Obese outbred mice only partially benefit from diet normalization or calorie restriction as preconception care interventions to improve metabolic health and oocyte quality.

STUDY QUESTION: Can diet normalization or a calorie-restricted diet for 2 or 4 weeks be used as a preconception care intervention (PCCI) in Western-type diet-induced obese Swiss mice to restore metabolic health and oocyte quality? SUMMARY ANSWER: Metabolic health and oocyte developmental competence was already significantly improved in the calorie-restricted group after 2 weeks, while obese mice that underwent diet normalization showed improved metabolic health after 2 weeks and improved oocyte quality after 4 weeks. WHAT IS KNOWN ALREADY: Maternal obesity is linked with reduced metabolic health and oocyte quality; therefore, infertile obese women are advised to lose weight before conception to increase pregnancy chances. However, as there are no univocal guidelines and the specific impact on oocyte quality is not known, strategically designed studies are needed to provide fundamental insights in the importance of the type and duration of the dietary weight loss strategy for preconception metabolic health and oocyte quality. STUDY DESIGN, SIZE, DURATION: Outbred female Swiss mice were fed a control (CTRL) or high-fat/high-sugar (HF/HS) diet. After 7 weeks, some of the HF mice were put on two different PCCIs, resulting in four treatment groups: (i) only control diet for up to 11 weeks (CTRL_CTRL), (ii) only HF diet for up to 11 weeks (HF_HF), (iii) switch at 7 weeks from an HF to an ad libitum control diet (HF_CTRL) and (iv) switch at 7 weeks from an HF to a 30% calorie-restricted control diet (HF_CR) for 2 or 4 weeks. Metabolic health and oocyte quality were assessed at 2 and 4 weeks after the start of the intervention (n = 8 mice/treatment/time point). PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in body weight were recorded. To study the impact on metabolic health, serum insulin, glucose, triglycerides, total cholesterol and alanine aminotransferase concentrations were measured, and glucose tolerance and insulin sensitivity were analyzed at PCCI Weeks 2 and 4. The quality of in vivo matured oocytes was evaluated by assessing intracellular lipid droplet content, mitochondrial activity and localization of active mitochondria, mitochondrial ultrastructure, cumulus cell targeted gene expression and oocyte in vitro developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: Significant negative effects of an HF/HS diet on metabolic health and oocyte quality were confirmed (P < 0.05). HF_CTRL mice already showed restored body weight, serum lipid profile and glucose tolerance, similar to the CTRL_CTRL group after only 2 weeks of PCCI (P < 0.05 compared with HF_HF) while insulin sensitivity was not improved. Oocyte lipid droplet volume was reduced at PCCI Week 2 (P < 0.05 compared with HF_HF), while mitochondrial localization and activity were still aberrant. At PCCI Week 4, oocytes from HF_CTRL mice displayed significantly fewer mitochondrial ultrastructural abnormalities and improved mitochondrial activity (P < 0.05), while lipid content was again elevated. The in vitro developmental capacity of the oocytes was improved but did not reach the levels of the CTRL_CTRL mice. HF_CR mice completely restored cholesterol concentrations and insulin sensitivity already after 2 weeks. Other metabolic health parameters were only restored after 4 weeks of intervention with clear signs of fasting hypoglycemia. Although all mitochondrial parameters in HF_CR oocytes stayed aberrant, oocyte developmental competence in vitro was completely restored already after 2 weeks of intervention. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we applied a relevant HF/HS Western-type diet to induce obesity in an outbred mouse model. Nevertheless, physiological differences should be considered when translating these results to the human setting. However, the in-depth study and follow-up of the metabolic health changes together with the strategic implementation of specific PCCI intervals (2 and 4 weeks) related to the duration of the mouse folliculogenesis (3 weeks), should aid in the extrapolation of our findings to the human setting. WIDER IMPLICATIONS OF THE FINDINGS: Our study results with a specific focus on oocyte quality provide important fundamental insights to be considered when developing preconception care guidelines for obese metabolically compromised women wishing to become pregnant. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO-SB grant 1S25020N and FWO project G038619N). The authors declare there are no conflicts of interest.

Maternal metabolic health and fertility: we should not only care about but also for the oocyte!

Metabolic disorders due to obesity and unhealthy lifestyle directly alter the oocyte's microenvironment and impact oocyte quality. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis. Acute effects on the fully grown oocytes are evident, but early follicular stages are also sensitive to metabolic stress leading to a long-term impact on follicular cells and oocytes. Improving the preconception health is therefore of capital importance but research in animal models has demonstrated that oocyte quality is not fully recovered. In the in vitro fertilisation clinic, maternal metabolic disorders are linked with disappointing assisted reproductive technology results. Embryos derived from metabolically compromised oocytes exhibit persistently high intracellular stress levels due to weak cellular homeostatic mechanisms. The assisted reproductive technology procedures themselves form an extra burden for these defective embryos. Minimising cellular stress during culture using mitochondrial-targeted therapy could rescue compromised embryos in a bovine model. However, translating such applications to human in vitro fertilisation clinics is not simple. It is crucial to consider the sensitive epigenetic programming during early development. Research in humans and relevant animal models should result in preconception care interventions and in vitro strategies not only aiming at improving fertility but also safeguarding offspring health.

Effect of overconditioning on the hepatic global gene expression pattern of dairy cows at the end of pregnancy.

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 ± 0.29 cm; least squares means ± standard error) and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to overconditioned cows (3.14 ± 0.43 cm and 0.38 ± 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

Capacity of Trolox to improve the development and quality of metabolically compromised bovine oocytes and embryos invitro during different windows of development.

Trials to improve oocyte developmental competence under metabolic stress by using antioxidants may start before or after oocyte maturation. In the present conceptual study, we aimed to identify the most efficient timing of antioxidant application in relation to a metabolic insult using a bovine invitro embryo production model. Pathophysiological concentrations of palmitic acid (PA) were used to induce metabolic stress during oocyte maturation or embryo development. Trolox (TR; antioxidant) treatment prior to, during or after the PA insult was tested to evaluate the protective, neutralising and rescuing capacity of TR respectively. Changes in embryo developmental competence, mitochondrial activity, reactive oxygen species (ROS) concentrations, blastocyst cell allocation and apoptosis and cell stress-related gene expression were monitored. The improvement in developmental capacity was most obvious when oocytes were preloaded with TR before the PA insult. This protective effect could be explained by the observed combination of increased mitochondrial activity with reduced ROS production. This resulted in blastocysts with normal cell counts and apoptosis, as well as increased nuclear factor erythroid 2-related factor 2 (NRF2) expression (a marker for redox regulatory processes) and normalised the expression of the mitochondrial transcription factor A (TFAM), a marker of mitochondrial biogenesis. These results indicate that 'pretreatment' of oocytes with antioxidants produces embryos that seem to be more resilient to a metabolic stress insult.

Altered embryotrophic capacities of the bovine oviduct under elevated free fatty acid conditions: an in vitro embryo--oviduct co-culture model.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

Is the pre-antral ovarian follicle the 'holy grail'for female fertility preservation?

Fertility preservation is not only a concern for humans with compromised fertility after cancer treatment. The preservation of genetic material from endangered animal species or animals with important genetic traits will also greatly benefit from the development of alternative fertility preservation strategies. In humans, embryo cryopreservation and mature-oocyte cryopreservation are currently the only approved methods for fertility preservation. Ovarian tissue cryopreservation is specifically indicated for prepubertal girls and women whose cancer treatment cannot be postponed. The cryopreservation of pre-antral follicles (PAFs) is a safer alternative for cancer patients who are at risk of the reintroduction of malignant cells. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation, or research purposes. Vitrification is being used more and more as it seems to yield better results compared to slow freezing, although protocols still need to be optimized for each specific cell type and species. Several methods can be used to assess follicle quality, ranging from simple viability stains to more complex xenografting procedures. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. This review will focus on recent developments in (human) fertility preservation strategies, which are often accomplished by the use of in vitro animal models due to ethical considerations and the scarcity of human research material.

The effect of elevated non-esterified fatty acid concentrations on bovine spermatozoa and on oocyte in vitro fertilisation.

Elevated non-esterified fatty acid (NEFA) concentrations, present in follicular and oviductal fluid, have been postulated as a causative link between metabolic disorders and subfertility. High NEFA conditions can directly disrupt oocyte maturation and developmental capacity after fertilisation. However, their influence on sperm function and the fertilisation process is not known. This study investigated the fertilisation process under high NEFA conditions. To differentiate between effects on both spermatozoa and oocytes or on spermatozoa only, different experiments were conducted. In the first experiment both gametes were simultaneously incubated during IVF under different conditions: (1) NEFA-free, solvent-free control conditions, (2) solvent control, (3) physiological concentrations of oleic (OA), palmitic (PA) and stearic (SA) acids or (4) pathophysiological concentrations of OA, PA and SA. In the second experiment spermatozoa were incubated (4h) under the same treatment conditions prior to routine IVF. Gamete co-incubation resulted in reduced fertilisation and cleavage rates and increased prevalence of polyspermy. In the second experiment embryo developmental capacity and quality were not affected, although sperm motility and plasma membrane integrity were decreased. In conclusion, lipolytic conditions affected the fertilisation process mainly through an effect on the oocyte. Spermatozoa were still able to fertilise even though these conditions reduced sperm function.

The Importance of the Periconception Period: Immediate Effects in Cattle Breeding and in Assisted Reproduction Such as Artificial Insemination and Embryo Transfer.

In livestock breeding, the successful outcome is largely depending on the "periconception environment" which, in a narrow sense, refers to the genital tract, where gametogenesis and embryogenesis occur. During these early stages of development, gametes and embryos are known to be particularly sensitive to alterations in their microenvironment. However, as the microenvironment somehow reflects what is going on in the external world, we must widen our definition of "periconception environment" and refer to all events taking place around the time of conception, including metabolic state and health and nutrition of the dam. In modern dairy cows that have to manage an optimal reproductive performance with continued growth and high milk yield, the periconception period is particularly challenging. The metabolic priority for growth and lactation is known to generate adverse conditions hampering optimal ovarian function, oocyte maturation, and development of embryo/fetus. In addition, by using artificial reproductive technologies (ARTs), gametes and/or embryos of livestock are exposed to unnatural conditions outside the male and female genital tract. Artificial insemination, the most widely used technique, is currently yielding pregnancy rates similar to natural mating, and calves produced by AI are equally viable after natural mating. In contrast, other ART, such as multiple ovulation and embryo transfer, have been reported to induce changes in gene expression and DNA methylation patterns with potential consequences for development.Finally, the "periconceptional" environment has been shown to not only influence the successful establishment of pregnancy but also the long-term health and productivity of the offspring. Hence, the optimization of management around the time of conception might open doors to improve animal production and product quality.

Differential effects of high and low glucose concentrations during lipolysis-like conditions on bovine in vitro oocyte quality, metabolism and subsequent embryo development.

Lipolytic metabolic conditions are traditionally associated with elevated non-esterified fatty acid (NEFA) concentrations, but may also be accompanied by hyperglycaemia in obesity or by hypoglycaemia during a negative energy balance status. Elevated NEFA concentrations disrupt oocyte and embryo development and quality, but little is known about whether the effects of lipolytic conditions on oocyte developmental competence are modulated by glucose availability. To answer this, bovine cumulus-oocyte complexes (COCs) were matured under different conditions: physiological NEFA (72µM) and normal glucose (5.5mM), pathophysiologically high NEFA (420µM) and normal glucose, high NEFA and high glucose (9.9mM), high NEFA and low glucose (2.8mM). Developmental potential, cumulus expansion and metabolism of COCs exposed to high NEFA and low glucose were affected to a greater extent compared with COCs matured under high NEFA and high glucose conditions. High NEFA and high glucose conditions caused a moderate increase in oocyte reactive oxygen species compared with their high NEFA and low glucose or control counterparts. Blastocyst metabolism and the transcriptome of metabolic and oxidative stress-related genes were not affected. However, both lipolytic conditions associated with hyper- or hypoglycaemia led to surviving embryos of reduced quality with regards to apoptosis and blastomere allocation.

Maternal metabolic stress may affect oviduct gatekeeper function.

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC-albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts.

BACKGROUND: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. RESULTS: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. CONCLUSIONS: Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.

The effect of a negative energy balance status on ß-carotene availability in serum and follicular fluid of nonlactating dairy cows.

Maternal metabolic pressure due to a cow's negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of ß-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocyte's micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.

Morphometrical analysis of preantral follicular survival of VEGF-treated bovine ovarian cortex tissue following xenotransplantation in an immune deficient mouse model.

The increasing number of cancer survivors the past decades, has sparked the need for fertility preservation strategies. Due to predominantly ethical constraints, human research material is scarce. A bovine in vitro model is a valuable alternative. Therefore, the following objectives were defined: 1) to xeno-graft bovine ovarian cortex tissue in immune deficient mice as a study-model for female fertility preservation strategies; 2) to stereologically quantify vascularization in Vascular Endothelial Growth Factor (VEGF)-treated and non-treated tissue; 3) to study preantral follicular survival in situ, after xenotransplantation. Bovine ovarian tissue strips were incubated with or without VEGF prior to grafting into female, neutered BALB/c-nu mice (n=16). Non-transplanted cortical tissue was used as a control. At time zero (control), two (2 weeks) and four (4 weeks) weeks after transplantation, grafts were retrieved and assessed by von Willebrand Factor and caspase-3 immunostaining. Data were analyzed using a linear mixed model. In the VEGF+ grafts, 31% of the follicles were considered 'alive' 2 weeks after transplantation, compared to only 17% in the VEGF- grafts (P<0.05). However, no difference could be detected 4 weeks after transplantation (P=0.76) with less follicles being considered 'alive' after transplantation (22%), compared to the control (47.5%) (P<0.05). Finally, the vascular surface density was significantly less in the grafts, irrespective of the transplantation period or the use of VEGF. Although the transplantation process overall negatively influenced the number of viable follicles and vascular density, VEGF exposure prior to transplantation can favor follicle survival during a 2 weeks transplantation period.

Elevated non-esterified fatty acid concentrations hamper bovine oviductal epithelial cell physiology in three different in vitro culture systems.

Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12-based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 µM NEFA) and a solvent control (0 µM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm(2)) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even associated with a significant TER reduction (-15 ± 10 Ω.cm(2); P < 0.05). Cell proliferation capacity showed a decreased cell migration with increasing NEFA concentrations but was irrespective of the exposure side. Bovine oviductal epithelial cell morphology was not affected. In conclusion, in an in vitro setting, NEFAs exert a negative effect on BOEC physiology but not morphology. Ultimately, these physiological alterations in its microenvironment may result in suboptimal development of the pre-implantation embryo and a reduced reproductive outcome in dairy cattle.

Morphologic characterization of isolated bovine early preantral follicles during short-term individual in vitro culture.

To provide new insights in the molecular mechanism controlling preantral follicular development and to unravel the needs to support in vitro follicular development of early-stage preantral follicles (PAFs), there is a need for alternative in vitro bovine follicle culture methods. In this study, we aimed to characterize follicular dynamics using an IVC system of isolated and individually cultured bovine early PAFs during 10 days to generate individual follicle follow-up data. Preantral follicles (<50 µm) were isolated from slaughterhouse ovaries and cultured individually for 10 days. Individual follicle morphology, growth, survival, quality, and cell proliferation were evaluated in time by combining noninvasive and invasive assessment methods. The PAFs were light microscopically evaluated during culture to assess follicular dynamics, stained with neutral red to determine follicle viability, stained with 4',6-diamidino-2-phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling to evaluate cell proliferation and follicle quality, and processed for histologic evaluation to assess follicle morphology. On the basis of their morphology, follicles were subdivided in three categories, with category 1 follicles showing the best morphologic features. On Day 0, only category 1 follicles were selected, but follicle categories were reassigned on evaluation Days 1, 2, 4, 7, or 10. Although 67% of the follicles survived 10 days of IVC, the number of follicles exhibiting a normal morphology decreased significantly from Day 7 onward and the apoptotic index increased significantly from Day 10. Both category 1 and 2 follicles showed a significant increase in follicular diameter (Day 10: 21.80 ± 0.86 and 11.82 ± 0.80, respectively). This increase in follicular diameter showed to be correlated with an increase in the total cell number. In conclusion, this culture system showed to support follicular development until Day 10, although the proportion of follicles showing normal morphologic features and the follicular quality decreased after 10 days of IVC. Follicles maintaining their category 1 morphologic features over time seem to be of a better quality and show a higher developmental competence as compared to category 2 and 3 follicles.

Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations.

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 µm for primordial follicles, 47 ± 6.3 µm for primary follicles and 67.1 ± 13.1 µm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles.

PURPOSE: Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles. METHODS: Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham's F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5% CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h. RESULTS: Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 µg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture. CONCLUSIONS: In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 µg/ml for 30 min, being perfectly compatible with PAF vitrification.

Dietary fat supplementation and the consequences for oocyte and embryo quality: hype or significant benefit for dairy cow reproduction?

In many countries, fat supplementation in the diet has become common in the dairy industry. There are several ideas as to how dietary fat could influence reproductive performance. Saturated fatty acids, such as palm oil, can increase milk yield but may aggravate negative energy balance and thus may impair fertility when fed during the first week post-partum. However, priming the lipid oxidation in the liver by feeding saturated fats during the dry period has recently been shown to be a potentially promising strategy to mitigate fat mobilization and liver accumulation post-partum. Furthermore, polyunsaturated fats (omega-3 fatty acids and conjugated linoleic acids) are fed to reduce the 'de novo' fat synthesis in the udder and thus the milk fat content, which may be of modest benefit for overall energy balance. Furthermore, omega-6 and omega-3 polyunsaturated fatty acids are reported to alter follicular growth, steroid synthesis and prostaglandin metabolism in the ovary and endometrium, respectively. Omega-6 fatty acids are believed to have pro-inflammatory and thus PGF2α-stimulating properties rendering them extra value as 'nutraceutical' early post-partum, while omega-3 fatty acids can weaken this inflammatory potency, leading to a higher chance of survival of the embryo when supplemented during the periconceptual period. Unfortunately, research results rarely provide a consensus in this perspective. The consequences of these fat-feeding strategies on oocyte and embryo quality remain an intriguing issue for debate. Fat feeding may alter the microenvironment of the growing and maturing oocyte of the early and older embryo and thus may affect reproductive outcome. We recently reported that dietary-induced hyperlipidaemic conditions can be harmful for embryo development and metabolism. However, to date, research results remain somewhat conflicting most probably due to differences in fat sources used, in diet and duration of supplementation and in experimental set-up in general.

Milk fat saturation and reproductive performance in dairy cattle.

Unsaturated fatty acids (UFA) cannot be synthesized by mammalian cells due to a lack of desaturase enzymes. Combined with their limited supply to the small intestines, UFA have been proposed as nutraceuticals to ameliorate dairy cow fertility. However, field studies based on a large number of animals are lacking on this subject. Therefore the aim of the present study was to analyze a large dataset containing individual cow fertility records from dairy herds and link fertility key-performance-indicators like conception rate to first insemination (CRFI), days in milk to first insemination (DIMFI) and days in milk to conception (DIMCONC), to the level of UFA in bulk tank samples, the latter being a proxy for the dietary fatty acid profile on these herds. Within the two year study period, information from 15,055 lactations and 35,433 bulk tank milk samples was collected on 90 herds. The multilevel logistic regression model used, revealed a decreased CRFI on herds with a higher bulk tank UFA level. The decrease in CRFI was larger for higher producing herds. Increased bulk tank UFA was furthermore associated with higher DIMFI which, together with the lower CRFI, subsequently increased DIMCONC. Interestingly, higher variability in UFA, expressed by an increased coefficient of variation, was associated with an increased CRFI and decreased DIMFI and DIMCONC. In conclusion, the present study demonstrates that increasing the UFA content of milk should not be a goal as such when supplementing UFA to dairy cows as higher bulk tank UFA are associated with worsened fertility results.