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“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
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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 n 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 Ren 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


    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 1Chapter 2 Aims of the Study 13Chapter 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 -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 197Summary 219Samenvatting 229Acknowledgments- Dankwoord 239Curriculum Vitae - Publicaties 247

  • List of Abbreviations

    AI artificial insemination ANOVA analysis of variance BB Belgian Blue beef cows BCS body condition score -OHB -hydroxybutyrate BSA bovine serum albumine COC cumulus oocyte complex DMEM dulbeccos modified eagle medium DMSO dimethyl sulfoxide E estradiol 17 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


  • General Introduction


    Bovine milk and dairy products have always been an appreciated part of mans 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; Lpez-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 Grhn, 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 cows adaptation

    during a period of NEB is to shift the bodys 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


    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 -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







    Adipose tissue


    Alternative energy source in peripheral tissue


    Acetyl CoA


    Fatty Acids

    Triglycerides VLDL Lipid


    Oxaloacetate Krebs Cycle



    DIET: Proprionate



  • General Introduction


    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


    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). OCallaghan 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 oocytes 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 -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 -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


    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


  • General Introduction


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    embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Molecular Reproduction and Development 61: 5766.

    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.

    Bilodeau-Goeseels S, Kastelic JP. 2003. Factors affecting embryo survival and strategies to reduce embryonic mortality in cattle. Canadian Journal of Animal Science 83: 659-671.

    Boland MP, Lonergan P, OCallaghan 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 Mdecin Vtrinaire 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, OFarrell 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, OSullivan 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


    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.

    Lpez-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: 415427.

    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, Qubec, 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.

    OCallaghan D, Boland MP. 1999. Nutritional effects on ovulation. Animal Science 68: 299314. 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, Grhn 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


    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 researchers 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, OFarrell KJ, Diskin M, Wylie ARG, OCallaghan 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, Overstrm EW, Duby RT, Herrmann D, Watson AJ, Niemann H, OCallaghan 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


  • Aims of the Study


    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 -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


  • Chapter 3: Reduced Fertility in Dairy Cows A Review



    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 (OCallaghan 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


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

    Author Reduced oocyte quality (yes/no)


    Kruip et al., 1995

    Yes After a more profound NEB oocytes 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 oocytes 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


    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


    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 oocytes 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


    stage and ooplasm (Revah and Butler, 1996; Yaakub et al., 1997; OCallaghan 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 oocytes 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


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



    NEBMetabolic Endocrine

    Elongated conceptus

    Expanded blastocyst

    Compact morula


    NUTRITIONEnergy and Protein

    Metabolic Endocrine

    60 90 days

    Carry over effects on oocyte and follicle quality

    Carry over effects on CL function

    High progesterone metabolism in the liver





    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


    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


    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; Lequarr

    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


    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 oocytes developmental competence (Yding-Anderson,


    During normal follicular growth 17-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


    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 (Bge, 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


    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 oocytes nuclear and cytoplasmatic maturation and can enhance cumulus cell health and

    proliferation (Klle 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


    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, -hydroxybutyrate (-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


    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


    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 oocytes 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 oocytes 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


    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 -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. -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 PGF2 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


    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


    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


    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 (OCallaghan 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


    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


    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: OCallaghan

    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

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    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 (OCallaghan 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


    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.


    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.

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    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


    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


    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 PGF2 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


    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


    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 hypothalamuspituitaryovary

    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.


    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


    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



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