x number of embryos Reference - LHFC (1) x number of embryos 1.44 1.09 – 1.89

EMBRYO COLOUR4

Independent variable Odds ratio 95% CI Serum total protein 0.11 0.03 – 0.41 Physiological status

NLHFH Reference - LHFC 110.28 13.84 – 878.87

1Embryo quality: (0) for excellent embryos and (1) for the other (good, fair, poor) embryos. 295% confidence interval around odds ratio. 3Interaction term 4Embryo colour: (0) for pale embryos and (1) for the other (medium, dark) embryos.

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0

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grade I grade II grade III grade IVEmbryo quality

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early morula compact morula blastocystEmbryo developmental stage

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Figure 2. The distribution of embryo quality, colour and developmental stage for embryos recovered from lactating Holstein Friesian cows (n = 328 embryos) (pale bars), non-lactating Holstein Friesian heifers (n = 168 embryos) (grey bars) and Belgian Blue beef cattle (n = 231 embryos) (black bars). a, b, c Bars with different superscripts differ significantly between the three groups (P < 0.05).

b

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Discussion

The purpose of the present study was to evaluate the effects of breed or physiological

status (producing milk or not) on embryo quality by comparing the quality and colour of

embryos from LHFC, NLHFH and BB. Furthermore, the intriguing fact that the world widely

reported fertility decline has only been mentioned in lactating dairy cows and not in maiden

heifers needs a further clarification. Therefore, in a second analysis only the data from

NLHFH and LHFC were used to identify factors associated with embryo quality and colour.

In the first part of this study, an important finding was that significantly more embryos

from LHFC were of inferior quality and darker colour compared to embryos of BB. This

resulted in a lower proportion of embryos frozen per ER (61% for HF cows compared to 75%

in BB). Breed differences in embryo colour have been described earlier (Visintin et al., 2002).

In our study, the quality and colour of NLHFH embryos were, however, comparable to those

of the BB. The latter implies that, factors other than breed or genetic background, such as

physiological status related to high milk production, may be responsible for reduced

morphological embryo quality in high yielding Holstein Friesian cows. Regarding the level of

milk yield, it is important to mention that the LHFC included in our study, had an 11% higher

milk production compared to the average production of their respective herds. In the second

part of the study, there were no significantly unconditional interactions between quality or

colour of LHFC embryos and the level of milk production as such. Based on the regression

analysis, however, “physiological status” (i.e. lactating or not) turned out to be significantly

associated with both, embryo quality and colour. Recently, Sartori et al. (2002) reported

similar results, using the same high fertility semen to exclude any sire effect. In contrast to

their study, we found no difference in the number of unfertilized ova following embryo

recovery, suggesting that fertilization rate, as such, was not affected by breed or

“physiological status”.

In bovine embryo transfer, evaluation of embryo morphology remains the method of

choice to select viable embryos (Van Soom et al., 2003). This method, however, can be

subjective, it may depend on the embryologist, and the morphological appreciation is not

always in accordance with the ultrastructural quality (Van Soom et al., 1996; Farin et al.,

1999; Aguilar et al., 2002). In our study, however, all embryos were scored for quality and

colour by the same experienced embryologist. Since the embryos were commercially used

after evaluation, invasive techniques were impossible to carry out and pregnancy results after

embryo transfer were impossible to obtain. Several other studies have already demonstrated

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the relationship between morphological embryo quality/colour and pregnancy rates after

transfer (Lindner and Wright, 19883; Hill and Kuehner, 1998; Hasler, 2001). Furthermore, it

has also been proven that the recipient is of major importance in determining the final

pregnancy rates (McMillan, 1998).

In embryo morphology evaluation, embryo colour, indicative of intracellular lipid

accumulation, is an important quality parameter (Sata et al., 1999; Abe et al., 2002; Abe and

Hoshi, 2003). The correlation between embryo colour and lipid content has recently been

confirmed in our lab, showing that dark embryos contain significantly more lipids in lipid

droplets (on average 45% more) compared to pale embryos (unpublished data, 2005). Similar

results were found ultrastructurally, comparing embryos from Nelore beef cows and HF dairy

cows (Visintin et al., 2002). To our knowledge, the observed differences in colour between

LHFC and NLHFH embryos have never been described before. Furthermore, we also found

that the colour of embryos from LHFC is similar to that of embryos produced in vitro, in

serum containing media (unpublished data, 2005). Such embryos are known to be dark in

appearance, due to accumulation of high amounts of lipids (Abe et al., 1999; Ferguson and

Leese, 1999; Leroy et al., 2005). A greater content of intracellular lipids impairs the quality of

the embryos by increasing their sensitivity to oxidative stress, chilling and cryopreservation

(Abe et al., 1999, Reis et al., 2003). The increased lipid accumulation is associated with

suboptimal mitochondrial function and a deviation in the relative abundance of

developmentally important gene transcripts, what impedes embryo quality and viability (Abe

et al., 2002, Rizos et al., 2003). However, whether or not this lipid accumulation is also

detrimental for the viability of an embryo that spends its whole life in vivo is not known.

Further research in this area is required.

The physiological explanation behind the observation that milk production is linked

with lipid accumulation in embryos is not known. This accumulation of lipid droplets is

possibly due to an altered lipid metabolism in high yielding dairy cows. Similarly, increased

levels of TG have been found in embryos of diabetic rats, which energy and lipid metabolism

is also disturbed (Sinner et al., 2003). High energy diets in combination with high milk

production have been shown to lead to elevated serum TC concentrations in dairy cows

(Varman and Schultz, 1968; Wehrman et al., 1991). This is in accordance with the results

obtained in the first part of our study, showing significantly greater TC concentrations in

LHFC compared to NLHFH or BB. The multivariable model of the second part of the study,

however, showed no association of TC and TG levels with embryo colour and quality, both in

the NLHFH and LHFC. Ryan et al. (1992) fed high lipid diets to beef heifers, but there were

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no apparent effects of the concomitant high TC concentrations on embryo recovery and

quality, without taking embryo colour into account. Hill and Kuehner (1998), on the other

hand, demonstrated that donors high in serum TC yielded darker embryos and transfer of such

embryos after cryopreservation resulted in significantly lower pregnancy rates. In that

univariable study, no other (possibly confounding) factors (e.g. physiological status) were

taken into account, which may explain the differences between the results of the multivariable

analysis in the second part of our work. After all, serum TC in the present study, when tested

separately for association with embryo quality, also appeared to be a significant factor.

Finally, diets high in energy, which are typically fed to high yielding dairy cows, may alter

the intrafollicular IGF and insulin system, leading to altered oocyte metabolism and a reduced

developmental capacity (Boland et al., 2001; Armstrong et al., 2001).

Sata et al. (1999) and Kim et al. (2001) demonstrated that oocytes and embryos

cultured in vitro, are able to accumulate fatty acids from their environment. Whether this

excessive lipid accumulation in the LHFC embryos occurs already in oocytes and/or in

embryos is not known. During prematuration and maturation in vivo, there is a physiological

lipid accumulation in the oocyte (Fair, 2003). Studies focusing on the lipid exchange between

blood serum, luminal fluids and the embryo in vivo are very scarce. Henault and Killian

(1993) investigated the lipids and their distribution in the oviductal epithelium and found a

high concentration of free cholesterol and TG in the cells of the preampulla and ampulla.

Particularly cholesterol and phospholipids are released into the lumen of the oviduct. Their

concentrations are substantially different from the blood serum levels and depend on the stage

of the estrous cycle (Killian et al., 1989). A recent study of Adamiak et al. (2004a) showed

that feeding heifers a diet with 6% of protected lipid resulted in elevated total fatty acid

content in both plasma and oocytes. When the serum from these heifers was added to embryo

culture media, the resulting embryos had an increased total fatty acid content, altered energy

metabolism and a higher incidence of apoptosis (Adamiak et al., 2004b).

It is important to mention that factors related to the feeding and management may have

acted as confounders when comparing embryo quality in our study. The higher serum urea

concentrations in the LHFC compared to the NLHFH and BB in the first part of the study are

probably caused by the protein rich rations, which are typically fed to high yielding dairy

cows and can be a possible explanation for the observed differences in embryo quality. Such

diets with concomitant elevated levels of urea and ammonia in serum may reduce embryo

viability, possibly by lowering uterine pH (Elrod et al., 1993; McEvoy et al., 1997; Dawuda

et al., 2002). Recent studies, however, demonstrated that the deleterious effects of very high

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urea and ammonia concentrations on embryo quality could be due to alterations in the oviduct

or follicular rather than uterine environment affecting the oocyte (Armstrong et al., 2001;

Papadopoulos et al., 2001; Leroy et al., 2004b), as has been confirmed in vitro (De Wit et al.,

2001; Ococn and Hansen, 2003; Rooke et al., 2004). Also, in the univariable screening in the

second part of our study, serum urea appeared to be significantly associated with embryo

quality. Despite this, serum urea dropped out as a significant factor in the final multivariable

model. Finally, Kenny et al. (Kenny et al., 2001) did not see any effect of high crude protein

diets on embryo survival.

In addition to a reduced embryo quality and a darker appearance, differences in the

developmental stage of the flushed embryos may be a possible explanation for the reduced

fertility in high yielding dairy cows. The LHFC yielded significantly more compact morulae

and fewer blastocysts on day 7 compared to NLHFH or BB. A delayed ovulation and/or a

retarded development together with a delayed blastulation can be responsible for this

observation. Whether this is related to decreased embryo quality and viability is not certain.

Contradictory results concerning pregnancy rates obtained after transfers of morulae or

blastocysts have been reported (Hasler et al., 1987; Hasler, 2001).

By means of the final multivariable analysis in the second part of the study, we tried to

differentiate the mechanism(s) producing embryos with lower quality and colour intensity in

the dairy group (NLHFH and LHFC). As discussed earlier, “physiological status” or in other

words “producing milk” turned out to be a crucial parameter influencing both embryo quality

and colour. The only blood parameter that unexpectedly showed to be significantly linked

with embryo quality and colour was TP. Higher TP concentrations were associated with an

improved embryo quality and colour. The sole link found in the literature was a study of Oh et

al. (1999), which reported that recipients with higher serum TP showed a significantly higher

pregnancy rate. Preliminary results of ongoing research suggest that especially the gamma

globulin concentration is well correlated with the TP level. The latter may imply that cows

with a well developed immunity status are more likely to yield good quality embryos.

Interpretation of this finding is difficult, as it may be confounded by secondary effects which

may not have been taken into account. Further research should focus on this in order to reveal

a potentially causal relationship.

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Conclusions

In the present study, we demonstrated that LHFC yielded embryos with a significantly

reduced quality and a darker appearance compared to NLHFH and BB. The darkness in the

lactating HF cow embryos is most likely caused by an accumulation of lipid droplets. Based

on the results of the multivariable model, producing milk and low serum concentrations of

total protein were associated with impaired embryo quality and a darker colour of the embryo.

These findings may suggest that reduced embryo quality on day 7 following AI can be an

important factor in the widely described fertility decline in high yielding dairy cows.

Acknowledgments

The authors thank Dr. M. Coryn and Dr. K. Moerloose for the critical reading of the

manuscript and all the farmers who collaborated. This research was funded by the Institute for

the Promotion of Innovation by Science and Technology in Flanders (Grant no° 13236).

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Walters AH, Bailey TL, Pearson RE, Gwazdauskas FC. Parity-related changes in bovine follicle and oocyte populations, oocyte quality, and hormones to 90 days postpartum. J Dairy Sci 2002;85:824-832.

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Wehrman ME, Welsh TH, Williams GL. Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity. Biol Reprod 1991;45:514-522.

Wiebold JL. Embryonic mortality and the uterine environment in first-service lactating dairy cows. J Reprod Fertil 1988;84:393-399.

Chapter 8

General Discussion and Conclusions

J.L.M.R. Leroy

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University, Merelbeke, Belgium

Chapter 8: General Discussion and Conclusions

198

Chapter 8: General Discussion and Conclusions

199

Introduction

The main scope of this thesis was to study the role and the importance of the oocyte

and the embryo proper in the complex problem of reproductive failure in modern, high

yielding dairy cows. The comprehensive documentation on reduced conception rates and on

the increased incidence of early embryonic mortality (Dunne et al., 1999; Bousquet et al.,

2004) clearly indicates that the healthy growth and maturation of the female gamete and/or

the normal development of the early embryo may be compromised. In literature, many

speculations and suggestions on the causes of these observations have been made, as

expressed in the following questions:

Are oocyte growth and maturation hampered well before ovulation due to biochemical

alterations in the intrafollicular environment (O’Callaghan and Boland, 1999; Lozano et

al., 2003)?

Has the microenvironment of the oviduct or the uterus been changed due to dietary and

metabolic changes in the modern dairy cow, creating a hostile environment for the early

embryo (Elrod and Butler, 1993; McEvoy et al., 1995; Kenny et al., 2002)?

Is there something going wrong with the genetic information of modern dairy cow oocytes

due to the consecutive years of rigorous genetic selection towards milk yield (Snijders et

al., 2000)?

In contrast with the extensive knowledge of the disturbed endocrine signalling and ovarian

function, clear evidence concerning the impact of hampered oocyte and/or embryo quality on

the final reproductive performance in high producing dairy cows is almost lacking. In the

present thesis attention was focused on possible pathways linking the negative energy balance

(NEB) and oocyte quality (Chapter 4 and 5). Furthermore, in case of successful fertilization

and embryo formation, it is not known whether the quality of this early life is impaired or not.

Therefore, we compared the embryo quality between lactating high yielding dairy cows, non-

lactating dairy heifers and beef cows by means of a field trial (Chapter 7). One of the

parameters used to evaluate embryo quality is the lipid content. Since current techniques to

evaluate lipid content require a large number of oocytes or embryos, a completely new and

highly sensitive technique needed to be developed in order to evaluate the lipid content of

single bovine oocytes or embryos (Chapter 6).

Chapter 8: General Discussion and Conclusions

200

Follicular fluid, the link between blood and gamete

Linking the status of NEB and oocyte quality is not an easy task. It is well known that

the NEB is featured by some typical endocrine and biochemical changes in the blood of

modern dairy cows (Herdt, 2000). Some studies associated these metabolic changes in serum

already with oocyte quality, without investigating the physiological connection between blood

and oocyte: the follicle and the follicular fluid (Hashimoto et al., 2000; De Wit et al., 2001;

Jorritsma et al., 2004).

Follicular cavity is an avascular ‘compartment’ in which the oocyte undergoes the fine

tuned process of oocyte growth, prematuration and final maturation (Bagavandoss et al.,

1983; Gosden et al., 1988). The physiological origin of follicular fluid has been reviewed by

Gosden et al. (1988). During the process of follicular growth, the physicochemical properties

of the blood-follicle barrier change thoroughly, suggesting that the oocyte’s environment

undergoes compositional changes (Edwards, 1974; Wise, 1987; Gosden et al., 1988). Also the

active transport mechanisms through the follicular wall may alter during follicular growth.

Argov et al. (2004) for example recently demonstrated that while lipoproteins are

predominantly internalized by endocytosis in small follicles, this is not the case in large

follicles in which circulating lipoproteins contribute their cholesterol esters by selective

uptake and without internalization of the lipoprotein as such. Hence, to learn more about the

rather unexplored biochemical world of the oocyte before ovulation, we analysed the

composition of follicular fluid originating from three differently sized follicles and compared

it with the composition of serum of 30 dairy cows shortly post mortem (Chapter 4A) . The

data of this first experiment confirmed indeed that follicular fluid composition is changing

from small to large follicles. However, to be physiologically correct, the ideal experimental

setup would have been a repeated aspiration of micro volumes (up to 20 μl) of follicular fluid

of the same follicle at different time points during its growth as has been done by others

(Ginther et al., 1997). In our experimental set-up, this was unfortunately not possible because

of the higher number of metabolites we wanted to analyse (Argov et al., 2004; Orsi et al.,

2005). Another important finding from this first experiment is the fact that the follicular fluid

composition is correlated with the composition of the serum. The given correlations are

however ‘static’ and some clarification is needed. It means that when a cow has, for example,

a higher glucose serum concentration compared to another cow, this will also be the case for

the follicular glucose concentrations. So, strictly spoken, it is wrong to conclude from these

Chapter 8: General Discussion and Conclusions

201

data that when glucose levels rise in serum of a cow, this rise will be paralleled in the

follicular fluid. For the confirmation of this particularly ‘dynamic’ correlation, we decided to

explore the follicular fluid composition in living high yielding dairy cows by means of a

repeated transvaginal follicle puncture.

Thus, in the second experiment (Chapter 4B) we concentrated on compositional

changes over time in follicular fluid of high yielding dairy cows early post partum. In this

way, we were able to compare these data with the already extensively studied biochemical

alterations in blood during the NEB (see above). According to our knowledge this had never

been studied before. A NEB typically causes some obvious changes in serum such as high

non-esterified fatty acids (NEFA) and β-hydroxybutyrate (β-OHB) concentrations or low

glucose concentrations (Baird, 1982; Chilliard et al., 1998; Duffield, 2000; Herdt, 2000). In

addition, an increased amino acid metabolism for gluconeogenesis or the intake of protein

rich diets can lead to high urea concentrations (Butler 1998; Sinclair et al., 2000). Britt (1992)

hypothesized that these features of the NEB can directly affect the follicle and the enclosed

oocyte, leading to the ovulation of an inferior oocyte. This hypothesis is plausible since it is

generally accepted that oocytes are highly vulnerable to any disruption in their environment

(O’Callaghan and Boland, 1999; Armstrong et al., 2001; Boland et al., 2001). We are

convinced that the data of our second experiment were essential to introduce the first

scientific evidence for this generally accepted ‘Britt hypothesis’: ‘How does the NEB directly

influence the micro-environment which is most intimately linked with the oocyte?’

An adapted ovum pick-up technique (Bols et al., 1995) turned out to be a perfect

method to collect follicular fluid from the dominant follicle at 6 different time points post

partum. Since we already knew from our first experiments that follicular size can influence

follicular fluid composition, it was important to aspirate similar sized follicles throughout the

whole study. Because of the reduced approachability of the ovaries in the puerperium,

follicular fluid was only collected from day 14 post partum onwards.

By analogy with the first experiment, ‘static’ correlations per time point post partum

between serum and follicular fluid composition were calculated and confirmed the results of

the first study. Especially for glucose, β-OHB, urea and total cholesterol, good correlations

were found. Based on the results of the repeated measurement design (dynamic correlations)

(Chapter 4B), we can say now that those typical postpartum serum fluctuations are more or

less reflected in the follicular fluid of the dominant follicle. For urea and β-OHB, no

concentration differences between serum and follicular fluid could be detected. It is important

to mention though that the follicle is able to maintain higher glucose and lower NEFA

Chapter 8: General Discussion and Conclusions

202

concentrations compared to serum. Or in other words, it can be suggested that the oocyte is

isolated (or even protected?) from extreme glucose or NEFA concentrations present in the

blood. Leese and Lenton (1990) described the opposite for glucose in human follicular fluid.

They concluded that follicular glucose concentrations are a function of glycolysis in the

granulosa cells, which is possibly superimposed on the flux of intact molecules from blood

into the fluid. No satisfying explanation for this observation could be found, but active

transport through the follicular wall could be possible. Further research concerning the

contribution of the follicular wall to the composition of follicular fluid is certainly needed but

was beyond the scope of the present thesis.

In spite of the follicle’s buffering capacities, glucose concentrations do decrease and

NEFA concentrations significantly rise in follicular fluid during the NEB. Also Jorritsma et

al. (2003) and Comin et al. (2002) described a NEFA rise in follicular fluid concentrations

parallel with an increase in the serum concentrations, due to an acute dietary restriction.

However, no concentration gradients between serum and follicular fluid have been

mentioned. It is only recently that Hammon et al. (2005) confirmed our findings concerning

follicular urea concentrations in high producing dairy cows early post partum. Conclusively,

there is now enough evidence to say that the growing and maturing oocyte is directly exposed

to the typical biochemical changes in high yielding dairy cows early post partum. It is

furthermore demonstrated that high urea concentrations can be toxic for oocytes during

maturation through an inhibition of the polymerization of tubulin into microtubules (De Wit

et al., 2001; Ocon and Hansen, 2003; Iwata et al., 2005). The same is true for the observed

low glucose concentrations. Adequate glucose supplies are necessary to support normal

cumulus expansion and nuclear maturation (Krisher and Bavister, 1998; Sutton-McDowall et

al., 2004). Similarly, high NEFA and β-OHB concentrations are probably harmful for the

oocyte’s developmental competence, but this has, to our knowledge, never been substantiated.

Therefore, we subsequently concentrated on possibly adverse effects of high NEFA or

β-OHB concentrations as has been described for urea and glucose (Chapter 5). As in other

studies, we were also obliged to use in vitro maturation models to get answers to these

questions. Since in vitro media are only an approach of the real in vivo conditions, results

should always be interpreted with caution!

Chapter 8: General Discussion and Conclusions

203

The negative energy balance and the direct consequences for oocyte quality: an in vitro

model

First of all, attention was paid on the NEFA fraction of the follicular fluid. Since

NEFA are a family of all kinds of fatty acids, new and specialized techniques to analyse the

follicular fluid of high yielding dairy cows during the NEB were needed. Not only the

absolute NEFA concentration but also the NEFA composition needed to be analysed by

means of a combined thin layer and gas chromatography. Since at least 1ml of follicular fluid

was needed, a new series of animals (9) was subjected to transvaginal follicular fluid

aspiration as explained above. The results surprisingly revealed that not only the NEFA

concentration (see above) but also the NEFA composition significantly differs between serum

and follicular fluid. A different concentration of albumin (on which NEFA are predominantly

bound) in the two compartments could be suggested as the most probable explanation (Yao et

al., 1980). However, we were not able to confirm this by additional albumin analyses

(unpublished results). Edwards (1974) documented that albumin and other proteins can enter

the follicle very easily, suggesting a paracellular transport, and thus a high permeability of the

follicular wall (Gosden et al., 1988). The latter confirms our findings for albumin but offers

no clue for explaining the observed differences in NEFA concentration and composition. Also

the described dynamic interchange of NEFA between serum and follicular fluid (Moallem et

al., 1999) is not really in line with our findings. It was a study of Chung et al. (1995) that

reported a possible useful clarification for our results. In the presence of high NEFA levels, a

substantial portion of the NEFA in serum is partitioned to low density lipoproteins (LDL).

Especially the saturated fatty acids are bound on LDL, while the unsaturated are preferably

bound on albumin (Chung et al., 1995). Because LDL are absent in FF, these findings could

account for the differences in concentration and composition of NEFA in FF compared to

serum early post partum (Wehrman et al., 1991). Further research to confirm this hypothesis

is however desirable.

Whatever the mechanisms are, we now know the concentrations of the three most

important fatty acids present in follicular fluid of the dominant follicle during the NEB: oleic,

palmitic and stearic acid. These concentrations were applied during an in vitro maturation

model to evaluate their effect on oocyte quality (Chapter 5A). While oleic acid had no effect,

exposing oocytes to palmitic and stearic acid at concentrations comparable to those assessed

in vivo under NEB conditions, resulted in a reduced maturation, leading to disappointing

Chapter 8: General Discussion and Conclusions

204

fertilization and cleavage rates. In addition, cumulus expansion was hampered, although no

lipid accumulation in the oocytes could be observed. In cumulus cells, a significantly higher

rate of apoptosis and even necrosis could be detected after 24 hours of exposure to high

stearic or palmitic concentrations. Similar toxic effects on bovine or human granulosa cells in

vitro have been shown in other studies (Mu et al., 2001; Jorritsma et al., 2004; Vanholder et

al., 2005). An optimal granulosa and cumulus cell function is indispensable for oocyte

maturation, because they are responsible for endocrine and paracrine signalling (Bilodeau-

Goeseels and Panich, 2002; Tanghe et al., 2002). Therefore, it is most likely that the toxic

effect of NEFA on oocyte quality is particularly an indirect effect, mediated through impaired

cumulus cell function.

In contrast with our results, Jorritsma et al. (2004) did find detrimental effects of oleic

acid. In their study however, oleic acid was bound on albumin and was added in

supraphysiological concentrations to an undefined in vitro maturation medium (addition of

fatty acids containing fetal calf serum). The more, it is unclear whether these adverse effects

were caused by the addition of BSA or by oleic acid. Homa and Brown (1992) showed that

albumin bound linoleic acid in IVM medium inhibits germinal vesicle breakdown in denuded

oocytes. Similar toxic effects of NEFA have also been described for Leydig cells, muscle

cells and pancreatic β-cells. Especially the induction of apoptosis and/or insulin resistance and

changes in membrane properties have been suggested as potential mechanisms explaining the

observed toxic effects (Shimabukuro et al., 1998; Maedler et al., 2001; Hirabara et al., 2003;

Lu et al., 2003; Jorritsma et al., 2004).

The results of our study are not only important concerning the subfertility issue in

modern dairy cows, but may also carefully be proposed as a valuable model for human

research. Obesity and diabetes is featured by increased concentrations of NEFA due to a high

adipose sensitivity for lipolytic triggers (Herdt, 2000; Cnop et al., 2001). Our data may

suggest that the frequently reported fertility disorders in obese or diabetic women (Pasquali et

al., 2003) are not only due to toxic effects of NEFA on granulosa cells, predominantly leading

to amenorrhea (Mu et al., 2001), but may also originate from the direct harmful effects on the

cumulus oocyte complex. The latter could explain the disappointing IFV or ICSI results and

the higher risk for early pregnancy loss in obese women as has been documented by

Fedorcsak et al. (2000; 2004) and Pasquali et al. (2003). Further research should confirm the

appropriateness of this bovine model in human medicine.

Not only high NEFA but also elevated ketone concentrations are a distinctive

characteristic of the NEB (Sato et al., 1999). High ketone concentrations mostly go together

Chapter 8: General Discussion and Conclusions

205

with hypoglycaemia (Herdt, 2000). In a second IVM model (Chapter 5B), we therefore

investigated the effect of combined high β-OHB and low glucose concentrations which were

based on the measurements in follicular fluid of dairy cows during the NEB (Chapter 4B).

The main conclusion of this study was that the in vitro model imitating subclinical ketosis,

had no effect on the oocyte’s developmental capacity in vitro. Clinical ketosis however,

turned out to be harmful to oocyte quality in vitro and this was due to the low glucose

concentrations rather than being the effect of high β-OHB concentrations. Thus the toxicity of

β-OHB as has been described for cells of the immune system (Hoeben et al., 1997; Sartorelli

et al., 2000) could not be confirmed for oocytes. Conclusively, it can be stated that inadequate

glucose supplies may compromise oocyte developmental competence which is in line with

other studies (Krisher and Bavister, 1998; Cetica et al., 2002; Sutton-McDowall et al., 2004).

When interpreting these in vitro results and translating them in terms of subfertility in

high producing dairy cows, some prudence is in order. In our study, we hypothesized that

elevated NEFA or β-OHB concentrations, in combination with low glucose concentrations,

may contribute to reduced fertility in high yielding dairy cows by exerting detrimental effects

on oocyte developmental competence. Furthermore, our findings are more or less in line with

the hypothesis of Britt (1992) who hypothesized that follicles grown during the period of

NEB early post partum could be affected by the unfavourable metabolic changes and

therefore contain a developmentally incompetent oocyte. Subsequently, after a growing and

maturation phase of several weeks, this inferior oocyte will be ovulated at the moment of the

first insemination (Lucy, 2003). This hypothesis has more or less been confirmed in recent in

vivo studies (Gwazdauskas et al., 2000; Snijders et al., 2000; Sartori et al., 2002). It is

however important to mention that the combined in vitro and in vivo model used in the

present thesis was not entirely appropriate to investigate the described carry-over effect on

oocyte quality as hypothesized by Britt (1992). Our results only document on the follicular

fluid composition in the dominant follicle during the NEB to be mimicked in vitro. Quiescent

follicles, which embed the oocytes of interest, however, provide less isolation of the oocyte

from the extrafollicular environment and blood serum. As a consequence, such oocytes are

probably exposed to even higher NEFA concentrations (Zamboni, 1974). Another possibility

is that oocytes of primordial follicles are completely insensitive to all these metabolic

disruptions. Moreover, in the present study, the cumulus oocyte complexes were exposed to

elevated NEFA or β-OHB and low glucose concentrations for only 24 h, whereas in vivo the

oocytes are exposed to such concentrations for several days or even weeks. In the ideal

model, primordial follicles should be cultivated in high NEFA conditions for several weeks.

Chapter 8: General Discussion and Conclusions

206

However, such long term cultures of primordial follicles still have major drawbacks and

growing primordial (bovine) follicles upon to the preovulatory stage is impossible so far

(Gutierrez et al., 2000). Nevertheless, we do believe that the model used in the present study

revealed for the first time possible toxic effects of high follicular fluid NEFA and low glucose

concentrations on the developmental competence of bovine oocytes in vitro.

The need for a new technique to evaluate the lipid content of oocytes and embryos

Lipid metabolism is altered in dairy cows during the first weeks post partum (Chilliard

et al., 1998). As has been explained earlier, there is a massive lipid mobilization in the

adipocytes and the resultant NEFA are transported to the liver. It is known that these NEFA

can be internalized by several cell types (Dutta-Roy, 2000). Also later in lactation, dairy cows

typically display high total cholesterol concentrations, which are positively correlated with

milk yield (Blum et al., 1983). At the same time, it is known that oocytes (Kim et al., 2001;

Adamiak et al., 2005) and embryos can accumulate lipids originating from their culture

environment (Abe et al., 1999; Ferguson and Leese, 1999; Sata et al., 1999; Abe et al., 2002).

This lipid accumulation results in a darker appearance of the cytoplasm leading to reduced

oocyte or embryo quality and cryotolerance (Abe et al., 1999; Abe et al., 2002). The

increased lipid content has been associated with a higher sensitivity to oxidative stress, with

suboptimal mitochondrial function and a deviation in the relative abundance of

developmentally important gene transcripts (Abe et al., 2002; Reis et al., 2003; Rizos et al.,

2003). For all these reasons, it was decided to introduce lipid content as potential quality

parameter in the research of oocyte and embryo quality in high yielding dairy cows. However,

an appropriate lipid evaluation technique, applicable to a single oocyte or embryo was

lacking. With this in mind, a new technique was developed, based on the fluorescent staining

of intracellular lipid droplets and the consequent evaluation of the amount of emitted

fluorescence light (Chapter 6). The fluorescent dye Nile red demonstrated to be highly

specific for the intracellular lipid droplets. Repeated measurements within one oocyte gave

very reproducible results and most importantly, the technique is designed to be applied on a

single oocyte or embryo. By means of this technique, we were able to confirm that bovine

oocytes contain more lipids than murine oocytes and less intracellular lipids in lipid droplets

compared to porcine oocytes (Loewenstein and Cohen, 1964; Ferguson and Leese, 1999;

Sturmey and Leese, 2003). The amount of emitted light was related to the transparency of the

Chapter 8: General Discussion and Conclusions

207

oocytes under the stereomicroscope. We could also confirm in single bovine embryos that

culture in the presence of serum resulted in an increased intracellular lipid content (Abe et al.,

1999; Ferguson and Leese, 1999; Kim et al., 2001). To our knowledge, this has never been

done before. However, the main drawback of this technique is that, because of the absence of

standards, only the relative amount of lipids present in lipid droplets can be estimated.

Furthermore, it does not allow the evaluation of different lipid fractions or individual fatty

acids.

In conclusion, the main advantage of this technique is that we can compare the lipid

content of single oocytes or embryos originating, from different donors or from different

treatments in vitro as well as in vivo.

Back to the field: a closer look at embryo quality

Until now, we predominantly focused on oocyte quality in relation to NEB. From the

above, it can be stated that the oocyte is vulnerable to some of the metabolic alterations

associated with a NEB. At least in vitro, obvious adverse effects on oocyte quality were

observed. Logically, the next step would be to investigate the consequences for embryo

quality. As has been suggested by Rizos et al. (2003), the conditions prior to fertilization are

determinant for embryo yield while the embryo culture environment is crucial for embryo

quality. Based on that theory, the toxic effects of high NEFA and low glucose concentrations

during oocyte maturation, as has been demonstrated in Chapter 5, will particularly lead to low

fertilization and thus conception rates in our high producing dairy cows. And based on the

reports of Bousquet et al. (2004) this seems to be the case. However, high energy and/or

protein diets can alter the microenvironment of the embryo in the oviduct and uterus (Kenny

et al., 2002; Elrod and Butler, 1993; McEvoy et al., 1995). Such changes are expected to be

pernicious for embryo quality (Rizos et al., 2003) as was experimentally confirmed by

Wrenzycki et al. (2000) in heifers. However, it has never been demonstrated whether this is

also the case in high yielding dairy cows. Therefore, we have set up a field trial to gain more

insight into the embryo quality of high producing dairy cows, in comparison with non

lactating dairy heifers and beef cows. In this way, we were able to investigate both the effect

of milk production and of breed (or genetic background). The results of this study have

extensively been discussed in Chapter 7. Briefly, lactating dairy cows clearly displayed an

inferior embryo quality as assessed by morphological evaluation, compared to dairy heifers or

Chapter 8: General Discussion and Conclusions

208

beef cows. Furthermore, we were able to demonstrate by means of a multivariable regression

model that producing milk or not was significantly associated with embryo quality. It is also

important to mention that no differences were found in fertilization rate or in the number of

transferable embryos per embryo collection.

Since the embryos of the lactating dairy cows were on average collected around day

230 post partum, it is very unlikely that a carry over effect of the NEB, as has been

hypothesized by Britt (1992), is responsible for this observation. This could have been the

case when embryo collection was performed on average around two to three months after

calving as has been done by Sartori et al. (2002), who also found an obvious difference in

embryo quality between lactating dairy cows and maiden heifers. In contrast with our results,

Sartori and coworkers (2002) reported not only an inferior embryo quality but also a lower

fertilization rate expressed as a higher proportion of unfertilized oocytes present in the uterine

flushing of lactating dairy cows. They probably described the adverse influences of a carry

over effect of the NEB on oocyte quality (reduced fertilization rates) combined with possible

negative effects of lactation, management or diet on the microenvironment of the oviduct or

the uterus (reduced embryo quality), like we have found in our field trial.

As a summary, all suggested mechanisms potentially hampering embryo quality, are

represented in Figure 3.

Further research should reveal the exact mechanism through which embryo quality is

hampered in lactating dairy cows:

Some of the physiological adaptations associated with milk production may have adverse

effects on embryo quality. One of the typical features of high milk production are high

total cholesterol and low triglyceride concentrations in blood which has been confirmed in

our study (Varman and Schultz, 1968; Blum et al., 1983). However, no direct associations

of these parameters with embryo quality could be found.

As has been stated above, the typical milk stimulating rations high in energy and protein

have been linked with reduced embryo quality (McEvoy et al., 1997; Yaakub et al.,

1999). This has already extensively been discussed in Chapter 3.

Chapter 8: General Discussion and Conclusions

209

Figure 3. Representation of possible mechanisms by which embryo quality can be impaired in high

yielding dairy cows.

One of the major morphological embryo characteristics we evaluated was colour or

opacity. We were able to confirm with our new lipid evaluation technique that embryo colour

is correlated with lipid content, as has previously been suggested by others (Sata et al., 1999;

Abe and Hoshi, 2003). Lactating dairy cow embryos were generally dark and contained as

much lipids as in vitro produced embryos which are known to accumulate excessive amounts

of lipids (Abe et al., 1999). This has never been shown before. Such a high lipid content has

obviously been linked with impaired embryo quality (Reis et al., 2003; Rizos et al., 2003).

The underlying mechanism linking the production of milk or nutrition with embryo colour is

not known, but recently Adamiak et al. (2004) handed us an interesting clue. They added

serum of heifers which received a diet containing 6% of protected fat, to an embryo culture

medium and reported that the resulting embryos had an increased lipid content, an altered

metabolism and a higher incidence of apoptosis compared to controls.

??

-

Embryo quality

Physiological status: lactation

Breed or genetic merit

Lipolysis

Ketogenesis

Lactose synthesis

Protein rich diet

Energy rich diet

Peripheral metabolism

Ovulation

Dominant follicle

NEFA

Β-OHB

GLUCOSE

UREA

Total CHOLESTEROL

? -

- -

NEFA

β-OHB

GLUCOSE

UREA

Total CHOLESTEROL

Blood

?

Embryo mortality

Fertilization failure

Chapter 8: General Discussion and Conclusions

210

Perspectives for future research

As has been mentioned above, future research should learn us more about the

interactions between blood, micro-environment in the oviduct or the uterus and embryo

metabolism. Furthermore, several studies indicated that NEB is also associated with

depressed immunity during the first weeks post partum, leading to an increased susceptibility

to infectious diseases, such as mastitis and metritis (Hoeben et al., 2000; Lacetera et al.,

2005). Bearing this knowledge in mind, it is important to consider not only a direct link

between the NEB and fertility, as has been discussed extensively in this thesis, but that

reproductive functions are also affected indirectly by the increased incidence of infectious

diseases. Mastitis for example, which is together with a disappointing fertility a major reason

for culling among dairy cows, has been demonstrated to be directly linked with a retarded

onset of ovarian activity post partum (Loeffler et al., 1999; Rajala-Schultz and Gröhn, 2001;

Huszenicza et al., 2005). Whether infectious diseases can affect the oocyte and/or the embryo

in a direct way is poorly studied and certainly needs further research (Hansen et al., 2004).

Also environmental pollution has been associated with direct harmful effects on oocyte

quality through the generation of endocrine disrupters (Brevini et al., 2005).

Some food for thought

Is there still a need for high producing dairy cows? Yield maximization per animal is

preferable from an economical and environmental point of view. However, only an

outstanding herd management can guarantee the animal welfare in such high producing cows.

But even then, the pressure on these animals remains high since they are rapidly culled for

reasons as reduced fertility, metabolic disorders and infectious diseases. The present thesis

revealed that even the oocyte and the embryo may suffer from this high productivity.

What about the demand for milk? Despite the fact that milk is an excellent calcium

source, some nutritionists regard milk lipids (mostly saturated ones) as a contribution to

atherogenesis.

And last but not least. How can the continuous striving for a high milk production be

reconciled with overproduction at the European level and with supported dumping of surplus

dairy products on third world markets?

Chapter 8: General Discussion and Conclusions

211

Conclusions

Conclusively, it can be said that the typical biochemical serum changes during the

NEB early post partum are well reflected in the follicular fluid of the dominant follicle

exposing the granulosa cells and the maturing oocyte. In vitro maturation models revealed

that NEB associated NEFA and glucose concentrations are indeed toxic for the oocyte,

resulting in a hampered oocyte maturation and less developmental competence.

Even when the period of NEB is over and when no carry over effects of the NEB are

present anymore, high yielding dairy cows produced siginificantly inferior embryos in

comparison with dairy heifers and beef cows. With a newly developed lipid evaluation

technique, we were able to demonstrate that high producing dairy cow embryos contained up

to 45% more lipids compared to the embryos of non-lactating animals. These findings imply

that not the genetic merit for milk production or breed has an impact on embryo quality but

that all kinds of factors associated with milk production as such (metabolism, nutrition,

management) induce hostile conditions preventing optimal embryo development. Further

research is needed to learn more how milk production and nutrition of the dairy cow can

influence embryo health and metabolism by altering its environment in the oviduct and the

uterus.

The results of the present thesis may be crucial in the understanding of the

pathogenesis of subfertility in high producing dairy cattle through an affected oocyte and

embryo quality. We got evidence that the occurrence of a NEB has harmful consequences for

the quality of the female gamete and that the fact a cow is producing a high amount of milk

(physiological mechanism sustaining milk secretion, nutrition, and management) is

detrimental for a normal embryo quality.

Yes, oocytes and embryos in high producing dairy cows are really in danger!

Chapter 8: General Discussion and Conclusions

212

References

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Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. 1999. Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Molecular Reproduction and Development 53: 325-335.

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Summary

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It has frequently been reported that, along with the continuously increasing milk

production, dairy cow fertility has been declining. Maintaining dairy cows fertile is, however,

of capital importance to guarantee an optimal milk yield during the cows’ life. Chapter 1

summarizes the major characteristics of this failing reproductive function such as reduced

oestrus symptoms or anoestrus, cyst formation, delayed first ovulation. The pathways leading

to this have extensively been investigated. The negative energy balance early post partum and

the associated endocrine and metabolic changes particularly play an important role. Once an

ovulation is established, disappointing fertilization rates and early embryonic mortality have

been proposed as major factors in the problem of subfertility. And it is only recently that

studies have started to focus on oocyte and subsequent embryo quality. Oocytes and embryos

are suggested to be highly sensitive to any disruption in their environment caused by

metabolic (negative energy balance), dietary or other factors thereby having fatal

consequences for final fertility. However, knowledge about the oocyte’s microenvironment

and about the oocyte and embryo quality in high producing dairy cows is extremely limited.

Hence, the main scope of this thesis was to study the role and the importance of the oocyte

and the embryo proper in the complex problem of reproductive failure in modern high

yielding dairy cows. In Chapter 2 the specific aims of the present thesis are described.

In Chapter 3 possible mechanisms, through which oocyte and embryo developmental

competence in high yielding dairy cows could be hampered, are reviewed. Firstly the effects

of a negative energy balance and the associated endocrine and metabolic changes on oocyte

quality are discussed in detail. Secondly, attention is paid to the corpus luteum and the uterine

environment supporting early embryo development. Finally, the review focuses on possible

consequences of milk yield stimulating rations (high starch, fat and protein content) typically

fed to dairy cows on the success rate of an oocyte to become a healthy embryo.

In the original studies of the present thesis attention was focused on the pathway

linking a negative energy balance and oocyte quality (Chapter 4 and 5). Furthermore, in the

case a successful fertilization takes place and an embryo is formed, it is not known whether

the quality of this early life is impaired or not. Therefore, by means of a field trial, we

compared the embryo quality between lactating high yielding dairy cows, non-lactating dairy

heifers and beef cows (Chapter 7). One of the parameters used to evaluate embryo quality is

the lipid content. Since current techniques to evaluate lipid content all require a large number

of oocytes or embryos, a completely new and highly sensitive technique needed to be

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developed in order to evaluate the lipid content of single bovine oocytes or embryos (Chapter

6).

Literature about the biochemical composition of follicular fluid, which is the oocyte’s

microenvironment, and the interrelation with the composition of blood serum in dairy cows is

almost absent. The purpose of the study described in Chapter 4A was to examine the

biochemical composition of follicular fluid harvested from different sized follicles and to

investigate its relationship with that of blood serum in dairy cattle. Following slaughter, blood

samples were collected from dairy cows (n = 30) and follicular fluid aspirated from three size

classes of non-atretic follicles (< 4 mm, 6 to 8 mm and > 10 mm diameter). Serum and

follicular fluid samples were assayed for ions (sodium, potassium and chloride) and

metabolites (glucose, β-hydroxybutyrate, lactate, urea, total protein, triglycerides, non-

esterified fatty acids and total cholesterol). Results showed that follicular fluid concentrations

of glucose, β-hydroxybutyrate and total cholesterol increased from small to large follicles and

decreased for potassium, chloride, lactate, urea and triglycerides. We also found a significant

concentration gradient for all variables between their concentrations in serum and follicular

fluid. Significant correlations were observed between serum and follicular fluid for chloride (r

= 0.40), glucose (r = 0.56), β-hydroxybutyrate (r = 0.85), urea (r = 0.95) and total protein (r =

0.60) for all three follicle size classes and for triglycerides (r = 0.43), non-esterified fatty acids

(r = 0.50) and total cholesterol (r = 0.42) for large follicles (P< 0.05). These findings suggest

that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical

environment that changes from small to large follicles. Furthermore, the significant

correlation between the composition of serum and follicular fluid for the above mentioned

metabolites suggests that metabolic changes in serum levels will be reflected in the follicular

fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.

In Chapter 4B a study is described about the oocyte’s environment in living high

producing dairy cows in the period early post partum. The aim was to examine to what extent

some of the typical metabolic changes that occur in early postpartum high yielding dairy cows

are reflected in the follicular fluid of the dominant follicle (> 8mm). Nine blood samples were

taken per cow from 9 high yielding dairy cows between 7 days before and 46 days after

parturition. From day 14 post partum on and together with blood sampling, follicular fluid

samples of the largest follicle were collected from the same cows by means of transvaginal

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follicle aspiration. Serum and follicular fluid samples were analysed for glucose, β-

hydroxybutyrate, urea, total protein, triglycerides, non-esterified fatty acids and total

cholesterol. All cows lost body condition during the experimental period, illustrating a

negative energy balance during the experimental period. In follicular fluid, glucose

concentrations were significantly higher and the total protein, triglycerides, non-esterified

fatty acids and total cholesterol concentrations were significantly lower than in serum. The

concentrations of glucose, β-hydroxybutyrate, urea and total cholesterol in serum and in

follicular fluid changed significantly over time. Throughout the study, changes of all

metabolites in serum were reflected by similar changes in follicular fluid. Especially for

glucose, β-hydroxybutyrate and urea such ‘dynamic’ correlations were remarkably high. The

results from this study demonstrate for the first time that the typical metabolic adaptations

which can be found in serum of high yielding dairy cows shortly post partum, are reflected in

the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa

cells.

The next step was to focus on non-esterified fatty acid, β-hydroxybutyrate and glucose

concentrations in follicular fluid and to investigate direct possible toxic effects of these in vivo

concentrations on oocyte quality. We therefore concentrated on the concentration and

composition of non-esterified fatty acids in follicular fluid of high yielding dairy cows during

the period of negative energy balance early post partum (Chapter 5A). At 16 and 44 days

post partum, follicular fluid of the dominant follicle and blood were collected from 9 high

yielding dairy cows. Samples were analysed for non-esterified fatty acids concentration and

composition. Non-esterified fatty acids concentrations in follicular fluid (0.2 - 0.6 mmol/l)

during the negative energy balance remained ± 40% lower compared to serum (0.4 – 1.2

mmol/l). The non-esterified fatty acids composition differed significantly between serum and

FF, with oleic acid, palmitic acid and stearic acid being the predominant fatty acids in

follicular fluid. The observed FF concentrations of these three fatty acids were imitated in an

in vitro serum-free maturation model to investigate their effects on the oocyte’s

developmental capacity. Addition of palmitic or stearic acid during oocyte maturation had

negative effects on maturation, fertilization and cleavage rate and blastocyst yield. More (late)

apoptotic cumulus cells were observed in cumulus oocyte complexes matured in the presence

of palmitic or stearic acid. Ethanol (the fatty acid carrier) or oleic acid had no effect. These in

vitro results suggest that a negative energy balance may hamper fertility of high yielding dairy

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cows through increased non-esterified fatty acid concentrations in follicular fluid affecting

oocyte quality.

As mentioned above, also β-hydroxybutyrate (BHB) and glucose are very important

metabolites in the follicular fluid of high yielding dairy cows early post partum. Therefore, we

tested the effect of two different β-hydroxybutyrate and glucose concentrations, which are

associated with subclinical or clinical ketosis, during in vitro maturation on the developmental

competence of bovine oocytes (Chapter 5B). In experiment 1, subclinical ketosis conditions

were imitated (2.75 mmol/l glucose and 1.8 mmol/l BHB). In experiment 2, clinical ketosis

conditions were mimicked by using 1.375 mmol/l glucose and 4.0 mmol/l BHB. After in vitro

maturation and fertilization, presumptive zygotes were cultured for 7 days in SOF (5% FCS).

At respectively 48h and 8 days after fertilization, cleavage rate and number of blastocysts

were recorded. Simultaneous exposure to subclinically low glucose and high BHB

concentrations decreased blastocyst yield compared to the other groups. Clinical ketosis

conditions were harmful for the oocyte’s developmental capacity. Especially the very low

glucose concentrations but not the high BHB levels were responsible for a hampered cumulus

expansion, cleavage and subsequent blastocyst formation. Conclusively, these results may

suggest that especially clinical ketosis can affect the oocyte’s developmental competence

most likely through a directly adverse effect of the very low glucose concentrations on oocyte

maturation.

Lipid metabolism is altered in high producing dairy cows and it is known that oocytes

and embryos are able to accumulate lipids from their environment, which can hamper their

quality. For this reason it was decided to introduce lipid content as a potential quality

parameter in the research of oocyte and embryo quality in high yielding dairy cows. However,

an appropriate lipid evaluation technique, applicable on a single oocyte or embryo was

lacking. Therefore, a new technique was developed, based on the fluorescent staining (Nile

red) of intracellular lipid droplets in single bovine oocytes and embryos and the consequent

evaluation of the amount of emitted fluorescence light (Chapter 6). It was hypothesized that

a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount

of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes,

the fluorescence of the whole oocyte was visualized by fluorescence microscopy and

quantified by means of a photometer and a photomultiplier connected to the microscope. The

peak of fluorescence was observed in the yellow spectrum (590nm) and the fluorescence was

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restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging

from 0.1 to 10 μg/ml yielded similar results. After fixation, a minimum of 2h staining was

necessary to reach maximal fluorescence which remained stable for several hours. The

position of the microscopic focus within the oocyte had no influence on the amount of

measured fluorescence. This very new method proved to be very repeatable. Finally, the

technique was validated by comparing the lipid content of bovine, porcine and murine

immature oocytes which are known to contain different amounts of lipids. After staining, the

fluorescence of murine oocytes was 2.8 fold lower than the fluorescence of bovine oocytes

which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this technique

we were also able to demonstrate that the lipid content of immature bovine oocytes is

correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark

cytoplasm contained significantly more intracellular lipids in lipid droplets compared to

oocytes with a granulated or pale cytoplasm. Furthermore, this lipid analysing technique was

applied for the first time on single bovine in vitro produced embryos showing a significant

increase of the lipid content in lipid droplets after culture in the presence of serum.

This fast and easy technique allows for the relative quantification of the lipid content (present

in lipid droplets) of one single oocyte or embryo.

In the last part of the present thesis we focused on the embryo quality of high

producing dairy cows (Chapter 7). The purpose of this study was to compare embryo quality

of lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH)

and Belgian Blue beef cows (BB) and to identify factors that are associated with embryo

quality in LHFC and NLHFH. After superovulation and embryo recovery at day 7, embryos

were scored morphologically for quality, colour and developmental stage. Blood samples and

data concerning parity, age, milk production and management were collected. Data were

compared univariably between the three groups. A multivariable regression model was built

with quality and colour of the LHFC and NLHFH embryos as dependent variables. Only

13.1% of LHFC embryos were categorized as excellent compared to 62.5% and 55.0% of the

embryos from NLHFH and BB, respectively. Almost none of the NLHFH or BB embryos

displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC embryos.

These dark LHFC embryos contained up to 45% more lipids compared to pale embryos. Only

4% of all LHFC embryos reached the blastocyst stage compared to 23.2% and 17.3% in

NLHFH and BB, respectively. Based on the multivariable regression analysis, “physiological

status” (lactating or not) together with the serum total protein concentration of LHFC and

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NLHFH was significantly associated with embryo quality and colour. Thus, LHFC display an

inferior embryo quality compared to NLHFH and BB. Producing milk or not seems to be

significantly associated with embryo quality. Therefore, reduced embryo quality on day 7

following AI could be an important factor in the subfertility problem in modern high yielding

dairy cows.

Finally, in Chapter 8, the main results are summarized and discussed. From these

results, the following conclusions are drawn:

1. The typical biochemical serum changes during negative energy balance early post partum

are well reflected in the follicular fluid of the dominant follicle.

2. In vitro maturation models revealed that such negative energy balance associated non-

esterified fatty acid and glucose concentrations are indeed toxic for the oocyte, resulting in

a hampered oocyte maturation and developmental competence.

3. High yielding dairy cows produced siginificantly more inferior embryos in comparison

with non-lactating dairy heifers and beef cows.

4. A new lipid analysis technique was developed to evaluate the lipid content of single

bovine oocytes and embryos.

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Het is algemeen bekend dat de vruchtbaarheid van hoogproductieve melkkoeien sterk

is afgenomen terwijl de melkproductie almaar blijft stijgen. Een goede vruchtbaarheid is

echter noodzakelijk om een optimale melkopbrengst te garanderen. In Hoofdstuk 1 worden

de belangrijkste kenmerken van deze relatieve onvruchtbaarheid samengevat en worden de

mogelijke mechanismen die tot subfertiliteit kunnen leiden besproken. Hierbij zouden vooral

de negatieve energiebalans vroeg post partum en de daarmee gepaard gaande endocriene en

metabole veranderingen een belangrijke rol spelen. Eenmaal de ovariële activiteit goed op

gang komt en een ovulatie optreedt, kunnen er nog andere factoren zoals een verminderde

bevruchtingskans of vroeg embryonale sterfte een optimale vruchtbaarheid in de weg staan.

Bij dit laatste spelen vooral de eicel- en de embryokwaliteit een belangrijke rol. Eicellen en

embryo’s zijn heel gevoelig voor wijzigingen in hun omgeving. De kennis omtrent de eicel-

en de embryokwaliteit en het folliculaire milieu waarin de eicel groeit en matureert, is echter

heel beperkt tot zelfs afwezig. Het hoofddoel van dit doctoraat bestond erin om klaarheid te

scheppen in de rol en het belang van de eicel en van het embryo in het complexe vraagstuk

van de verminderde vruchtbaarheid bij hoogproductieve melkkoeien. De specifieke

doelstellingen worden in detail uiteengezet in Hoofdstuk 2.

In het overzichtsartikel van Hoofdstuk 3 worden alle mogelijke mechanismen die de

eicel- en de embryokwaliteit bij hoogproductieve koeien kunnen beïnvloeden, overlopen. De

effecten van een negatieve energiebalans, voor zover gekend, en de invloeden van de corpus

luteum werking op de eicel-, respectievelijk de embryokwaliteit, worden uiteengezet.

Tenslotte wordt in hoofdstuk 3 dieper ingegaan op de mogelijke gevolgen van een typisch

melkdrijvend (heel eiwit- en energierijk) dieet voor de ontwikkelingscompetentie van de

eicel en het embryo.

In de daaropvolgende onderzoeken wordt het verband tussen de negatieve

energiebalans en de eicelkwaliteit verder onderzocht (Hoofdstukken 4 en 5). Daarnaast

wordt ook dieper ingegaan op de embryokwaliteit van hoogproductieve melkkoeien door

middel van een veldstudie (Hoofdstuk 7). Eén van de kwaliteitsparameters die gebruikt

worden om de embryokwaliteit in te schatten, is het vetgehalte. Aangezien de huidige

technieken enkel toepasbaar zijn op grote aantallen eicellen of embryo’s, werd een nieuwe en

gevoelige techniek ontwikkeld om het vetgehalte van één enkele eicel of embryo te bepalen

(Hoofdstuk 6).

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De kennis over de samenstelling van het follikelvocht, het micromilieu van de eicel, is

heel beperkt. Omtrent de relatie met de serumsamenstelling is zo goed als niets bekend. Het

doel van het onderzoek dat in Hoofdstuk 4A is beschreven, was dan ook de biochemische

samenstelling van het folliculaire milieu van drie verschillende follikelklassen te onderzoeken

en na te gaan hoe die gecorreleerd is met de samenstelling van het serum. Dadelijk na het

slachten werd bloed genomen van 30 melkkoeien en werd het follikelvocht geaspireerd uit de

drie follikelklassen (klein: < 4 mm, middelgroot: 6 – 8 mm en groot: > 10 mm). Het serum en

het follikelvocht werden geanalyseerd op natrium-, kalium- en chloorionen, glucose, β-

hydroxyboterzuur, lactaat, ureum, totaal eiwit, triglyceriden, vrije vetzuren en totaal

cholesterol. Tijdens de groei van de follikel bleken de glucose-, β-hydroxyboterzuur- en totaal

cholesterolconcentraties te stijgen terwijl de concentraties van kalium- en chloorionen en van

lactaat, ureum en triglyceriden opmerkelijk daalden. Er was een duidelijke verschil tussen de

concentraties in het serum en het follikelvocht voor alle gemeten parameters. Tevens werden

er duidelijke correlaties gevonden tussen de samenstelling van serum en follikelvocht voor

wat betreft glucose, β-hydroxyboterzuur, ureum, totaal eiwit en vrije vetzuren. De resultaten

van dit onderzoek suggereren dat de eicel groeit en matureert in een biochemisch dynamische

omgeving waarvan de samenstelling goed gecorreleerd is met de samenstelling van het

bloedserum. Met andere woorden, belangrijke biochemische veranderingen in het serum

worden weerspiegeld in het follikelvocht en kunnen de integriteit van de eicel dus

rechtstreeks beïnvloeden.

In Hoofdstuk 4B werd verder ingegaan op de samenstelling van het folliculaire milieu

bij levende melkkoeien in de periode vroeg postpartum. Het doel van dit tweede onderzoek

bestond erin na te gaan in welke mate de belangrijkste metabole veranderingen die vroeg post

partum optreden, weerspiegeld worden in de samenstelling van het follikelvocht van

hoogproductieve melkkoeien. Van 9 melkkoeien werden negen bloedstalen per koe genomen

tussen dag 7 vóór en dag 46 na het afkalven. Vanaf dag 14 werd er om de 6 dagen

follikelvocht geaspireerd van de dominante follikel met behulp van de ovum pick-up

techniek. Dezelfde metabolieten als beschreven in hoofdstuk 4A werden bepaald. Alle koeien

vermagerden tijdens de experimentele periode, wat wijst op het negatief zijn van de

energiebalans bij deze dieren. De glucoseconcentraties waren duidelijk hoger en de totaal

eiwit-, triglyceriden-, vrije vetzuren- en totaal cholesterolconcentraties duidelijk lager in het

follikelvocht dan in het serum. De gehalten aan glucose, β-hydroxyboterzuur, ureum en totaal

cholesterol veranderden tijdens de experimentele periode, zowel in het serum als in het

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follikelvocht. De concentratieveranderingen van alle metabolieten in het serum werden

duidelijk weerspiegeld in het follikelvocht. Vooral voor glucose, β-hydroxyboterzuur en

ureum waren deze dynamische correlaties opvallend hoog. De resultaten van deze tweede

studie tonen duidelijk aan dat de typisch metabole veranderingen die koeien ondergaan tijdens

de eerste weken na het afkalven hun weerslag hebben op de samenstelling van het

follikelvocht. De eicel kan hierdoor dus rechtstreeks beïnvloed worden.

De logisch volgende stap in het onderzoek was na te gaan wat de mogelijke directe

gevolgen zijn van de in het follikelvocht geobserveerde verhoogde gehalten aan vrije vetzuren

en β-hydroxyboterzuur en de verlaagde glucoseconcentraties voor de

ontwikkelingscompetentie van de eicel. Daarom werd er in Hoofdstuk 5A verder ingegaan op

de concentratie en de samenstelling van de vrije vetzurenfractie in het follikelvocht tijdens de

periode van negatieve energiebalans. Voor het in vivo deel van dit onderzoek werden 9

hoogproductieve koeien gebruikt waarvan het follikelvocht werd geaspireerd op dag 16 en 44

post partum. Het verzamelde follikelvocht werd geanalyseerd voor de vrije

vetzurenconcentratie en –samenstelling. De vrije vetzurenconcentraties in het follikelvocht

tijdens de periode van negatieve energiebalans (0,2 – 0,6 mmol/l) bleven ± 40% lager dan die

in het serum (0,4 – 1,2 mmol/l). De samenstelling van de vrije vetzurenfractie verschilde

significant tussen serum en follikelvocht waarin palmitine-, stearine- en oleïnezuur de

belangrijkste vetzuren waren. Toevoeging van palmitine- en stearinezuur in het in vitro

maturatiemedium in concentraties zoals in het follikelvocht gemeten, had nefaste effecten op

de graad van eicelmaturatie, -fertilisatie, -klieving en op de blastocystontwikkeling. De hoge

mate van apoptose die werd opgemerkt in het cumulus oocyte complex na maturatie in de

aanwezigheid van palmitine- of stearinezuur, zou een mogelijke verklaring kunnen vormen.

Toevoeging van oleïnezuur had geen effect. Deze in vitro resultaten tonen voor de eerste maal

aan dat het vóórkomen van een negatieve energiebalans directe schadelijke gevolgen kan

hebben op de eicelkwaliteit via toegenomen vrije vetzurenconcentraties in het follikelvocht.

Zoals hierboven reeds werd vermeld zijn ook glucose en β-hydroxyboterzuur (BHB)

belangrijke parameters in het serum en het follikelvocht van hoogproductieve koeien vroeg

postpartum. Daarom werd in een volgend onderzoek (Hoofdstuk 5B) in een in vitro

maturatiemedium het effect op de eicelkwaliteit getest van twee verschillende β-

hydroxyboterzuur- en glucoseconcentraties die geassocieerd worden met subklinische en

klinische ketose. In experiment 1 werd de subklinische ketose nagebootst (2,75 mM glucose

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en 1,8 mM BHB). In experiment 2 werd de klinische ketose nagebootst door gebruik te

maken van 1,375 mmol/l glucose en 4,0 mmol/l BHB. Op respectievelijk 48 uur en 8 dagen

na de fertilisatie werden de klieving en het aantal gevormde blastocysten genoteerd.

Simultane blootstelling aan subklinisch lage glucose- en hoge β-

hydroxyboterzuurconcentraties resulteerde in duidelijk minder blastocysten.

Cultuuromstandigheden die de klinische ketose nabootsten bleken toxisch te zijn voor de

ontwikkeling van eicellen. Hierbij bleken vooral de lage glucoseconcentraties en niet zozeer

de zeer hoge β-hydroxyboterzuurconcentraties de gedaalde ontwikkelingscompetentie van de

eicellen te verklaren. Samenvattend kan gesteld worden dat vooral klinische ketose een

rechtstreeks toxisch effect kan uitoefenen op de eicelkwaliteit en dát vooral door de lage

glucoseconcentraties die daarmee gepaard gaan.

Hoogproductieve melkkoeien hebben een gewijzigd vetmetabolisme en tegelijkertijd

is bekend dat eicellen en embryos in staat zijn om vetten uit hun omgeving op te nemen wat

tot een kwaliteitsdaling kan leiden. Daarom werd besloten om het vetgehalte van eicellen en

embryo’s als een extra kwaliteitsparameter op te nemen. Tot op de dag van ons onderzoek

was er echter geen enkele techniek voorhanden om het vetgehalte van één enkele eicel of

embryo te evalueren. Daarom werd een nieuwe techniek ontwikkeld waarmee dit wel

mogelijk is (Hoofdstuk 6). Deze techniek is gebaseerd op het laten fluoresceren van

intracellulaire vetdruppeltjes met behulp van de kleurstof Nile red. De hypothese hierbij was

dat de aanwezigheid van een groter aantal vetdruppels zou resulteren in een grotere

hoeveelheid uitgestraald fluorescent licht. Eicellen werden ontdaan van alle cumuluscellen,

gefixeerd en gekleurd. De fluorescentie van de volledige eicel werd gevisualiseerd met een

fluorescentiemicroscoop en het uitgestraalde licht werd gekwantificeerd met een fotometer en

een lichtversterker die met de microscoop waren verbonden. Het fluorescentiemaximum werd

geobserveerd in het gele lichtspectrum (590 nm) en deze fluorescentie bleef beperkt tot de

vetdruppeltjes (apolaire vetten). Nile red concentraties variërend tussen 0.1 en 10 μg/ml

bleken afdoende te zijn voor een betrouwbaar resultaat. Na de fixatie moest een

kleurstofincubatieduur van minimaal 2 uur in acht worden genomen. De positie van de focus

van de microscoop bleek van ondergeschikt belang te zijn. Deze nieuwe techniek bleek tevens

heel herhaalbaar te zijn. Om de test verder te valideren werd het vetgehalte van boviene

eicellen vergeleken met dat van varkens- en muizeneicellen. De verhoudingen van het

vetgehalte van de eicellen tussen deze drie diersoorten bleken goed overeen te komen met de

literatuurgegevens. Met de nieuwe techniek was het daarenboven mogelijk om aan te tonen

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dat eicellen met een donker ooplasma significant meer vet in vetdruppeltjes bevatten dan

blekere eicellen. Tenslotte werd de techniek ook toegepast op boviene embryo’s (morula’s) en

kon bevestigd worden dat embryo’s meer vet bevatten na een cultuurperiode in aanwezigheid

van foetaal kalfserum. Ook dit bevestigt wat in de literatuur reeds herhaaldelijk werd

beschreven. Gebaseerd op deze testresultaten kan gesteld worden dat dit de eerste techniek is

die uitermate geschikt is voor de semiquantitatieve vetgehaltebepaling (in vetdruppels) van

één eicel of embryo.

In het laatste deel van het onderzoek werd dieper ingegaan op de embryokwaliteit van

hoogproductieve melkkoeien (Hoofdstuk 7). De embryokwaliteit van lacterende Holstein

Friesian koeien (LHFK) werd vergeleken met die van niet-lacterende Holstein Friesian

vaarzen (NLHFV) en dikbilkoeien (DB). Eveneens werd er gezocht naar risicofactoren die de

embryokwaliteit bij de Holstein Friesian koeien en vaarzen zouden kunnen beïnvloeden. Na

superovulatie en het uitspoelen van de embryo’s op dag 7, werden de embryo’s morfologisch

gescoord voor kwaliteit, kleur en ontwikkelingsstadium. Van elke donor werd een

bloedmonster genomen en een groot aantal donorgegevens (pariteit, leeftijd, melkproductie,

management, ...) werden genoteerd. Al deze gegevens werden univariabel vergeleken tussen

de drie donorgroepen. Daarenboven werd er gebruik gemaakt van een multivariabel

regressiemodel voor identificatie van de belangrijkste factoren die de kwaliteit en de kleur van

de Holstein Friesian embryo’s beïnvloeden. Slechts 13,1% van de LHFK embryo’s konden als

uitmuntend gecatalogeerd worden, terwijl dat voor de NLHFV 62,5% en voor de DB 55,0%

was. Bijna geen van de NLHFV en de DB embryo’s vertoonden een donker cytoplasma

terwijl dat voor 24,1% van de LHFK embryo’s wel het geval was. Deze donkere embryo’s

bleken tot 45% meer vet te bevatten dan de bleke NLHFV en DB embryo’s. De LHFK

embryo’s ontwikkelden zich ook opvallend trager dan de embryo’s uit de andere groepen. En

tenslotte bleek uit de multivariabele regressie analyse dat de “fysiologische status” (het feit of

een dier lacteert of niet) en het totaal eiwitgehalte in het serum de belangrijkste factoren zijn

die significant geassocieerd zijn met embryokwaliteit en -kleur. Concluderend kan dus gesteld

worden dat LHFK duidelijk minder goede embryo’s produceren dan de NLHFV en de DB en

dat het al dan niet lacteren hierin een belangrijke rol speelt. Een verminderde embryokwaliteit

zou dus een mogelijke verklaring kunnen vormen voor de ontgoochelende

vruchtbaarheidsresultaten bij hoogproductieve melkkoeien.

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Tenslotte worden in Hoofdstuk 8 de belangrijkste resultaten opgesomd en

bediscussieerd. Uit het bovenstaande kunnen de volgende, korte conclusies getrokken

worden:

1. De typisch biochemische veranderingen in het serum die optreden tijdens de periode van

de negatieve energiebalans vroeg post partum worden duidelijk weerspiegeld in het

follikelvocht en kunnen dus de eicel rechtstreeks beïnvloeden.

2. Op basis van in vitro maturatiemodellen werd het duidelijk dat vrije vetzuren- en

glucoseconcentraties die geassocieerd worden met zo’n negatieve energiebalans, nefast

zijn voor de correcte ontwikkeling van eicellen.

3. De embryo’s van hoogproductieve melkkoeien zijn van inferieure kwaliteit in vergelijking

met die van niet-lacterende melkveevaarzen of dikbilkoeien.

4. Er werd een volledig nieuwe en betrouwbare techniek ontwikkeld om het vetgehalte van

individuele eicellen of embryo’s te evalueren.

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Doctoreren, PhD, doctor in philosophy... Zit er eigenlijk wel veel filosofie in eicellen, ovaria en embryo’s van melkkoeien die zodanig veel melk produceren dat ze niet meer vruchtbaar zijn? Vier jaar geleden dacht ik van niet. Maar nu, ja, nu weet ik het wel zeker: de studie van eicel en embryo heeft me aan het denken, of waarom ook niet, aan het filosoferen gezet. Literatuur, het eerste schuchtere protocol, wetenschappelijke en minder wetenschappelijke discussies met promotoren en collega’s, de eerste laboproeven, de eerste mislukkingen die de eerste successen aankondigden, samenwerken, mensenkennis, internationale congressen, samen een pint drinken, vergaderingen, wachtregelingen, compromissen sluiten, monikkenwerk, twijfelen aan het nut van je onderzoek, fier je resultaten presenteren, ...

De Vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde heeft me altijd geboeid door de aangename werksfeer en de mengelmoes van verschillende disciplines waardoor het doctoreren in de volledige betekenis van het woord pas echt mogelijk was. Ik heb erg genoten van de combinatie van studie en praktijk. Daarom zou ik in eerste instantie het volledige team van de vakgroep willen bedanken.

Ann, ik weet dat ik 4 jaar geleden een beetje onverwacht je labo kwam ‘ingesprongen’. Je hebt me echter altijd bijgestaan met woord en daad. De grote vrijheid en het daarmee gepaard gaande vertrouwen dat je me schonk, hebben me echt deugd gedaan. Nog steeds heb ik heel veel bewondering voor de manier waarop jij een meer dan fulltime job combineert met een groot gezin. Ook de vertrouwelijkere gesprekken tijdens ons Brazilië avontuur, vaak geïnspireerd door de heerlijke caipirinha, zal ik nooit vergeten. Ik ben blij dat we samen de wereldwinkelkoffie op onze dienst hebben binnengeloodst. Dank je wel voor dit alles.

Geert, hartelijk dank voor je promotorschap van de voorbije jaren. De positieve energie die je steeds uitstraalde, ook als het eens wat minder ging, heeft me aangezet om vol te houden. Dank je wel voor de vrijheid die je me hebt gelaten en voor de vele contacten die je voor ons hebt gelegd. Ik heb veel geleerd van je lesgeverskunst en je gave om compromissen te sluiten. Hopelijk wordt ons Fertiliteitssymposium een ongelooflijk succes.

Tom, eigenlijk reken ik jou ook tot mijn promotoren. Puur wetenschappelijk heb ik heel wat aan jou gehad. De vele kleine maar vruchtbare discussies rond artikels, protocols, nieuwe ideeën, dierenproeven... hebben dit doctoraat een

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impuls gegeven. Lacunes werden hierdoor opgevuld en we trokken letterlijk aan hetzelfde zeel. Vaak genoeg heb ik gebruik gemaakt van je ongelooflijke literatuurkennis.

Professor de Kruif, als copromotor en vakgroepvoorzitter hebt u me steeds enorm geboeid door de manier waarop u onze vakgroep runt. De open geest, het vertrouwen en de vrijheid die er heerst samen met uw realistische visie op de wereld rondom ons, zorgen voor een ‘vruchtbare’ werksfeer. Het is dankzij jou dat we onze resultaten op talrijke congressen mochten gaan presenteren. Dank je wel.

Doctoreren was natuurlijk niet mogelijk geweest zonder de ‘begeleiding achter d’ uren’. Pien, dank je wel voor de motiverende gesprekken, de knuffels, het stimuleren van m’n buitenlandse bezoeken. Misschien ben jij wel mijn grootste promotor. Moe en Va, uit het allerdiepste van mijn hart wil ik jullie bedanken voor de gelukkige kindertijd die ik heb gehad, voor de niet vanzelfsprekende kansen die jullie beiden voor ons hebben gecreëerd. Tien kinderen aan de Unief laten studeren… je moet het maar doen. Ik zou hiervoor het ultieme toverwoord wel willen kennen. Moe, jij creëerde voor ons de ideale studeersfeer terwijl Va de mentale coach was: “als je aan iets begint moet je het beste van jezelf geven, anders begin je er beter niet aan”. In één lange adem wil ik dan ook m’n broers en zussen bedanken voor de gezellige sfeer thuis, de vele leerrijke gesprekken, de soms oeverloze discussies over pietluttige details. Bij ons moest ‘je het wel goe kunnen uitleggen’ …

Walter en Marina, dank je wel voor de enorme gastvrijheid, de gezellige tafelmomenten en het opvangen van Helene als Katrien en ik nog maar eens op weekend gingen.

Een heel speciaal woord van dank zou ik graag tot de Cachacero Certificado Professor Peter Bols (UA) richten. Peter, jij hebt voor mij heel wat hermetische deuren geopend. Je hebt me geleerd dat veel zaken bespreekbaar zijn als je zelf maar genoeg initiatief neemt. Jouw ‘recht voor de raap zijn’, heeft me van bij onze eerste ontmoeting sterk aangesproken. Dank je wel voor de wetenschappelijke en minder wetenschappelijke discussies, de motiverende gesprekken, de bruikbare adviezen en de fijne e-mail communicatie. Onze spectaculaire rit naar Parma en onze boeiende Brazilië-studie-reizen zal ik nooit vergeten. Vooral de gevleugelde woorden “zie ons hier nu zitten …” zullen eeuwig blijven nazinderen. Hopelijk kunnen we in de toekomst nog heel wat projecten samen tot een goed eind brengen. Bedankt Peter!

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Ook de andere leden van de begeleidings- en examencommissie zou ik willen bedanken voor hun engagement. Zonder de hulp van Professor Delanghe en Professor Christophe zou dit doctoraat niet mogelijk zijn geweest. Vele honderden stalen kon ik op hun labo tegen een gunsttarief laten analyseren. Die zeer goede service was van een onschatbare waarde voor dit werk. Steeds kon ik bij jullie terecht met mijn soms heel rare vragen, problemen of opmerkingen. Dank je wel hiervoor.

And of course, it is an honour for me to acknowledge Dr. Rizos from the INIA. I have always been a great admiror of your work concerning embryo quality. Thank you for your willingness to become a member of my PhD committee.

Louis Goossens en Wies Geldhof wil ik hartelijk danken voor de enorme hulp die ze geboden hebben bij onze veldproef. Zij hebben letterlijk bijna al het werk gedaan. Zij waren het die me voorzichtig de term ‘embryokleur’ leerden kennen. Louis, dank je wel voor je blijvende interesse, de leerrijke gesprekken en de lange tijd die je stak in het opzoeken van al die ingevroren donkere en bleke embryo’s. Ik hoop dat we nog vaak kunnen samenwerken en wie weet, gaan we nog eens samen op congres.

Joke, Griet en Janina … de ware fundamenten van m’n onderzoek, dank je wel voor de vele uren werk die jullie in m’n doctoraat hebben gestoken. Al die ritten naar het slachthuis, al dat gepuncteer, honderden eicellen opzoeken die dan niet per 100 maar per 60 moesten worden gematureerd. Ik heb altijd genoten van de gezellige sfeer in het labo, het streven naar hoge blastocystpercentages, het opzetten van serumvrije culturen en het bestrijden van besmettingen. Duizendmaal dank voor de leuke baby-gesprekken die we voerden!

Je veux aussi remercier Gaetan Genicot et Professeur Isabelle Donnay pour l’agréable coopération dans le lab de Louvain la Neuve.

Sarne en Jeroen, bedankt voor de hulp bij het statistisch verwerken van m’n gegevens. Het waren alleen de lastigste datasets die ik aan jullie doorspeelde … Marc, bedankt voor het minutieus napluizen van m’n artikels en ellenlange referentielijsten, op zoek naar een ontbrekend punt. Van monnikenwerk gesproken … Leila en Nadine, dank je wel voor het regelen van allerlei administratieve details.

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Liefste bureaugenoten, Tom, Stefaan en Liesbeth. Bedankt voor het gezelschap en de fijne werksfeer. Collega’s van het IVF labo, Bart, Tom, Leen en Sofie. Ik heb genoten van de fijne samenwerking en de zeer gezellige momenten op de verschillende congressen. Bart, ik zal jouw bedgeheimen nooit verklappen. Wat zou doctoreren toch zielig zijn zonder fijne collega’s. Aan de jonge, beginnende collega’s Liesbeth, Mirjan, Philippe, Sofie en Jo B wil ik alvast het allerbeste wensen. Ga er voor jongens …

Mens sana in corpore sano: een mentale inspanning vraagt een fysieke uitlaatklep. Tijdens het laatste jaar van dit doctoraat legden we ons niet alleen toe op het schrijven maar ook op het lopen. Jo V en Liesbeth, bedankt voor het trouwe gezelschap tijdens de looptochten die steeds langer werden.

Tevens wil ik alle collega’s van de buitenpraktijk bedanken voor de heel aangename werksfeer, het vlot regelen van de wachtdienst, de geanimeerde vergaderingen, de casussen. Ik ben blij dat ik nog enkele maanden kan en mag blijven meedraaien.

Ook de veehouders die hebben meegewerkt aan m’n onderzoek wil ik langs deze weg bedanken. De mensen van de proefhoeve (Professor Christiaens, Dr. Eeckhout, Karel en André) hebben onze aanwezigheid heel vaak moeten tolereren. Tientallen keren hebben ze geholpen bij het apart zetten van de koeien en bij het uitvoeren van de transvaginale follikelvocht aspiraties.

Juliën van Overbeke, Etienne De Wilde en Dr. Marc Van Hoye dank jullie wel omdat ik van jullie vele jaren geleden de ware stiel geleerd heb: omgaan met koeien, melken, voederen, met de traktor rijden, dieren leren observeren, ziektes diagnosticeren, enz ...

Dankzij het vertrouwen van de lamaklanten kon ik af en toe m’n gedachten eens verzetten. Toon en Jeroen (Ablynx), dank je wel voor de heel aangename samenwerking. Ik hoop dat die nog lang kan blijven bestaan. Maar ook Ignace en Annemarie wil ik bedanken voor het vertrouwen en voor de steun bij het zoeken naar een goed gebouwd huis. Gelukkig werd er sinds onze weddenschap geen lamaveulen meer geboren!

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Buurman, bedankt om tijdens mijn afwezigheden het reilen en zeilen in onze Weldadigheidstraat in de gaten te houden. Veiligheid voor alles. De zwakken moeten er zowiezo van tussen. Sam, bedankt voor de vele prachtige foto’s en de fijne leuke whisky-babbels. Een dank je wel voor al m’n vrienden en familie voor de vele gezellige momenten tijdens de voorbije jaren.

En tenslotte, m’n Pien, m’n lief, m’n vrouw, m’n metgezel, m’n zielsverwant en onvoorwaardelijke reisgenoot. Vele jaren geleden heb je m’n gezicht terug naar de warme zonnestralen gericht en me met zachte druk in m’n rug verder richting een veelbelovende toekomst geduwd. Samen hebben we letterlijk heel wat hoogtes en laagtes overwonnen, heel wat spannende avonturen beleefd, ja zelfs voor ons leven gevreesd ... En zie waar we nu staan, al die jaren later ... prachtig toch. Dank je wel voor je enorm doorzettingsvermogen, je ‘volharden in de boosheid’, je nuchter relativeringsvermogen. Ik hoop uit het diepst van m’n hart dat ik ook de volgende jaren evenveel tijd met m’n vrouwen kan blijven doorbrengen, evenveel leuke reisen kan ondernemen. Enfin, we zien wel.

Dank je wel voor onze prachtige dochter Helene, voor de fijne opvoeding die we onze pientere krullekop willen geven en voor de vaak ondergewaardeerde opofferingen die je er voor doet. En die mooie, sexy bolle buik die je onder je kleren draagt, belooft heel wat goeds ... dat weet ik wel zeker.

Katrien, ik zie je graag.

Jo

vrijdag 16 september 2005

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Curriculum vitae

Jo (Leo Moniek René) Leroy werd op 6 juni 1977 geboren te Deinze. Na het behalen

van zijn diploma van het hoger secundair onderwijs als “primus perpetuus” aan het Sint-

Hendrikscollege te Deinze (Grieks-Latijn), begon hij zijn studies Diergeneeskunde in Gent.

Hij behaalde zijn diploma van Dierenarts in 2001 met grootste onderscheiding en kreeg

hiervoor de Prijs van de Faculteit Diergeneeskunde voor de bijzondere wijze van

onderscheiden tijdens de studies in de Diergeneeskunde (6 juli 2001). Zijn afstudeerwerk

handelde over het voorkomen en het belang van (sub)klinische mastitis bij vaarzen prepartum.

In een eigen onderzoek werd er gezocht naar het belang en voorkomen van Staphylococcus

chromogenes in de tepeltopkolonisatie en intramammaire infectie bij jonge, drachtige en pas

afgekalfde vaarzen (Vakgroep Voortplanting Verloskunde en Bedrijfsdiergeneeskunde,

Promotor: Dr. S. De Vliegher). Dit eindwerk werd bekroond met een prijs geschonken door

de firma Pharmacia Animal Health (6 juli 2001).

Na wekenlange omzwervingen op het Zuidamerikaanse continent trad hij in dienst van

de Vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde op 1 oktober 2001.

Sinds 1 januari 2002 werkte hij als doctoraatsbursaal op een specialisatiebeurs die werd

gefinancieerd door het Instituut voor de Aanmoediging van Innovatie door Wetenschap en

Technologie in Vlaanderen (IWT-Vlaanderen). Deze doctoraatsbeurs leidde een kleine 4 jaar

later tot dit proefschrift. In het najaar van 2005 werd tevens het postgraduaatsdiploma van de

doctoraatsopleiding in de diergeneeskundige wetenschappen behaald.

Gedurende de 4 jaar op de Vakgroep was Jo Leroy werkzaam in de buitenpraktijk en

participeerde hij in de dag-, nacht- en weekenddiensten. Eveneens legde hij zich toe op de

geneeskunde van de kleine cameliden. Daarnaast was hij meerdere malen spreker op

studiedagen in binnen- en buitenland. Zijn studieresultaten heeft hij 7 maal mondeling mogen

verdedigen op internationale symposia. In 2004 won hij de Student Competition voor de beste

abstract, poster en orale presentatie op de twintigste meeting van de ‘European Society of

Embryo Transfer’ in Lyon (10-12 September 2004). Tevens werd hij twee maal uitgenodigd

als spreker op een internationaal congres (‘International Congress on Animal Reproduction’

2004, Porto Seguro, Brazilië; het congres van de ‘Brasilian Society of Embryo Technologies’

2005, Angra dos Reis, Brazilië). Tenslotte is hij auteur en co-auteur van 19 publicaties in

nationale en internationale tijdschriften en van 32 abstracts in de proceedings van

internationale congressen.

Curriculum Vitae - Publicatons

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Publications

International Journals

o S De Vliegher, H Laevens, LA Devriese, G Opsomer, JLMR Leroy, HW Barkema, A de

Kruif. 2003. Prepartum teat apex colonization with Staphylococcus chromogenes in dairy

heifers is associated with low somatic cell count in early lactation. Veterinary

Microbiology 92: 245-52.

o PEJ Bols, JLMR Leroy, T Vanholder, A Van Soom. 2004. A comparison of a

mechanical sector and a linear array transducer for ultrasound-guided transvaginal oocyte

retrieval (OPU) in the cow. Theriogenology 62: 906-914.

o JLMR Leroy, T Vanholder, JR Delanghe, G Opsomer, A Van Soom, PEJ Bols, A de

Kruif. 2004. Metabolite and ionic composition of follicular fluid from different-sized

follicles and their relationship to serum concentrations in dairy cows. Animal

Reproduction Science 80: 201-211.

o A Van Soom, B Mateusen, J Leroy, A de Kruif. 2003. Assessment of mammalian embryo

quality: what can we learn from embryo morphology? Reproductive Biomedicine Online

www.rbmonline.com/article/982. 7: 96-102.

o JLMR Leroy, T Vanholder, JR Delanghe, G Opsomer, A Van Soom, PEJ Bols, J Dewulf,

A de Kruif. 2004. Metabolic changes in follicular fluid of the dominant follicle in high-

yielding dairy cows early post partum. Theriogenology 62: 1131-1143.

o T Vanholder, JLMR Leroy, G Opsomer, A Vansoom, A de Kruif. 2005. Effect of non-

esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

Animal Reproduction Science 87: 33-44.

o YQ Yuan, A Van Soom, JLMR Leroy, J Dewulf, A Van Zeveren, A de Kruif, LJ

Peelman. 2005. Apoptosis in cumulus cells, but not in oocytes, may influence bovine

embryonic developmental competence. Theriogenology 63: 2147-2163.

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251

o G Genicot*, JLMR Leroy*, A Van Soom, I Donnay. 2005. The use of a fluorescent dye,

Nile Red, to evaluate the lipid content of single mammalian oocytes. Theriogenology 63:

1181-1194. *Both authors equally contributed to this work.

o JLMR Leroy, G Genicot, I Donnay, A Van Soom. 2005. Evaluation of the lipid content

in bovine oocytes and embryos with Nile red: a practical approach. Reproduction in

Domestic Animals 40: 76-78.

o T Vanholder, JLMR Leroy, J Dewulf, L Duchateau, M Coryn, A de Kruif. 2005.

Hormonal and metabolic profiles of high-yielding dairy cows prior to ovarian cyst

formation or first ovulation post partum. Reproduction in Domestic Animals 40: 460-467.

o JLMR Leroy, PEJ Bols, G Opsomer, A Van Soom, A de Kruif. 2005. Aspiração de

oócitos (ovum pick-up), a técnica ideal para o estudo do ambiente intra-follicular e da

qualidade oocitária em vacas de leite de alta produção. Acta Scientiae Veterinariae 33

(Suppl 1): 5-18.

o PEJ Bols, JLMR Leroy, JHM Viana. 2005. Aspectos técnicos e biológicos na

recuperação de oócitos via trans-vaginal guiada por ultra-som em vacas. Acta Scientiae

Veterinariae 33 (Suppl 1): 103-118.

o JLMR Leroy, G Opsomer, S De Vliegher, T Vanholder, L Goossens, A Geldhof, PEJ

Bols, A de Kruif, A Van Soom. Comparison of embryo quality in high-yielding dairy

cows, in dairy heifers and in beef cows. Theriogenology, In Press.

o JLMR Leroy, T Vanholder, B Mateusen, A Christophe, G Opsomer, A de Kruif, G

Genicot, A Van Soom. 2005. Non-esterified fatty acids in follicular fluid of dairy cows

and their effect on developmental capacity of bovine oocytes in vitro. Reproduction 130:

485-495.

o T Vanholder, JLMR Leroy, A Van Soom, D Maes, M Coryn, T Fiers, A de Kruif, G

Opsomer. Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and

proliferation in vitro. Animal Reproduction Science, In Press.

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252

o T Vanholder, JLMR Leroy, A Van Soom, M Coryn, A de Kruif, G Opsomer. Effects of

β-OH butyrate on bovine granulosa and theca cell function in vitro. Reproduction in

Domestic Animals, In Press.

o T Vanholder, K Goossens, LJ Peelman, JLMR Leroy, M Coryn, A de Kruif, G Opsomer.

mRNA transcription levels of insulin receptor isoforms A and B, insulin-like growth

factor receptors 1 and 2, and luteinizing hormone receptor in follicular cysts and dominant

follicles in the bovine. Reproduction, Submitted.

o JLMR Leroy, T Vanholder, G Opsomer, A Van Soom, A de Kruif. The in vitro

development of bovine oocytes after maturation in glucose and β-hydroxybutyrate

concentrations associated with negative energy balance in dairy cows. Reproduction in

Domestic Animals, In Press.

National Journals

o JL Leroy, T Geurden, G Meulemans, K Moerloose, A de Kruif. 2003. Severe Sarcoptes

scabiei infection in the llama. Flemish Veterinary Journal 72: 359-363.

o JL Leroy, T Flahou, K Moerloose, A de Kruif. 2004. Reproduction in the llama and

alpaca mare: a review. Flemish Veterinary Journal 73: 310-316.