Follicular metabolic alterations are associated with obesity in mares and can be mitigated by dietary supplementation.

Obesity is a growing concern in human and equine populations, predisposing to metabolic pathologies and reproductive disturbances. Cellular lipid accumulation and mitochondrial dysfunction play an important role in the pathologic consequences of obesity, which may be mitigated by dietary interventions targeting these processes. We hypothesized that obesity in the mare promotes follicular lipid accumulation and altered mitochondrial function of oocytes and granulosa cells, potentially contributing to impaired fertility in this population. We also predicted that these effects could be mitigated by dietary supplementation with a combination of targeted nutrients to improve follicular cell metabolism. Twenty mares were grouped as: Normal Weight [NW, n = 6, body condition score (BCS) 5.7 ± 0.3], Obese (OB, n = 7, BCS 7.7 ± 0.2), and Obese Diet Supplemented (OBD, n = 7, BCS 7.7 ± 0.2), and fed specific feed regimens for ≥ 6 weeks before sampling. Granulosa cells, follicular fluid, and cumulus-oocyte complexes were collected from follicles ≥ 35 mm during estrus and after induction of maturation. Obesity promoted several mitochondrial metabolic disturbances in granulosa cells, reduced L-carnitine availability in the follicle, promoted lipid accumulation in cumulus cells and oocytes, and increased basal oocyte metabolism. Diet supplementation of a complex nutrient mixture mitigated most of the metabolic changes in the follicles of obese mares, resulting in parameters similar to NW mares. In conclusion, obesity disturbs the equine ovarian follicle by promoting lipid accumulation and altering mitochondrial function. These effects may be partially mitigated with targeted nutritional intervention, thereby potentially improving fertility outcomes in the obese female.

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation.

BACKGROUND: Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI. OBJECTIVES: We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI. MATERIALS AND METHODS: Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry. RESULTS: Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI. DISCUSSION AND CONCLUSION: Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

Phospholipase C Zeta 1 (PLCZ1): The Function and Potential for Fertility Assessment and In Vitro Embryo Production in Cattle and Horses.

Phospholipase C Zeta 1 (PLCZ1) is considered a major sperm-borne oocyte activation factor. After gamete fusion, PLCZ1 triggers calcium oscillations in the oocyte, resulting in oocyte activation. In assisted fertilization, oocyte activation failure is a major cause of low fertility. Most cases of oocyte activation failures in humans related to male infertility are associated with gene mutations and/or altered PLCZ1. Consequently, PLCZ1 evaluation could be an effective diagnostic marker and predictor of sperm fertilizing potential for in vivo and in vitro embryo production. The characterization of PLCZ1 has been principally investigated in men and mice, with less known about the PLCZ1 impact on assisted reproduction in other species, such as cattle and horses. In horses, sperm PLCZ1 varies among stallions, and sperm populations with high PLCZ1 are associated with cleavage after intracytoplasmic sperm injection (ICSI). In contrast, bull sperm is less able to initiate calcium oscillations and undergo nuclear remodeling, resulting in poor cleavage after ICSI. Advantageously, injections of PLCZ1 are able to rescue oocyte failure in mouse oocytes after ICSI, promoting full development and birth. However, further research is needed to optimize PLCZ1 diagnostic tests for consistent association with fertility and to determine whether PLCZ1 as an oocyte-activating treatment is a physiological, efficient, and safe method for improving assisted fertilization in cattle and horses.

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

BACKGROUND: The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). METHODS: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. RESULTS: An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. CONCLUSION: These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

L-carnitine enhances developmental potential of bovine oocytes matured under high lipid concentrations in vitro.

Maternal obesity elevates non-esterified fatty acids (NEFA) follicular concentrations. Bovine cumulus-oocyte complexes (COCs) matured in vitro under high NEFA have altered metabolism and reduced quality. Systemically, obesity promotes altered mitochondrial metabolism linked to L-carnitine insufficiency. We hypothesized that L-carnitine supplementation during IVM of bovine COCs in the presence of high NEFA would lessen the negative effects of exposure to excessive lipids on embryonic development and oxidative stress. COCs were collected from abattoir ovaries and matured in four groups: CON (control), LC (3 mM L-carnitine), HN (high NEFA: 200uM oleic, 150uM palmitic and 75uM stearic acid), and HNLC (HN and LC). Mature oocytes were assayed for aerobic and anaerobic metabolism utilizing oxygen and pH microsensors or fertilized in vitro (D0). Cleavage (D3) and blastocyst (D7, D8) rates were assessed. D3 embryos with ≥ 4 cells were stained for cytosolic and mitochondrial ROS. D8 blastocysts were assayed for gene transcript abundance of metabolic enzymes. Oocyte metabolism was not affected by IVM treatment. D3 formation of embryos with ≥ 4 cells were lower in LC or HN than CON or HNLC; blastocyst rates were greater for CON and lower for HN than LC and HNLC. D3 embryo mitochondrial and cytosolic ROS were reduced in HNLC when compared to other groups. IVM in HN altered blastocyst gene transcript abundance when compared to CON, but not LC or HNLC. In conclusion, supplementation with L-carnitine protects oocytes exposed to high NEFA during IVM and improves their developmental competence, suggesting that high lipid exposure may lead to L-carnitine insufficiency in bovine oocytes.

Oocyte metabolic function, lipid composition, and developmental potential are altered by diet in older mares.

Dietary supplementation is the most feasible method to improve oocyte function and developmental potential in vivo. During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant support (reproductive support supplement (RSS)) intended to increase antioxidant activity and lipid oxidation. RSS was modified for individual experiments with additional antioxidants or altered concentrations of n-3 to n-6 fatty acids. Oocytes from mares supplemented with RSS when compared to COB had higher basal oxygen consumption, indicative of higher aerobic metabolism, and proportionately more aerobic to anaerobic metabolism. In the second experiment, oocytes collected from the same mares prior to (CON) and after approximately 8 weeks of RSS supplementation had significantly reduced oocyte lipid abundance. In the final experiment, COB was compared to RSS supplementation, including RSS modified to proportionately reduce n-3 fatty acids and increase n-6 fatty acids. The ability of sperm-injected oocytes to develop into blastocysts was higher for RSS, regardless of fatty acid content, than for COB. We demonstrated that short-term diet supplementation can directly affect oocyte function in older mares, resulting in oocytes with increased metabolic activity, reduced lipid content, and increased developmental potential.

Effect of Mare Age on Transcript Abundance of Connexins-37 and -43, Zona Pellucida Proteins, and Sperm Binding.

Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment 1, oocytes, corona radiata, cumulus, and granulosa cells were collected from mares classified as young (4-12 years) or old (> 20 years). Transcript abundance was evaluated for connexins -37 (GJA4) and -43 (GJA1); zona pellucida glycoproteins 1, 2, 3, and 4 (ZP1, ZP2, ZP3, ZP4); Tubulin (TUBA1A), and equine chorionic gonadotropin ß. For experiment 2, oocytes that failed to cleave following intracytoplasmic sperm injection (ICSI) were stored in salt solution for up to 4 years and used for sperm binding assays. Transcript abundance for GJA1 was decreased in oocytes, corona radiata, and granulosa cells while GJA4 was decreased in cumulus cells from old compared to young mares. Additionally, ZP1 tended to be decreased in corona radiata and cumulus cells from old mares. Oocytes from old mares tended to bind less spermatozoa compared young mares. Oocytes that failed to cleave following ICSI can be used for sperm binding studies for up to 2 years without losses in sperm binding. Our findings suggest that maternal age may contribute to changes in cellular communication and the ZP that could impact sperm binding.

Equine maternal aging affects oocyte lipid content, metabolic function and developmental potential.

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.

Equine Aging and the Oocyte: A Potential Model for Reproductive Aging in Women.

Numerous similarities in reproductive aging have been documented between the mare and woman. Aging is associated with a decline in fertility. In mares and women, oocyte transfer procedures were initially used to establish that oocyte donor age is associated with oocyte quality. Age-associated differences in oocytes include altered morphology, gene expression, and developmental potential. Reactive oxygen species and mitochondrial dysfunction are thought to be important contributors to loss of oocyte quality. In the woman, aneuploidy is a primary consideration with maternal aging. Although misalignment of chromosomes during meiosis has been observed in the mare, less is known in this area. Reproductive aging will be reviewed in the mare and compared with the woman with emphasis on factors that affect oocyte quality and developmental potential. Areas in which the mare could be used as a research model to study reproductive aging in women will be highlighted.

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos.

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

Association of equine oocyte and cleavage stage embryo morphology with maternal age and pregnancy after intracytoplasmic sperm injection.

In this retrospective study the morphological characteristics of oocytes and cleavage stage embryos were associated with pregnancy results from clinical intracytoplasmic sperm injection (ICSI) in mares. Oocytes were collected from preovulatory follicles, and images (×200; n=401) were captured for measurements of ooplasm, the perivitelline space and zona pellucida. After ICSI and before transfer into recipients' oviducts, cleavage stage embryos were imaged (n=178). Oocyte donor ages (3-13, 14-19, 20-23, 24-27 years) were compared, as were mares aged 3-13 years without versus with recent histories of performance or injury stress. Cleavage rates did not differ with age. However, pregnancy rates declined and pregnancy loss rates (11-50 days gestation) increased with mare age. Young mares with performance or injury stress had significantly lower pregnancy rates than young mares under management typical for broodmares. No morphological oocyte characteristic was consistently associated with age or pregnancy outcome. Cleavage stage embryo morphology was not associated with pregnancy outcome; however, the rate of embryo development before oviductal embryo transfer was faster (P<0.05) for embryos that resulted in an early pregnancy (≤17 days) and tended (P ≤ 0.1) to be higher for embryos that produced a 50-day pregnancy. Embryonic vesicles that had a more rapid increase in diameter were more often (P<0.05) maintained until 50 days gestation.

Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro.

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

In Vitro Culture of Embryos from Horses.

Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species. In part, this delay was caused by the failure of standard in vitro fertilization techniques in horses. However, the development of intracytoplasmic sperm injection (ICSI) for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and, typically, with the addition of serum. Although systems specifically for horse embryo culture still have not been established, ICSI-produced embryos are developmentally competent and capable of producing live offspring.

Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses.

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.

Use of microfluidics to sort stallion sperm for intracytoplasmic sperm injection.

We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF sorting resulted in a sperm subpopulation with greater motility, morphology, viability, and membrane as well as DNA integrity. After sorting by MF, SLC and SU in Experiment 2, motility, viability, and membrane integrity were similar for sperm sorted using MF and SLC; however, morphology and DNA integrity were greater in sperm sorted using MF when compared with SLC. Swim-up was the least effective sorting method. In Experiment 3, sperm were processed using MF and SLC prior to ICSI. Motility, morphology and DNA integrity were similar for sperm subpopulations sorted using either method; but viability was greater for sperm sorted using MF than SLC. Sorting did not improve sperm membrane integrity. Sorting with MF prior to ICSI resulted in similar cleavage and blastocyst development rates as SLC. We concluded that MF separation of stallion sperm resulted in a subpopulation with improved sperm population parameters, comparable or better than SLC and SU. Embryos were produced after ICSI using MF sperm sorting.

Association of equine sperm population parameters with outcome of intracytoplasmic sperm injections.

Limited clinical information is available regarding sperm population parameters that are important for use with equine intracytoplasmic sperm injection (ICSI). Therefore, the appropriateness of a sample of sperm is typically not known before ICSI. The aim of our study was to determine which sperm population characteristics were predictive of ICSI outcome. Frozen-thawed sperm samples (n = 114) from 37 stallions in a clinical program were analyzed after ICSI for percentages of normal morphology (MORPH+), live as assessed by eosin/nigrosin stain (LIVE+), membrane intact as assessed by hypoosmotic swelling test (HOS+), and DNA fragmentation determined by sperm chromatin dispersion (DNA-). ICSI was performed on 147 oocytes, and cleavage (≥2 cells), embryo development (morula or blastocyst), and pregnancy status after embryo transfer were determined. Among the examined sperm parameters, LIVE + correlated positively with MORPH+ and HOS+, and MORPH + negatively with DNA-; no other significant correlations were observed. When used for ICSI, sperm population percentages for MORPH+ and DNA- were not predictive of ICSI outcome, including cleavage, embryo development, and establishment of a pregnancy. Sperm population percentages significantly affecting ICSI outcomes were LIVE+ and HOS + for oocyte cleavage, LIVE + for embryo development, and HOS + for establishment of a pregnancy. The probability of a pregnancy was significantly higher for sperm populations having HOS+ ≥40% than populations having HOS+ ≤20%. The mean age of the donor mare per sperm-injected oocyte did not differ for oocyte cleavage, embryo production, or establishment of pregnancy. In our study, the probability of sperm-injected oocytes to develop into an embryo (morula or blastocyst) improved when sperm were selected from a population with higher indicators of membrane integrity (LIVE+ and HOS+).

Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis.

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry. Obese mares had elevated concentrations of insulin, leptin, and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates that increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.

Impact of Equine and Bovine Oocyte Maturation in Follicular Fluid From Young and Old Mares on Embryo Production in Vitro.

Equine follicular fluid (FF) provides autocrine and paracrine factors from theca, granulosa, and cumulus cells, both reflecting and impacting oocyte and follicle maturation. We hypothesized that maturation of oocytes in FF from old versus young mares has a deleterious effect on oocyte maturation and their subsequent developmental potential. Follicular fluid was collected from the large, dominant follicle from young mares (4-13 years) or old mares (21-26 years) and classified as: (1) Noninduced follicular fluid (NFF), FF from noninduced follicle 33 ± 3 mm, or (2) Induced follicular fluid (IFF), FF collected ∼24 hours after administration of ovulation-inducing drugs when a follicle 33 ± 3 mm was observed. In experiment 1, immature equine oocytes were collected, matured in vitro for 30 ± 2 hours in 100% IFF, collected from young or old mares, with the addition of follicle stimulating hormone (5 mU/mL), then fertilized by intracytoplasmic sperm injection. In experiment 2, immature bovine oocytes were collected, matured in 100% IFF or NFF, collected from young mares or old mares, then fertilized via in vitro fertilization. In experiment 1, more blastocysts tended (P = .08) to be produced from equine oocytes that were matured in old versus young mare FF. In experiment 2, when IFF and NFF groups were combined, cleavage rates were higher (P = .001) when bovine oocytes were matured in FF from young than old mares. In contrast to our hypothesis, we observed no conclusive evidence that FF from old mares has a deleterious impact on oocytes and their early developmental potential.

Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro.

Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/ß tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.