Jo Leroy

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology.

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.

Assessment of mammalian embryo quality: what can we learn from embryo morphology?

Embryo morphology assessment, however imperfect it may be, is at present the most popular method for embryo selection prior to transfer, both in human and bovine assisted reproduction. A major difference between human and bovine embryos is the fact that in the latter, assessment of morphology is jeopardized by the opacity of the blastomeres, which is caused by lipid droplet accumulation. This opacity makes it difficult to assess nuclear and nucleolar morphology, aspects which can easily be evaluated in human zygotes or early cleaving embryos. However, recent research which focused on correlation between bovine embryo morphology and embryonic ultrastructure, gene expression and cryoresistance, has provided evidence that much more can be deduced from mere embryo morphology than previously thought. Morphological features such as colour of the blastomeres, the extent of compaction, timing of blastocyst formation and expansion and diameter of the embryo at hatching can be linked with embryo quality. On the other hand, cattle embryos of deviant chromosomal constitution or with aberrant genetic make-up cannot be selected against by means of the current morphological techniques. Possible solutions include the visualization of bovine pronuclei at the zygote stage by means of ultracentrifugation or multiphoton laser scanning microscopy, and adjustment of genetic analysis in order to reconstruct embryo genetic make-up starting from the biopsy material.

Intrafollicular conditions as a major link between maternal metabolism and oocyte quality: a focus on dairy cow fertility

Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing fertility in high producing dairy cows. This review aims to shed more light on the importance of the intrafollicular environment in the subfertility problem in dairy cows. Metabolic disturbances associated with negative energy balance (NEB) early postpartum are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle during a period of NEB can indirectly affect oocyte quality. Furthermore, a maternal metabolic disorder (linked with NEB or nutritionally induced) may alter the endocrine and biochemical composition of the follicular fluid, the micro-environment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes reduce the oocytes developmental competence. Also, embryo quality is significantly reduced due to maturation in adverse conditions. Well balanced and timed oocyte metabolism and gene expression are crucial to safeguard an optimal oocyte development. In that perspective, metabolome and transcriptome parameters of the oocyte may serve to predict reproductive success rates. Finally, there is growing evidence that adverse conditions for oocyte growth and maturation may also jeopardise the health and performance of the offspring.

Occurrence of endocrine disrupting compounds in tissues and body fluids of Belgian dairy cows and its implications for the use of the cow as a model to study endocrine disruption

The reproductive performance of high producing dairy cows has dropped severely throughout the last decades. It has already been suggested that the presence of endocrine disrupting compounds (EDCs) in the environment could be one of the reasons for this declining fertility. Reliable data concerning tissue and body fluid concentrations of these chemicals are thus crucial, but currently only scarcely available. Therefore, we selected dairy cows (≥6years) from diverse locations in Belgium and analysed tissues (liver, adipose tissue, muscle, kidney, and ovaria) and body fluids (serum, follicular fluid, and milk) for their content of potential EDCs, such as polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs). Furthermore, we collected milk and serum samples from high producing dairy cows 2-3weeks post-partum to verify if the massive lipolysis required to sustain milk production is accompanied with an increase in EDC concentrations in milk and serum. Overall, contamination was very low (median sum PCBs liver: 11.7ngg(-1) lw), with follicular fluid samples showing no detectable contamination. CB 153 was present in each tissue sample. Strong correlations could be found between EDCs in the same tissue. The increased PCB concentrations observed in milk samples from high producing dairy cows could indicate that massive lipolysis can play a role in liberating and thereby increasing EDC concentrations in milk. Because concentrations of the most prevalent EDCs in dairy cow tissues and body fluids are very low, exposure to EDCs can hardly be considered as a major cause of declining fertility in high producing dairy cows in Belgium. As a result of this low contamination and the similarities between the female bovine and human reproductive physiology, in vitro studies based on Belgian dairy cow ovarian follicles can be considered as a valuable model to study the effects of EDCs on human reproduction.

The association between indicators of inflammation and liver variables during the transition period in high-yielding dairy cows : an observational study

During the transition period, cows are confronted with infectious and inflammatory challenges leading to an acute phase response (APR) marked by increased hepatic synthesis of positive acute phase reactants (+AP) and a decrease in negative acute phase reactants (-AP). The aim of this study was to quantify the APR in 21 high-yielding dairy cows studied from 9 days before until 42 days after calving, and to assess the association between the APR, disease incidence and indicators of liver function. Repeated blood samples were analyzed for -AP (retinol, albumin, cholesterol), +AP (haptoglobin, caeruloplasmin), paraoxonase, and liver-associated variables (aspartate aminotransferase, γ-glutamyl transferase, bilirubin). All cows displayed postpartum decreases in -AP and paraoxonase, and increases in +AP and liver variables. When retrospectively categorized, cows presenting a stronger -AP decline displayed higher +AP and liver variables, and a higher disease incidence compared to cows with a milder decline. Altogether, typical changes in -AP and +AP identify the transition period as a time of increased inflammatory load. Group differences in liver variables suggest that a more severe APR may be associated with altered liver function. However, no causal relationship can be proven based on this observational dataset, and results should be interpreted cautiously.

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.

Fatty acid composition of the follicular fluid of normal weight, overweight and obese women undergoing assisted reproductive treatment : a descriptive cross-sectional study

BackgroundIt has been well documented that the maturing oocyte is very vulnerable to changes in its micro-environment, the follicular fluid (FF). Recent research has focused on different components within this FF, like hormones, growth factors and metabolites, and how their concentrations are altered by diet and the metabolic health of the mother. It has been proposed that fatty acids (FAs) are potential factors that influence oocyte maturation and subsequent embryo development. However, a thorough study of the specific FF FA composition per lipid fraction and how this may be affected by BMI is currently lacking. Therefore, we investigated the BMI-related concentration of FAs in the phospholipid (PL), cholesteryl-ester (CHE), triglyceride (TG) and non-esterified (NE) lipid fraction in the FF of women undergoing assisted reproductive treatment (ART).MethodsIn this descriptive cross-sectional study, the FF of normal weight (18.5u2009≤u2009BMIu2009<u200925.0xa0kg/m(2), nu2009=u200910), overweight (25.0u2009≤u2009BMIu2009<u200930.0xa0kg/m(2), nu2009=u200910) and obese (BMIu2009≥u200930.0xa0kg/m(2), nu2009=u200910) women, undergoing ART, was sampled and analyzed for 23 specific FAs in the PL, CHE, TG and NEFA fraction, using a gas chromatographic analysis method. Differences between BMI-groups were studied by means of univariate general linear models and post hoc Sheffé tests.ResultsTotal FA concentrations in the PL and CHE fraction did not differ between BMI groups. Total TG concentrations tended to differ and total NEFA concentrations differed significantly between BMI groups. Interestingly, 42% and 34% of the total FAs was esterified in the PL and CHE fraction, respectively, while only 10% were present in both the TG and NEFA fraction. Only few individual FA concentrations differed in the PL, CHE and TG fraction between BMI groups, whereas abundant BMI-related differences were found in the NEFA fraction.ConclusionsOur data show that differences in BMI are associated with alterations in the FA composition of the FF, an effect most pronounced in the NEFA fraction. These BMI-related variations could possibly affect granulosa cell viability, oocyte developmental competence and subsequent embryo quality possibly explaining differences in oocyte quality in obese patients described by others.

Circadian rhythm of metabolic changes associated with summer heat stress in high-producing dairy cattle

The current study aimed to investigate the circadian rhythm of blood metabolic parameters associated with summer heat stress (HS) in dairy cows. Ten healthy lactating Holstein Friesian cows were followed during HS for three successive days at six different time points. Blood was sampled from each cow starting from 07:00u2009am; at 4-h intervals. Ambient air temperature and relative humidity were recorded, and temperature-humidity index (THI) was calculated as well. Respiration rate (RR) and rectal temperature (RT) were recorded for each cow at the time of blood sampling. Concentrations of glucose, non-esterified fatty acids (NEFA), total cholesterol (TC) and urea were measured in each blood sample. The THI values were >68 at all times of the day, and the highest values were recorded at 11:00u2009am, 03:00u2009pm and 07:00u2009pm (80.9, 83.7, and 80.8, respectively). All the cows showed a significantly higher RR and RT coinciding with higher THI values (93u2009±u20094 and 39.6u2009±u20090.1; 90.2u2009±u20093.4, and 40.1u2009±u20090.1; 87.6u2009±u20094.1, and 39.8u2009±u20090.1, respectively, Pu2009<u20090.05). The concentrations of glucose were the lowest at 11:00u2009am and 03:00u2009pm (3.75u2009±u20090.1 and 3.44u2009±u20090.1xa0mmol/L, respectively, Pu2009<u20090.05). Decreased glucose concentrations coincided with increased NEFA concentrations, (0.43u2009±u20090.01 and 0.56u2009±u20090.02xa0mmol/L, respectively, Pu2009<u20090.05), and were highly negatively correlated (ru2009=u2009−0.50, Pu2009<u20090.001). The highest urea and TC concentrations were registered at 11:00u2009am (6.11u2009±u20090.15xa0mmol/L and 109.9u2009±u20092.2xa0mg/dl, respectively) whereas the lowest urea and TC values were recorded at 03:00u2009am (4.97u2009±u20090.18xa0mmol/L and 99.5u2009±u20091.7xa0mg/dl, respectively, Pu2009<u20090.05). The results of the present study indicate that there was a circadian variation in glucose, NEFA, urea, and TC resulting in the most unfavorable metabolic condition during the hottest moment of the day in dairy cattle. Earlier work revealed that HS-metabolic changes are reflected in the follicular fluid. The circadian changes observed in the present study associated with HS may imply that also the microenvironment of the oocyte is affected.

Reduced oocyte and embryo quality in response to elevated non-esterified fatty acid concentrations: A possible pathway to subfertility?

Reproductive performance is increasingly considered as a barometer of general well-being of the mother. A normal maternal metabolic health status is essential to safeguard successful ovulation, conception and further embryo development. When alterations in serum metabolites are reflected in the oocyte and embryonic micro-environment, these metabolic changes can affect follicle health, oocyte development and even subsequent embryo physiology. The search continues for signals that may be critically affecting the early developmental stages in life. Years of expertise in animal in vitro embryo culture models contribute to the awareness on the influence of elevated non-esterified fatty acid (NEFA) concentrations on follicle cells, oocyte and embryo quality. High NEFA concentrations in the blood are known to alter the follicular micro-environment. The latter alterations in NEFA concentrations have been associated with a disappointing fertility outcome through disabled ovarian cell function and reduced oocytes developmental competence. Even more, elevated NEFA concentrations during bovine oocyte maturation influence the subsequent embryo characteristics. This review provides a cross-species overview on the consequences of elevated NEFA concentrations, originating from maternal lipolytic conditions, on female fertility, with particular focus on the early stages in life. Thereby, we will describe to what extent elevated serum NEFA concentrations are a potential threat around the period of conception.

A diet enriched in linoleic acid compromises the cryotolerance of embryos from superovulated beef heifers

Dietary rumen-protected fat rich in linoleic acid may affect the superovulatory response and embryo yield; however, its effects on in vivo embryo cryotolerance are unknown in zebu cattle. The present study evaluated the production and cryotolerance after freezing or vitrification of embryos from Nelore heifers supplemented with rumen-protected polyunsaturated fatty acids (PUFA). Forty heifers kept in pasture were randomly distributed into two groups according to the type of feed supplement (F, supplement with rumen-protected PUFA, predominantly linoleic; C, control fat-free supplement with additional corn). Supplements were formulated to be isocaloric and isonitrogenous. Each heifer underwent both treatments in a crossover design with 70 days between replicates. After 50 days feeding, heifers were superovulated. Embryos were evaluated morphologically and vitrified or frozen. After thawing or warming, embryo development was evaluated in vitro. There was no difference between the F and C groups (P>0.10) in terms of embryo production. Regardless of the cryopreservation method used, Group C embryos had a greater hatching rate after 72h in vitro culture than Group F embryos (44.3±4.2% (n=148) vs 30.9±4.0% (n=137), respectively; P=0.04). Moreover, vitrified and frozen embryos had similar hatching rates (P>0.10). In conclusion, dietary rumen-protected PUFA rich in linoleic acid did not improve embryo production and compromised the cryotolerance of conventionally frozen or vitrified embryos from Nelore heifers.