Surgical Closure of Equine Abdomen, Prevention, and Management of Incisional Complications.

Although rarely fatal, complications of ventral midline laparotomy incision in equine patients increase hospitalization cost and duration and may jeopardize return to athletic function. Therefore, many techniques have been developed to reduce their occurrence and expedite their resolution when they occur. Our technique of celiotomy incision closure includes the use of tension sutures (vertical U mattress) of polyglactin 910 on the linea alba, which is then apposed by polyglactin 910 interrupted sutures or a simple continuous pattern suture with a stop midway before routine closure of the superficial layers. The celiotomy incision is protected by an elastic bandage during the immediate postoperative period. This technique has been associated with favorable results: 5.3% confirmed incisional infections after a single celiotomy and 26.7% after repeat celiotomy. The overall incisional complication (serous/sanguineous discharge, hematoma, infection, hernia formation, and complete wound breakdown) occurrence was 9.5% and 33.3% after single and repeat laparotomy, respectively. In cases considered more susceptible to infection (early relaparotomy or laparotomy incisions longer than 30 cm), negative pressure therapy was found easy to apply on closed incisions. No detrimental effects were observed. However, the potential prophylactic benefit of this therapy needs to be confirmed in a larger group. In infected laparotomy wounds requiring drainage, the use of negative pressure therapy seemed to have a positive effect on the formation of granulation tissue. However, there was no control group to allow statistical confirmation. Finally, one case of complete breakdown of the laparotomy incision was managed by stainless steel retention sutures, the application of negative pressure therapy, and a hernia belt. At re-evaluation 15 months post-surgery, several small hernias were detected, but the horse had returned to his previous level of sports performance and had not shown any episode of colic.

Early Castration in Horses Does Not Impact Osteoarticular Metabolism.

The castration of stallions is traditionally performed after puberty, at around the age of 2 years old. No studies have focused on the effects of early castration on osteoarticular metabolism. Thus, we aimed to compare early castration (3 days after birth) with traditional castration (18 months of age) in horses. Testosterone and estradiol levels were monitored from birth to 33 months in both groups. We quantified the levels of biomarkers of cartilage and bone anabolism (CPII and N-MID) and catabolism (CTX-I and CTX-II), as well as of osteoarthritis (HA and COMP) and inflammation (IL-6 and PGE2). We observed a lack of parallelism between testosterone and estradiol synthesis after birth and during puberty in both groups. The extra-gonadal synthesis of steroids was observed around the 28-month mark, regardless of the castration age. We found the expression of estrogen receptor (ESR1) in cartilage and bone, whereas androgen receptor (AR) expression appeared to be restricted to bone. Nevertheless, with respect to osteoarticular metabolism, steroid hormone deprivation resulting from early castration had no discernable impact on the levels of biomarkers related to bone and cartilage metabolism, nor on those associated with OA and inflammation. Consequently, our research demonstrated that early castration does not disrupt bone and cartilage homeostasis.

Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions.

Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation.

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Characterization of Myeloperoxidase in the Healthy Equine Endometrium.

Myeloperoxidase (MPO), as a marker of neutrophil activation, has been associated with equine endometritis. However, in absence of inflammation, MPO is constantly detected in the uterine lumen of estrous mares. The aim of this study was to characterize MPO in the uterus of mares under physiological conditions as a first step to better understand the role of this enzyme in equine reproduction. Total and active MPO concentrations were determined, by ELISA and SIEFED assay, respectively, in low-volume lavages from mares in estrus (n = 26), diestrus (n = 18) and anestrus (n = 8) in absence of endometritis. Immunohistochemical analysis was performed on 21 endometrial biopsies randomly selected: estrus (n = 11), diestrus (n = 6) and anestrus (n = 4). MPO, although mostly enzymatically inactive, was present in highly variable concentrations in uterine lavages in all studied phases, with elevated concentrations in estrus and anestrus, while in diestrus, concentrations were much lower. Intracytoplasmic immunoexpression of MPO was detected in the endometrial epithelial cells, neutrophils and glandular secretions. Maximal expression was observed during estrus in mid and basal glands with a predominant intracytoplasmic apical reinforcement. In diestrus, immunopositive glands were sporadic. In anestrus, only the luminal epithelium showed residual MPO immunostaining. These results confirm a constant presence of MPO in the uterine lumen of mares in absence of inflammation, probably as part of the uterine mucosal immune system, and suggest that endometrial cells are a source of uterine MPO under physiological cyclic conditions.

Early castration in foals: Consequences on physical and behavioural development.

BACKGROUND: The impact of very early castration of foals has not yet been studied despite the many positive effects observed in dogs and cats. OBJECTIVES: The objective of the study was to compare castration at 3 days and 18 months and assess their subsequent morphological and behavioural development. STUDY DESIGN: This was a randomised, blinded clinical study. METHODS: Twenty-two Welsh ponies underwent either early (3-day old, EC group, n = 11) or traditional (18-month old, TC group, n = 11) castration. Animals were followed up to 3 years of age. All ponies were castrated using a primary closure technique under general anaesthesia. Weight and morphometric measurements were monitored monthly from birth until 8 months of age in both groups. Then, measurements were taken every 3 months until 2 years of age and then every 6 months until 3 years of age. Temperament tests were performed on all animals when they were 1- and 3-years old. RESULTS: No differences were observed between the EC and TC groups in terms of physical development from birth until 40 months of age or in terms of temperament and behaviour at either 1 or 3 years of age. MAIN LIMITATIONS: The study included only one breed (Welsh ponies) and only 22 animals that were castrated before 2 years of age, precluding comparison with castration performed at older ages. CONCLUSIONS: We demonstrate that early castration at 3 days does not interfere with morphological or behavioural development.

Evolution of 17-ß-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry.

This study describes 17-ß-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's parity and age on estrogens concentrations were investigated. Peak of E2 was observed at 5 months (median: 46.4 pg/mL; maximum: 201.5 pg/mL). A strong correlation was observed between E1 and E1S (p < 0.0001, r = 0.85). Peak of E1 (median: 571.0 pg/mL; maximum: 1641.9 pg/mL) and E1S (median: 573.6 ng/mL; maximum: 997.6 ng/mL) concentrations was observed at the 5th month and then E1S decreased quicker than E1 until the end of pregnancy. Higher E2 and E1 concentrations were observed in SJ than in SPB mares between the 6th and the 8th months. No difference between breeds was observed for E1S monthly evolution. Estrogen peak values were all observed at 5 months. Unlike recent LC-MS studies, E1S values observed here were in the same range than those previously established using immuno-assays. After the 6th month, E1S decreased quicker than E1. Effect of breed only observed on non-sulfonated estrogens should be further confirmed. These findings confirm that sulfonation activity of the allantochorion may be limited after the 6th month.

Investigation of equine testis contribution to vitamin D bioactivation.

Although vitamin D acts in various biological processes, it plays a critical role in the maintenance of bone health, and regulates calcium homeostasis. In humans and rodents, the main tissues involved in vitamin D metabolism are the liver and the kidneys, however it has been shown that the testis has strongly participated in its bioactivation. Indeed, in these different species, enzymes metabolizing vitamin D (CYP27A1, CYP27B1 and CYP2R1) have been demonstrated in this tissue. Moreover, men with hypogonadism have shown a decrease in circulating levels of vitamin D. In equine species, the castration of males is a regular practice to reduce the behavior of stallions deemed too aggressive. Castration is carried out at various ages: in foals during their growth or in adulthood once they have reached their optimum size. Although horses exhibit atypical vitamin D metabolism with low circulating levels of vitamin D, it was suggested that testis may contribute to its activation as has been described in rodents and humans; castration could therefore be likely to affect its metabolism. In this study, blood levels of bioactive form of vitamin D (1 α,25[OH] 2 vitamin D 3 ) were measured before and after castration at different ages: 1 wk, after puberty (2 yr) and at adulthood (6 yr). The gene expression of enzymes involved in vitamin D metabolism has been sought in the testis of different experimental groups. No change in bioactive vitamin D3 levels was observed after castration regardless of the age at the time of surgery. The exceptional status of equine species is confirmed with a low or a lack of testis contribution to vitamin D metabolism, regardless of testicular development. This is demonstrated by a low or a lack of signal from enzymes involved in vitamin D bioactivation. Therefore, horses constitute a unique model in comparative endocrinology.

Effect of Infant Presence on Social Networks of Sterilized and Intact Wild Female Balinese Macaques (Macaca fascicularis).

Contraception is increasingly used to control wild animal populations. However, as reproductive condition influences social interactions in primates, the absence of new offspring could influence the females' social integration. We studied two groups of wild macaques (Macaca fascicularis) including females recently sterilized in the Ubud Monkey Forest, Indonesia. We used social network analysis to examine female grooming and proximity networks and investigated the role of infant presence on social centrality and group connectivity, while controlling for the fertility status (sterilized N = 14, intact N = 34). We compared the ego networks of females experiencing different nursing conditions (young infant (YI) vs. old infant (OI) vs. non-nursing (NN) females). YI females were less central in the grooming network than other females while being more central in proximity networks, suggesting they could keep proximity within the group to protect their infant from hazards, while decreasing direct grooming interactions, involving potential risks such as kidnapping. The centrality of sterilized and intact females was similar, except for the proximity network where sterilized females had more partners and a better group connectivity. These results confirm the influence of nursing condition in female macaque social networks and did not show any negative short-term effects of sterilization on social integration.

Tubectomy of Pregnant and Non-pregnant Female Balinese Macaques (Macaca Fascicularis) With Post-operative Monitoring.

Worldwide, primates, and humans increasingly share habitats and often enter in conflict when primates thrive in human-dominated environments, calling for special management measures. Reproductive control is increasingly used to manage population growth but very few monitoring data are available. Therefore, the efficiency and implications of such programs require a careful examination. In the context of a contraception program in wild female long-tailed macaques in Ubud, Bali, conducted over four successive campaigns between 2017 and 2019, including 140 females (i.e., 41.9% of the reproductive females of the population in 2019), modifications of an endoscopic tubectomy procedure, a permanent sterilization method, clinical evaluation of this method, and the post-operative monitoring results of the neutered females after release are described. This surgical approach was applicable for pregnant females: 28.6% of the treated females were pregnant at the time of the surgery. The procedure used a single lateral port to reach and cauterize both oviducts in non-pregnant as well as in early to mid-term pregnant females. Pregnant females nearer to term required a second lateral port to access both oviducts masked by the size of the gravid uterus. Moreover, bipolar thermocauterization was utilized successfully without resection to realize the tubectomy. The average duration of the laparoscopic surgery was 14 min for non-pregnant females and 22 min for pregnant females. Animals were released 3 h 22 min in average following their capture. This short holding time, recommended for free-ranging primates, was made possible by the minimal invasiveness of the sterilization approach. A laparoscopic post-operative evaluation conducted on two patients during the following campaign confirmed that the oviducts were definitely disrupted and no longer patent. Moreover, no new pregnancies in sterilized females were recorded during the 3-year observation period. The survival rate of the treated females 6 months after sterilization was high (96.3%) with no major post-operative complications clinically recorded. Among females that were pregnant during surgery, 81.1% were confirmed to experience term delivery. This study demonstrates the safety and efficiency of endoscopic tubectomy, even for pregnant females, as a mean of wild macaques' population control.

Optimization and assessment of a novel gastric electrode anchoring system designed to be implanted by minimally invasive surgery.

A novel electrode anchoring design and its implantation procedure, aiming for a minimally invasive solution for gastric electrical stimulation, are presented. The system comprises an anchor made of a flexible body embedding two needle-shaped electrodes. The electrodes can easily switch from a parallel position - to pierce the stomach - to a diverging position - enabling them to remain firmly anchored into the muscular layer of the stomach. Key device parameters governing anchoring stability were assessed on a traction test bench, and optimal values were derived. The device was then implanted in six dogs by open surgery to assess its anchoring durability in vivo. Computed tomography images showed that the electrodes remained well placed within the dogs' gastric wall over the entire assessment period (more than one year). Finally, a prototype of a surgical tool for the minimally invasive device placement was manufactured, and the anchoring procedure was tested on a dog cadaver, providing the proof of concept of the minimally invasive implantation procedure. The use of our electrode anchoring system in long-term gastric electrical stimulation is promising in terms of implantation stability (the anchor withstands a force up to 0.81 N), durability (the anchor remains onto the stomach over one year) and minimal invasiveness of the procedure (the diameter of the percutaneous access is smaller than 12 mm). Moreover, the proposed design could have clinical applications in other hollow organs, such as the urinary bladder.

Microbial Prevalence and Antimicrobial Sensitivity in Equine Endometritis in Field Conditions.

Endometritis is one of the main causes of infertility in mares. In the present study, 363 mares with a history of repetitive infertility, and positive endometrial cytology and/or vaginal discharge were included. An endometrial swab for microbiological purposes plus sensitivity test was obtained from each mare. A positive culture was obtained in 89% of mares. The main isolated genera were Staphylococcus (25.1%), Streptococcus (18.2%), Escherichia (17.3%) and Pseudomonas (12.1%). With regard to species, the most isolated microorganism was Escherichia coli (17.3%), Staphylococcus spp. (15.6%) and Streptococcus spp. (13.5%). Sensitivity tests showed that the most efficient antimicrobial was amikacin (57.3% of cultures), followed by cefoxitin (48.6%) and gentamicin (48.3%). When sensitivity test was analyzed in terms of Gram+ and Gram- bacteria, Gram+ were highly resistant to cephaloridine (77.3% of cultures), apramycin (70.8%) and penicillin (62.3%), whereas Gram- were highly resistant to penicillin (85.8%), followed by cephaloridine (78.9%). In conclusion, the present study shows the most prevalent microorganisms isolated from equine endometritis, which were found to be resistant to ß-lactam antimicrobials. Likewise, these results highlight the significance of performing microbiological analyses as well as sensitivity tests prior to applying an antimicrobial therapy.

Case Report: Suppression of Harem Stallion Behavior and Fertility Following Anti-Gonadotropin-Releasing Hormone Vaccination of a Captive Wild Przewalski's Horse (Equus ferus przewalskii).

This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 µg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation was safe and effective to collect spermatozoa. Motility was low but was improved by a significant dilution of sample (1v/4v-1v/5v) after collection. Immuno-neutering resulted in a decrease of the total spermatozoa number and motility 1 month after primary vaccination. However, infertility could not yet be guaranteed. Six months post-vaccination, serum testosterone concentrations had decreased and the treated stallion had lost his harem stallion role. Moreover, at the same time, the total spermatozoa number was near zero with no motile spermatozoa, and offspring was no longer observed. As a conclusion, electro-ejaculation under general anesthesia is suitable on wild horses to obtain spermatozoa that should be washed or largely diluted before use for artificial insemination (AI) programs. Anti-GnRH immuno-neutering protocol led to a dramatic decrease of spermatozoa number, motility, and testosterone production. This also induced deep changes in the social structure of the band. Such technique could be considered as an alternative to surgical castration in wild horses.

Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares.

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. Historical treatments were invasive and had adverse effects on the endometrium. Hysteroscopically guided laser therapy is easy and effective for endometrial cysts resection, with no deleterious effects for the endometrium. A 110 cm long and 1.0 cm wide endoscope is sterilely introduced in the uterus through the open cervix of an estrous mare after vulvar cleaning. The uterus is slowly infused with less than 1 L of physiologic solution and the laser fiber is inserted in the biopsy canal of the endoscope. Cysts are then cauterized with the 980 nm diode laser with a contact fiber set at 20‒2 5W in continuous mode. Each cyst is punctured until complete voiding of the cyst and shrinking of the cyst wall around the fiber. Uterine lavages with sterile saline solution are performed directly after the surgery and for one or two days as non-inflammatory fluid can be observed. This procedure is easy and quickly performed, with no obvious deleterious effects. Cysts resection makes ultrasound pregnancy diagnosis easier and, in some cases, could restore proper embryo migration in the uterine horns between day 6.5 and 17. However, this treatment does not improve the underlying histological lesions related to endometriosis. These considerations should be clearly expressed to the breeder before this procedure.

Two-dimensional speckle tracking echocardiography in goats: repeatability, variability, and validation of the technique using an exercise test and an experimentally induced acute ischemic cardiomyopathy.

BACKGROUND: Two-dimensional speckle tracking (2DST) technique has been validated in numerous animal species, but neither studies of repeatability nor measurements after exercise or in animals with cardiac disease have been reported in goats. Goats are an attractive candidate for animal models in human cardiology because they are easy to handle and have a body and heart size comparable to that of humans. Therefore, the aim of this study was to validate this technique in goats for further clinical and experimental applications in this species. RESULTS: This study was divided into several steps. First, a standardized echocardiographic protocol was performed and 5 cineloops of a right parasternal short-axis view at papillary muscles level were recorded three times at one-day intervals in ten healthy adult unsedated Saanen goats to test repeatability and variability of 2DST measurements. Then, the same measurements were performed immediately before and after a standardized exercise on treadmill in seven of the goats, and at 24 h after induction of an experimental ischemic cardiomyopathy in five of the goats, to test the reliability of the technique to assess physiological and pathological changes. Average and regional measurements of radial and circumferential strain and strain rate, radial displacement, rotation and rotation rate were obtained. Comparisons were performed using two-way ANOVA (p < 0.05). Caprine 2DST average measurements have demonstrated a good repeatability with a low to moderate variability for all measurements except for the diastolic peaks of the circumferential strain rate, radial strain rate and rotation rate. Segmental 2DST measurements were less repeatable than average measurements. Time effect of two-way ANOVA was significant for anteroseptal segment diastolic peaks measurements, rotation and rotation rate measurements. Overall variability of segmental measurements was moderate or high. Segmental and average peak values obtained after exercise and after myocardial ischemia were significantly different than curves obtained at baseline. CONCLUSIONS: The results of this study are consistent with those previously described in other animal species and humans. 2DST echocardiography is a valid technique to evaluate physiological and pathological changes in myocardial function in goats, despite the technical limitations observed in this species.

First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods.

Most wild donkey breeds are severely threatened by poaching for meat, habitat loss, and competition with livestock for food resources. Moreover, due to the mechanization in agriculture and in transport, most domestic donkey breeds are at risk of extinction. Considering the importance of biodiversity and preservation of genetic resources, the creation of genetic banks for endangered donkey breeds is urgently needed. Cryopreservation of immature jennies oocytes would be an efficient tool to allow storage of female genetics. The aim of the present study was to establish conditions for immature donkey oocyte vitrification, using equine oocytes as a control. Asine and equine immature cumulus-oocyte complexes were collected by transvaginal ultrasound-guided follicular aspiration and flushed to obtain oocytes surrounded by only corona radiata. Oocytes were vitrified after exposure to increasing concentrations of dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectants in a solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin. Oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation. The recovery rate was 48% for jennies oocytes (4.8 oocyte per female) and 42% for mares oocytes (3.5 oocyte per female). When oocytes were exposed to cryoprotectants in INRA-Freeze™ medium none of the jennies re-warmed oocytes matured, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation. When oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured. In conclusion, we developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. Their competence for fertilization and development has to be ascertain.

In Vivo Validation of a Less Invasive Gastrostimulator.

Gastrointestinal stimulator implants have recently shown promising results in helping obese patients lose weight. However, to place the implant, the patient currently needs to undergo an invasive surgical procedure. We report a less invasive procedure to stimulate the stomach with a gastrostimulator. After attempting fully endoscopic implantation, we more recently focused on a single incision percutaneous procedure. In both cases, the challenges in electronic design of the implant are largely similar. This article covers the work achieved to meet these and details the in vivo validation of a gastrostimulator aimed to be endoscopically placed and anchored to the stomach.

Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse.

Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected. Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.