“de discussie van het nut van fundamenteel onderzoek …€œde discussie van het nut van...
TRANSCRIPT
“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
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
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
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General Introduction
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Lucy MC. 2003. Mechanisms linking nutrition and reproduction in postpartum cows. Reproduction Supplement 61: 415–427.
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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
10
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
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
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
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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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.
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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.
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Grummer, R.R., Carroll, D.J., 1988. A review of lipoprotein cholesterol metabolism: importance to ovarian function. J. Anim. Sci. 66, 3160-3173.
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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
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
91
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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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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
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
100
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
Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality
101
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
Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality
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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.
Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality
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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
Chapter 5A: Non-esterified Fatty Acids in Follicular Fluid and their Effect on Oocyte Quality
106
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
108
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
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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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Tanghe S, Van Soom A, Nauwynck H, Coryn M & de Kruif A 2002 Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev 61 14-24.
Vanholder T, Leroy JLMR, Vansoom 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.
Vernon RG 2002 Nutrient partitioning, lipid metabolism and relevant imbalances. In Proceedings of the 12th World Buiatrics Congress, 18-23 August 2002, Hannover, Germany, pp 210-223.
Walters AH, Pryor AW, Bailey TL, Pearson RE & Gwazdauskas FC 2002 Milk yield, energy balance, hormone, follicular and oocyte measures in early and mid-lactation Holstein cows. Theriogenology 57 949-961.
Wehrman ME, Welsh TH & Williams GL 1991 Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity. Biol Reprod 45 514-522.
Wise T 1987 Biochemical analysis of bovine follicular fluid: albumine, total protein, lysosomal enzymes, ions, steroids and ascorbic acid content in relation to follicular size, rank, atresia classification and day of estrous cycle. J Anim Sci 64 1153-1169.
Yao JK, Ryan RJ & Dyck PJ 1980 The porcine ovarian follicle. VI. Comparison of fatty acid composition of serum and follicular fluid at different developmental stages. Biol Reprod 22 141-147.
Zamboni L 1974 Fine morphology of the follicle cell-oocyte association. Biol Reprod 10 125-149. Zhou SL, Stump D, Isola L & Berk PD 1994 Constitutive expression of a saturable transport system
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postpartum dairy cows. J Dairy Sci 78 1909-1920.
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
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
128
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.
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.
Chapter 6A: Lipid Evaluation of Single Oocytes
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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
149
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
151
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
152
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
153
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
154
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
155
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
156
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
159
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).
Chapter 6A: Lipid Evaluation of Single Oocytes
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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.
Chapter 6A: Lipid Evaluation of Single Oocytes
162
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
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
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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184
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.
Chapter 7: Embryo Quality in Dairy Cows
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0
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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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Chapter 7: Embryo Quality in Dairy Cows
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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
Chapter 7: Embryo Quality in Dairy Cows
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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
Chapter 7: Embryo Quality in Dairy Cows
189
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
Chapter 7: Embryo Quality in Dairy Cows
190
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.
Chapter 7: Embryo Quality in Dairy Cows
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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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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
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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
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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
Summary
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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
Summary
225
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
Summary
226
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.
Samenvatting
231
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
Curriculum Vitae - Publications
249
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
250
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.
Curriculum Vitae - Publications
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.
Curriculum Vitae - Publicatons
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.