J. L. M. R. Leroy

Reduced Fertility in High-yielding Dairy Cows: Are the Oocyte and Embryo in Danger? Part I The Importance of Negative Energy Balance and Altered Corpus Luteum Function to the Reduction of Oocyte and Embryo Quality in High-yielding Dairy Cows*

Fertility in high yielding dairy cows is declining, and there is increasing evidence to presume that oocyte and embryo quality are major factors in the complex pathogenesis of reproductive failure. In this report we present an overview of possible mechanisms linking negative energy balance (NEB) and deficiencies in oocyte and embryo developmental competence; specifically, in the high producing dairy cow. Changes in follicular growth patterns during a period of NEB can indirectly affect oocyte quality. The endocrine and biochemical changes, which are associated with NEB, are reflected in the microenvironment of the growing and maturing female gamete, and likely result in the ovulation of a developmentally incompetent oocyte. Even after an oocyte is successfully ovulated and fertilized, a full-term pregnancy is still not guaranteed. Inadequate corpus luteum function, associated with reduced progesterone, and probably also low insulin-like growth factor concentrations, can cause a suboptimal microenvironment in the uterus that is incapable of sustaining early embryonic life. This may partly account for the low conception rates and the high incidence of early embryonic mortality in high yielding dairy cows.

Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part II. Mechanisms linking nutrition and reduced oocyte and embryo quality in high-yielding dairy cows.

Dairy cow fertility has been declining during since the mid-80s and this has given rise to numerous scientific studies in which important parts of the pathogenesis are elucidated. Reduced oocyte and embryo quality are acknowledged as major factors in the widely described low conception rates and in the high prevalence of early embryonic mortality. Apart from the importance of the negative energy balance (NEB) and the associated endocrine and metabolic consequences, there is a growing attention towards the effect of the milk yield promoting diets which are rich in energy and protein. Starch-rich diets can improve the energy status and thus the ovarian activity in the early postpartum period but the oocyte and embryo quality can suffer from such insulinogenic diets. Supplementation of dietary fat has a similar dual effect with a beneficial stimulation of the ovarian steroid production while the oocyte and the embryo display an altered energy metabolism and excessive lipid accumulation. High-protein diets can elevate the ammonia and urea concentrations in the blood, leading to changed intrafollicular, oviductal and uterine environments. Oocytes and embryos are highly sensitive to such changes in their microenvironment, possibly leading to a disturbed maturation, fertilization or early cleavage. Several nutrition-linked mechanisms, through which oocyte and/or embryo quality can be affected in modern dairy cows, well after the period of NEB, are proposed and comprehensively reviewed in the present report.

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

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, 6-8 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, beta-hydroxybutyrate (beta-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, beta-OHB 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), beta-OHB (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), NEFA (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.

Nutrient Prioritization in Dairy Cows Early Postpartum: Mismatch Between Metabolism and Fertility?

For several decades, researchers worldwide report a decrease in fertility in high-yielding dairy cows, most probably based on conflicting metabolic and reproductive needs. The dairy herd managers success at improving milk production has been accompanied by a negative trend for the most visible reproductive parameters such as calving intervals, number of days open and number of inseminations needed per pregnancy. In parallel, many research groups studied the metabolic and endocrine factors that influence follicular growth and the developmental competence of oocytes and embryos. In the past, herd managers and reproductive biologists each tried to tackle the same problems with limited consultation. More recently, the situation has improved significantly and theriogenologists, nutritionists and veterinarians now conduct research in multidisciplinary teams. This review paper starts in a general way by discussing nutrient prioritization towards the udder to guarantee milk production and by describing interactions between the somatotropic and gonadotropic axis. It then focuses on the consequences of the negative energy balance on follicular growth and environment, oocyte and embryo quality, not only by summarizing the currently accepted hypotheses but also based on clear scientific evidence at the follicular level. All this, with one question in mind: is there a mismatch between metabolism and fertility and what can the dairy manager learn from research to tackle the problem of reduced fertility?

Prepartum teat apex colonization with Staphylococcus chromogenes in dairy heifers is associated with low somatic cell count in early lactation

A high number of dairy heifers freshen with udder health problems. The prevalence of teat apex colonization (TAC) with Staphylococcus chromogenes, one of the most widespread coagulase-negative staphylococci (CNS) in milk samples from freshly calved dairy heifers, was measured cross-sectionally in non-lactating heifers on eight commercial dairy farms in Belgium. The influence of age on this prevalence, and the association between teat apex colonization with S. chromogenes prepartum and quarter milk somatic cell count (SCC) in early lactation were studied. In total, 492 teat apices were sampled from 123 heifers. The age of the heifers varied from 8 to 34 months. Overall, 20% of the heifers had at least one teat apex colonized with S. chromogenes. Of all teats sampled, 10% were colonized with S. chromogenes. The chance of having at least one teat apex colonized with S. chromogenes increased with age of the heifer. The presence of prepartum teat apex colonization with S. chromogenes was not associated with intramammary infection (IMI) early postpartum with the same bacterium. On the contrary, teat apex colonization with S. chromogenes prepartum appeared to protect quarters in the first few days of lactation from having somatic cell count >or=200000cells/ml milk, commonly accepted as the threshold for intramammary infection.

The effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality

BACKGROUND Obesity is associated with female reproductive abnormalities. Hyperlipidaemia might alter the embryonic micro-environment and potentially result in reduced fertility. We aimed to induce hyperlipidaemia nutritionally and investigate the consequences of hyperlipidaemic culture conditions on bovine in vitro embryo development, embryo quality and gene expression patterns. METHODS Bovine zygotes (n = 1545) were cultured in synthetic oviductal fluid medium supplemented with serum from heifers (n = 3), each fed three successive dietary treatments: (i) control serum, following a hay-based diet, (ii) hyperlipidaemic serum, following a carbohydrate and protected palm-oil-rich diet (FatCh) or (iii) hyperlipidaemic serum, following a protected palm-oil-rich diet (Fat). Blastocysts were evaluated for development, cell count, picnotic and mitotic indexes and cryotolerance. Selected mRNA transcripts were measured by quantitative RT-PCR. RESULTS FatCh and Fat diets approximately doubled the total cholesterol concentrations, compared with controls (167.1 +/- 11.9, 150.0 +/- 12.8 versus 83.4 +/- 13.7 mg/dl, respectively, P < 0.05), and fatty acid concentrations (8146.60 +/- 214.61, 6935.56 +/- 1081.04 versus 3944.0 +/- 425.07 micromol/l, respectively, P < 0.05). Supplementation of culture media with FatCh and Fat serum significantly reduced blastocyst rates, compared with controls (27.8, 23.4% versus 36.2%, respectively, P < 0.01), total cell number (103.3 +/- 30.1, 95.6 +/- 28.2 versus 146.9 +/- 34.2, respectively, P < 0.01), mitotic index (1.3 +/- 1.1, 1.7 +/- 2.4 versus 3.6 +/- 2.2%, respectively, P < 0.01) and hatching rates after vitrification (20.4, 13.8 versus 35.7%, respectively, P = 0.03). Embryos in FatCh and Fat groups exhibited significantly higher mRNA levels for genes related to apoptosis and metabolism, compared with controls. CONCLUSIONS This combined in vivo and in vitro model indicates that the exposure of preimplantation embryos to hyperlipidaemic conditions may result in reduced embryo quality and developmental potential, possibly resulting in poorer fertility.

Biochemical changes in the follicular fluid of the dominant follicle of high producing dairy cows exposed to heat stress early post-partum

High yielding dairy cows experience a negative energy balance (NEB) early post-partum and it was hypothesized that this may be aggravated under summer heat stress (HS) conditions. In this study, which was performed in Egypt, 20 Holstein cows were followed during summer (n=10) and winter (n=10) seasons. All cows were multiparous and kept at the same herd. Blood was sampled from each cow starting 1 week before the expected calving date and then at 1-week intervals until week 6 post-partum. From week 2 to 6 post-partum follicular fluid was collected through transvaginal follicular fluid aspiration at 6 days intervals. Ambient air temperature (AT) and relative humidity (RH) were recorded and temperature-humidity index (THI) was calculated as well. Respiration rate (RR), rectal temperature (RT), and body condition score (BCS) were recorded for each cow at the time of blood sampling. Concentrations of glucose, insulin like growth factor-1 (IGF-1), non-esterified fatty acids (NEFA), urea and total cholesterol (TC) were measured in each blood and follicular fluid sample. All the cows showed a significantly higher RR and RT in summer (95.5+/-1.1 and 39.88+/-0.06, respectively) than in winter (43.89+/-0.61 and 38.94+/-0.07, respectively) (P<0.001). Body condition score loss during the early post-partum period was higher in summer than in winter (1.1+/-0.07 vs. 0.85+/-0.06 point, respectively) (P<0.001). The average dominant follicle diameter was significantly lower in summer than in winter during the period of negative energy balance (11.6+/-0.7mm vs. 15.3+/-1.2mm, respectively) (P<0.01). Under summer heat stress, the concentrations of glucose (2.98+/-0.07 and 2.19+/-0.04mmol/L), IGF-1 (106.7+/-2.9 and 99.0+/-3.4ng/ml) and TC (137.3+/-5.3 and 62.2+/-5.1mg/dl) in blood and FF, respectively, were significantly lower than winter concentrations by (0.17+/-0.03mmol/L, P<0.001 and 0.26+/-0.06mmol/L, P<0.001), (12.3+/-3.6ng/ml, P<0.001 and 9.0+/-2.7ng/ml, P<0.001) and (20.7+/-1.8mg/dl, P<0.001 and 7.3+/-1.1mg/dl, P<0.01), respectively. However, the concentrations of NEFA (0.68+/-0.14 and 0.22+/-0.02mmol/L) and urea (9.27+/-0.34 and 9.96+/-0.25mmol/L) in blood and FF, respectively, were significantly higher in summer compared to winter (0.50+/-0.08mmol/L, P<0.001 and 0.20+/-0.02mmol/L, P<0.001) and (8.77+/-0.23mmol/L, P<0.05 and 8.96+/-0.29mmol/L, P<0.001), respectively, throughout the experimental period. The results of the present study indicate that heat stress early post-partum aggravates NEB in high yielding dairy cows, reduces BCS, dominant follicle diameter and alters the biochemical concentrations in the follicular fluid of the dominant follicle which may result in inferior oocyte and granulosa cell quality and hence poorer fertility.

Interrelations Between Glucose-Induced Insulin Response, Metabolic Indicators, and Time of First Ovulation in High-Yielding Dairy Cows

High-yielding dairy cows are more susceptible to metabolic and reproductive disorders than low-yielding cows. Insulin plays a pivotal role in the development of both problems. In the present study, we aimed to assess the glucose-induced insulin responses of dairy cows at different time points relative to calving and to relate this to the metabolic status and the time of first ovulation. Twenty-three healthy, multiparous Holstein-Friesian cows with a high genetic merit for milk yield were studied from 14 d prepartum to 42 d postpartum. Intravenous glucose tolerance tests were performed on -14, 14, and 42 d relative to calving to evaluate the plasma insulin and glucose responses to a glucose load, as estimated by the peak concentration, the area under the curve (AUC), and the clearance rates of insulin and glucose. Blood samples were obtained at 3-d intervals and analyzed for glucose, insulin, and nonesterified fatty acids (NEFA). The time of first ovulation was defined by transrectal ultrasonography and plasma progesterone analysis. Glucose-induced insulin AUC and peak concentration decreased and glucose clearance increased during lactation compared with the dry period. Plasma NEFA concentrations were negatively related to insulin AUC and peak concentrations. Fourteen cows ovulated within 42 d postpartum, and the remaining 9 cows suffered from delayed resumption of ovarian function. Survival analysis demonstrated that cows with lower NEFA concentrations during the dry period tended to have earlier resumption of ovarian activity. In conclusion, our data suggest a decreased plasma insulin response to glucose postpartum in high-yielding dairy cows, possibly contributing to metabolic stress during the early postpartum period. It is hypothesized that NEFA impair glucose-induced insulin secretion in dairy cows. Additionally, our results suggest the importance of lipolysis during the transition period as a risk factor for delayed ovulation.

BMI-related metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment and the consequences for oocyte and embryo quality

STUDY QUESTION Is the metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment (ART) related to serum composition and BMI and is it associated with oocyte and embryo quality? SUMMARY ANSWER We showed that metabolic alterations in the serum are reflected in the follicular fluid and that some of these alterations may affect oocyte quality, irrespective of BMI. WHAT IS KNOWN ALREADY Many studies have focused on the effect of metabolic disorders, such as obesity and type 2 diabetes, on assisted reproduction outcomes. There are, however, only few studies focusing on the importance of the correlation between serum and follicular fluid compositions and the composition of the follicular fluid as the oocytes micro-environment, affecting its development and subsequent embryo quality. DESIGN, PARTICIPANTS AND SETTING In this prospective cohort study, patient information, fertility treatment outcome data, follicular fluid and serum were obtained from women undergoing ART. Patients were categorized according to their BMI (kg/m(2)) as normal (n = 60), overweight (n = 26) or obese (n = 20). Serum and follicular fluid samples were analyzed for urea, total protein, albumin, cholesterol, high-density lipoprotein cholesterol, triglycerides, non-esterified fatty acids, apolipoprotein A1, apolipoprotein B, glucose, lactate, C-reactive protein, insulin-like growth factor -1 (IGF-1), IGF-binding protein 3 (only in follicular fluid), free carnitine and total carnitine. Metabolite concentrations in serum and follicular fluid samples were correlated and were associated with BMI and fertility treatment outcome. MAIN RESULTS Most serum metabolite differences between patients were reflected in the follicular fluid (P < 0.05). Follicular fluid apolipoprotein A1 and follicular fluid total protein concentrations negatively affected oocyte quality parameters (P < 0.05). However, overall BMI-related associations were poor. BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION In this study, we included every patient willing to participate. Within this cohort, women with a BMI transcending 35 kg/m(2) were scarce (n = 2), because extremely overweight women are mostly advised to lose weight before starting ART. Furthermore, the number of patients in each BMI group was different, possibly masking associations between the metabolic composition of serum and follicular fluid and oocyte quality parameters. GENERALIZABILITY TO OTHER POPULATIONS There were significant associations indicating that metabolic changes in the serum are reflected in the follicular fluid, potentially affecting oocyte quality, irrespective of the patients BMI. For ethical reasons, this study only focused on women already in need of artificial reproductive treatment. From a metabolic point of view, we consider this cohort as a representative sample of all women of reproductive age. STUDY FUNDING This study was funded by the special research fund, university of Antwerp (BOF UA). None of the authors has any conflict of interest to declare.

Effect of cumulus cell coculture and oxygen tension on the in vitro developmental competence of bovine zygotes cultured singly

The customary practice in bovine in vitro embryo production (IVP) is to handle oocytes and embryos in groups; although there are several reasons for establishing an IVP system for individual embryos that allows for following a single oocyte from retrieval through development to the blastocyst stage. To date, reports of individual IVP are inconsistent, and in most cases, resulted in unsatisfactory blastocyst rates. The objective of this study was to develop an efficient system for routine in vitro culture of individual bovine embryos. Single culture of zygotes in 2 different culture volumes (20 and 500microL) yielded less than 3% blastocysts in experiment 1. In an attempt to improve these results, cumulus cells were added to the culture medium in experiment 2, after which blastocyst rates increased from 2.9 to 21.8% (P<0.05). The third experiment revealed that an atmospheric oxygen tension, which is commonly used with somatic cell coculture, was not beneficial during individual embryo-cumulus cell coculture, because it resulted in lower blastocyst rates (Odds ratio 0.57, P<0.001) and in lower blastocyst cell numbers (P<0.05), when compared to culture in 5% oxygen. Grouped vs. single culture and reduced oxygen tension did not have a significant effect on cleavage and hatching rates. In experiment 4, three different cumulus cell coculture conditions during individual culture were tested and compared with the cleavage, blastocyst and hatching rates, and cell number of group culture (73.2%, 36.4%, 66.7% and, 155.1+/-7.26, respectively). The outcome variables after individual embryo culture on a 5-day-old cumulus cell monolayer (74.1%, 38.2%, 71.9% and 133.4+/-9.16, respectively), and single culture in the presence of added cumulus cells (69.9%, 31.9%, 66.7% and 137.3+/-8.01, respectively) were not significantly different from those obtained after group culture (P<0.05). Though, individual culture in a cumulus cell conditioned medium significantly reduced both the cleavage (59.0%) and blastocyst rates (6.3%). These results demonstrate that single culture of bovine zygotes can be fully sustained by coculture with cumulus cells in a low oxygen environment; implementation of these findings in our IVP system produced blastocysts comparable in quantity and quality to those obtained by group culture. These results were consistently achieved after acquiring experience and expertise in the handling of single zygotes.