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Contents Implantation is a pivotal step in the establishment of mammalian pregnancy. Although implantation strategies vary between species, many aspects of conceptus-maternal signalling necessary to induce uterine receptivity to implantation are conserved. The temporal 'window' for the initiation of implantation is short and precisely controlled by endocrine, paracrine and autocrine factors. An invariable prerequisite for the development of uterine receptivity to implantation is continuous exposure of the endometrium to progesterone which, after a species-specific interval, downregulates progesterone receptor ( PGR) expression in the epithelium and stimulates the production of progestamedins. Uterine receptivity involves temporal changes in the expression of genes, leading to modifications in surface, extracellular matrix and secretory characteristics that support growth, proliferation, migration and attachment of the conceptus. Moreover, a complex interplay between endometrial progestamedins and estramedins and conceptus-derived oestrogens, cytokines and interferons ( INFs), prostaglandins ( PGs) and cortisol is crucial to the preparation for implantation. Understanding the individual roles and combined actions of conceptus and endometrial autocrine and paracrine factors in the development of uterine receptivity to implantation is essential for translational research into strategies to reduce pregnancy loss in man and animals. [ABSTRACT FROM AUTHOR]

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P = .09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P > .05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19, IGF1, FGF2 and PGR gene expression, more conducive to embryo survival and further development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia, and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 × 106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and >20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Background: Management of twin pregnancy after conceptus vesicle fixation in the horse is challenging because the reduction techniques described are either invasive, difficult to perform or associated with disappointing success rates., Objectives: To evaluate the success of transrectal ultrasound-guided fetal thorax compression for reducing post-fixation twin pregnancy in mares., Study Design: Retrospective clinical study., Methods: Sixteen mares were presented for twin reduction between 51 and 79 days of gestation. History obtained from the owner and/or referring veterinarian detailed information regarding the mare (age, breed), pregnancy (day of gestation, dizygotic versus monozygotic twins, unilateral versus bilateral fixation), treatment and outcome (one live fetus at discharge; live singleton at foaling) after twin reduction. Transrectal fetal thorax compression was performed under ultrasound guidance by two experienced operators., Results: Overall 9 of 16 twin pregnancies were successfully reduced and the likelihood of success was significantly higher in dizygotic than monozygotic twins. The procedure was successful in 9 of 10 dizygotic twins but unsuccessful in all six cases of monozygotic twins. Among the dizygotic twins, two mares lost the pregnancy after discharge from the clinic, seven mares delivered a healthy foal of normal size., Main Limitations: Small case number., Conclusions: Transrectal ultrasound-guided fetal thorax compression is a minimally-invasive and successful technique for reducing dizygotic twin pregnancies at approximately 2 months of gestation, but does not lead to any live births in cases of monozygotic twins., (© 2024 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium., (© 2024. The Author(s).)

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts ( n = 3) and blastocysts ( n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro ( n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca 2+ ] i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca 2+ ] i decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca 2+ ] i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 μm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 μm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification., Competing Interests: Declaration of competing interest Authors have no conflict of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these 'fertilization supporting factors' would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7-28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gibson, de Ruijter-Villani and Stout.)

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0-13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5-10%), re-differentiated monolayers with 11-27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux ( p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maitan, Bromfield, Hoogendijk, Leung, Zeev-Ben-Mordehai, van de Lest, Jansen, Leemans, Guimarães, Stout, Gadella and Henning.)

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos., (© 2021 Schneider et al.)

Background: Changes in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown., Objectives: Determine the order in which EMS-associated changes in cardiovascular parameters arise., Animals: Twenty Shetland pony mares., Methods: High-energy (HE) diet mares were fed 200% of net energy requirements for 1 (n = 3) or 2 (n = 7) consecutive diet-years, with 17 weeks of hay-only between years. Noninvasive BP measurements and echocardiograms were performed during both years. Resting 24-hour ECGs and measurements of autonomic tone (splenic volume and packed cell volume [PCV]) were performed at the end of diet-year 1. Results were compared to control mares receiving a maintenance diet for 1 (n = 7) or 2 (n = 3) consecutive years., Results: In year 1, HE mares had significantly higher values than control mares for mean relative left ventricular wall thickness (P = .001). After 2 diet-years, mean systolic (P = .003), diastolic (P < .001) and mean arterial BP (P = .001), heart rate (HR; P < .001), and mean left ventricular wall thickness (P = .001) also were significantly increased in HE compared to control mares. No pathological arrhythmias or differences in splenic volume or PCV were detected., Conclusions and Clinical Importance: Ingesting a HE diet first induced minor changes in BP, and progressed to left-sided cardiac hypertrophy in Shetland pony mares. These findings are of interest given the increasing incidence of obesity in horses., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes ( IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN ), DNMTs ( DNMT1 and DNMT3B ), glucose ( SLC2A1 ), fructose ( SLC2A5 ) and amino acid ( SLC7A2 ) transporters following ET from an HE to a control mare. Expression of amino acid transporters ( SLC1A5 and SLC7A1 ) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prep TM ) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prep TM solution (20-60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prep TM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions ( n = 3) with high sperm DFI (>20%). The optimal Opti-prep TM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prep TM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased ( p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prep TM cushion and a 20% top layer.

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

Aneuploidy originating during meiosis in oocytes is the major cause of reduced fertility, implantation failure and miscarriage in women beyond their mid-thirties. Loss of chromosome cohesion, and defective microtubule dynamics and spindle assembly are, in turn, the major contributors to the error-prone nature of chromosome segregation in the oocytes of older women. However, the underlying molecular defects are not well understood. Altered function of MPS1 and AURKC have been shown to induce multipolar spindle phenotypes in murine oocytes and cancer cells, however, their role in reproductive aging associated oocyte aneuploidy is not known. Although age-related gamete and embryonic aneuploidy has been studied in female rodents, the horse may be a more appropriate animal model. Similar to women, aged mares suffer from reduced fertility and an increased incidence of oocyte aneuploidy. Moreover, mares show a long interval (decades) to reproductive senescence and, unlike rodents but similar to women, horse oocytes assemble the meiotic spindle in a slow and unstable manner, independent of microtubule organizing centers. In this study we found that oocytes from aged mares have lower expression of mRNA for Mps1, Spc25 and AurkC than oocytes from young mares while gene expression for other meiosis regulators did not differ. To assess the ability of horse oocytes to correctly form a bipolar spindle, in vitro matured MII oocytes were allowed to re-form their spindle after nocodazole-induced microtubule depolymerization. To investigate the importance of MPS1 and AURKC function in spindle (re)assembly, various concentrations of a MPS1 inhibitor (MPS1i, Compound 5) or an AURK inhibitor (AURKi, ZM447439) were included after nocodazole washout. MII oocytes from aged mares showed a higher incidence of spindle abnormalities after exposure to MPS1i. In contrast, Aurora kinase inhibition severely impaired microtubule organization and spindle formation in all oocytes, irrespective of mare age. In conclusion, gene expression for the kinases Mps1, Spc25 , and AurkC is reduced in oocytes from aged mares. Moreover, spindle (re)assembly in aged mares' oocytes is more unstable when Mps1 is inhibited. Overall, this suggests that compromised Mps1 activity predisposes to meiotic spindle instability in aged mare oocytes. This spindle instability could predispose to chromosome segregation errors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rizzo, Stout, Cristarella, Quartuccio, Kops and De Ruijter-Villani.)

Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of in vitro produced (IVP) embryos. However, this parameter is difficult to assess after blastocyst expansion. In this study, we used mechanical hatching to confirm the presence of cellular fragments in the perivitelline space of bovine IVP blastocysts. We further looked for associations between possible apoptosis within extruded cells/ cellular fragments and the quality of bovine blastocysts using quantitative RT-PCR and immunofluorescence. Surprisingly, more than 42% of expanded blastocysts had cellular fragments in the perivitelline space; however, more than 37% of extruded cells were TUNEL negative. We observed no significant difference in embryo quality between expanded blastocysts with and without cellular fragments in the perivitelline space. Overall, our data suggest that embryos extrude abnormal cells to maintain their developmental potential. The presence of fragmented cells is not an indicator of embryo quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yu, van Tol, Stout and Roelen.)

Aneuploidy of meiotic origin is a major contributor to age-related subfertility and an increased risk of miscarriage in women. Although age-related aneuploidy has been studied in rodents, the mare may be a more appropriate animal model to study reproductive aging. Similar to women, aged mares show reduced fertility and an increased incidence of early pregnancy loss; however, it is not known whether aging predisposes to aneuploidy in equine oocytes. We evaluated the effect of advanced mare age on (1) gene expression for cohesin components, (2) incidence of aneuploidy and (3) chromosome centromere cohesion (measured as the distance between sister kinetochores) in oocytes matured in vitro . Oocytes from aged mares showed reduced gene expression for the centromere cohesion stabilizing protein, Shugoshin 1. Moreover, in vitro matured oocytes from aged mares showed a higher incidence of aneuploidy and premature sister chromatid separation, and weakened centromeric cohesion. We therefore propose the mare as a valid model for studying effects of aging on centromeric cohesion; cohesion loss predisposes to disintegration of bivalents and premature separation of sister chromatids during the first meiotic division, leading to embryonic aneuploidy; this probably contributes to the reduced fertility and increased incidence of pregnancy loss observed in aged mares.

A high degree of inbreeding has been reported to negatively impact semen quality in Friesian horses and Shetland ponies. Both breeds are characterized by a closed studbook, small population size, and high incidence of inbreeding. The Dutch Warmblood studbook (KWPN: Koninklijk Warmblood Paardenstamboek Nederland) is a much larger studbook with two distinct populations: the KWPN-Riding horses, managed as an 'open' studbook, and the KWPN-Harness horses, representing a much smaller subpopulation within the KWPN breed and managed as an 'almost closed' studbook. It was recently reported that the degree of inbreeding in KWPN-Harness horses has increased in recent decades due to the small gene pool; however, the degree of inbreeding is still lower than that of Friesian horses and Shetland ponies. We hypothesized that a high or rising degree of inbreeding might negatively impact semen quality. In the present study, we retrospectively compared semen quality parameters of stallions from four different breeds or types (Friesian Horses, Shetland Ponies, KWPN-Riding horses, and KWPN-Harness horses), each reported with different degrees of inbreeding. Semen concentration, and percentages of motile, morphologically normal and live spermatozoa, and the total number of morphologically normal, progressive motile spermatozoa per ejaculate (TNM) were analyzed for 2832 semen evaluations performed over a 15-year period. KWPN-Harness horses had a significantly lower sperm concentration, % motile spermatozoa and % live spermatozoa than KWPN-Riding horses but the % motile and % morphologically normal spermatozoa and TNM in both KWPN-Harness and KWPN-Riding horses were significantly higher than in Friesian horses and Shetland ponies. These results suggest a lower semen quality in KWPN-Harness than KWPN-Riding horses, potentially as a result of a higher coefficient of inbreeding. The negative trend observed in the KWPN-Harness horses may be a warning sign, and breeders or stud books should monitor the degree of inbreeding carefully to avoid a further reduction in semen quality, to the levels observed in Friesian horses and Shetland ponies., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.

The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor mares (historically <25%; now >50%) and in the successful culture of zygotes to the blastocyst stage in vitro (historically <10%; now >20%). It has also been established that immature oocytes can be "held" at room temperature for at least 24 hours, allowing overnight transport to a laboratory with expertise in IVEP. Moreover, because in vitro-produced embryos can be cryopreserved with no appreciable reduction in viability, they can be shipped and stored until a suitable recipient mare is available for transfer. Most importantly, in an established equine ovum pick-up intracytoplasmic sperm injection (OPU-ICSI) program, blastocyst production rates now exceed 1 per procedure, and posttransfer foaling rates exceed 50%, such that overall efficiency betters that of either embryo flushing or oocyte transfer. Moreover, OPU-ICSI can be performed year round and allows embryo production from mares with severe acquired subfertility and extremely efficient use of scarce or expensive frozen semen. Cumulatively, these factors have stimulated rapid growth in demand for IVEP among sport horse breeders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

P-element induced wimpy testis (PIWIs) are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, we investigated the expression, distribution, and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex with Tudor and KH domain-containing protein (TDRKH) and poly(A)-specific ribonuclease-like domain containing 1 (PNLDC1), and demonstrated by mutagenesis that PIWIL3 N-terminal arginines are required for complex assembly. Finally, we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here that about 50% of these piRNAs map to transposable elements, recapitulating the important role of PIWIL3 in maintaining genome integrity in mammalian oocytes.

Background: Overfeeding is associated with obesity and insulin dysregulation (ID), which are both risk factors for equine metabolic syndrome. How chronic overfeeding affects development of these factors is poorly understood., Objectives: To examine the influence of long-term high-energy diet provision on body condition and ID., Animals: Eleven Shetland pony mares., Methods: In a 3-phase study, the high-energy group (n = 7) was fed 200% of net energy (NE) requirements (hay; concentrate: 36% sugar and starch, 13% fat) for 24 weeks, followed by 17 weeks hay-only feeding before resuming the high-energy diet (n = 4) for an additional 29 weeks. Mares were weighed weekly. Oral glucose tolerance tests were performed 3 to 4 times per dietary period. Results were compared with those of a control group (phase 1, n = 4; phases 2 and 3, n = 6) that received 100% NE requirements, using a general linear mixed model with post hoc Bonferroni testing., Results: The mean body weight of the high-energy group increased by 27% per high-energy feeding period. During both feeding periods, area under the curve (AUC) for plasma glucose concentration decreased (P < .01), whereas AUC for plasma insulin concentration increased. Mean basal plasma glucose concentration and peak plasma insulin concentrations were higher (P < .05) in the high-energy group than in the control group., Conclusion and Clinical Importance: Feeding a high-energy diet to healthy nonobese Shetland pony mares led to more efficient glucose metabolism within 5 weeks, followed by significant hyperinsulinemia and obesity. Hyperinsulinemic status was reversed during 17 weeks of hay-only feeding, regardless of body condition, but returned rapidly after restarting the high-energy diet., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to 'extracellular exosome'. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Objective: To describe perioperative management, surgical procedure, and outcome in mares with third-degree perineal lacerations (TDPL) treated with a single-stage repair, the Utrecht repair method (URM)., Study Design: Retrospective study., Animals: Twenty mares with TDPL., Methods: Medical records of mares with TDPL reconstructed with a URM were reviewed for perioperative management; surgical outcome; and postoperative fertility, athletic performance, and complications., Results: Mares ranged in age from 3.5 to 11 years. Long-term follow-up was available for 13 mares. Mean duration of follow-up was 9 years (median, 9.5; range, 2-215 months (17.9 years)). Standardized perioperative fasting and postoperative refeeding protocols were used. Only five mares received supportive gastric medication. Reconstruction of the rectovestibular shelf was successful in 18 of 20 mares. Two of 20 mares developed a small rectovestibular fistula after the initial repair, which was successfully repaired with a second surgery. Other postoperative complications were observed in 13 mares and consisted of mild postanesthetic myositis, facial nerve paralysis, esophageal obstruction, rectal obstipation, partial perineal dehiscence, and rectal or vestibular wind-sucking. Six of seven mares that were subsequently bred became pregnant. One mare was successfully used for embryo recovery, and five of six mares foaled without recurrence of a TDPL. Nine of 13 mares were used for riding at various levels., Conclusion: The alternative single-stage reconstruction for TDPL was successful in 18 of 20 mares after a single surgery. No major complications related directly to the technique were noted., Clinical Significance: The URM is a valid alternative surgical technique for repairing TDPL in mares., (© 2019 The American College of Veterinary Surgeons.)

Lumpy skin disease (LSD) is an important transboundary animal disease of cattle with significant economic impact because of the implications for international trade in live animals and animal products. LSD is caused by a Capripoxvirus, LSD virus (LSDV), and results in extensive hide and udder damage, fever and pneumonia. LSDV can be shed in semen of infected bulls for prolonged periods and transmitted venereally to cows at high doses. This study examined the effects of LSDV in frozen-thawed semen on in vitro embryo production parameters, including viral status of media and resulting embryos. Bovine oocytes were harvested from abattoir-collected ovaries and split into three experimental groups. After maturation, the oocytes were fertilized in vitro with frozen-thawed semen spiked with a high (HD) or a lower (LD) dose of LSDV, or with LSDV-free semen (control). Following day 7 and day 8 blastocyst evaluation, PCR and virus isolation were performed on all embryonic structures. After completing sufficient replicates to reach 1,000 inseminated oocytes, further in vitro fertilization (IVF) runs were performed to provide material for electron microscopy (EM) and embryo washing procedures. Overall, in vitro embryo yield was significantly reduced by the presence of LSDV in frozen-thawed semen, irrespective of viral dose. When semen with a lower viral dose was used, significantly lower oocyte cleavage rates were observed. LSDV could be detected in fertilization media and all embryo structures, when higher doses of LSDV were present in the frozen-thawed semen used for IVF. Electron microscopy demonstrated LSDV virions inside blastocysts. Following the International Embryo Transfer Society washing procedure resulted in embryos free of viral DNA; however, this may be attributable to a sampling dilution effect and should be interpreted with caution. Further research is required to better quantify the risk of LSDV transmission via assisted reproductive procedures., (© 2019 Blackwell Verlag GmbH.)

Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed using immunofluorescent staining, confocal microscopy and three-dimensional image analysis. Vitrification reduced the ability of oocytes to reach MII and resulted in ultrastructural changes to the meiotic spindle, including shortening of its long axis, and an increased incidence of chromosomes failing to align properly at the metaphase plate. We hypothesise that aberrant chromosome alignment is an important contributor to the reduced developmental competence of vitrified equine oocytes. Contrary to expectation, oocytes from young mares were more severely affected than oocytes from older mares; we propose that the reduced effect of vitrification on oocytes from older mares is related to pre-existing compromise of spindle assembly checkpoint control mechanisms in these mares.

Field data indicate that a longer period of estrus prior to ovulation correlates positively with fertility. To test the hypothesis that the duration of exposure to estrogens prior to progesterone dominance influences endometrial function, we used anestrous mares to simulate varying durations of estrus (3 groups of 5 mares): long (LE), short (SE), and no estrus (NE), as determined by the duration of estradiol priming prior to progesterone treatment: 7, 2 and 0 days for the LE, SE and NE, respectively. Endometrial biopsies were recovered 4 days after progesterone administration in all groups for real time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical analyses. A total of 17 genes believed to contribute to a "receptive endometrium" for embryo development and viability were evaluated by RT-qPCR. Of the genes evaluated, the expression of FGF-2 (fibroblast growth factor-2) decreased with increased length of preceding estrus, whereas P19 (uterocalin) expression was higher in the LE than in the SE or NE groups. In conclusion, a lower abundance of FGF-2 and higher abundance of uterocalin, a lipocalin protein known to play an important role in providing lipids to the embryo, could contribute to a more receptive endometrium in mares following a long estrus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chain reaction assay. Foetal membrane swabs and tissue samples were taken immediately post-partum, and venous blood samples and nasal swabs were obtained from both mare and foal 8 h after birth. Neither equid herpesvirus type 1 nor equid herpesvirus type 4 nucleic acid was detected in any sample, and it was concluded that there was no active shedding of equid herpesvirus types 1 and 4 at the time of sampling. Consequently, no evidence of vertical transmission of these viruses could be found on this stud farm during the sampling period.