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Topic:Oocyte
Oocytes in horses are the female gametes involved in reproduction, playing a fundamental role in equine fertility and breeding. These cells are essential for the development of embryos following fertilization and are a focus of study in reproductive biology and veterinary medicine. Research on equine oocytes includes their maturation, quality, and the factors influencing their developmental competence. Studies often explore techniques for in vitro maturation and fertilization, as well as the impact of various physiological and environmental factors on oocyte viability. This page compiles peer-reviewed research studies and scholarly articles that examine the characteristics, developmental processes, and technological advancements related to oocytes in horses.
Assisted reproduction techniques in the horse. This paper reviews current equine assisted reproduction techniques. Embryo transfer is the most common equine ART, but is still limited by the inability to superovulate mares effectively. Immature oocytes may be recovered by transvaginal ultrasound-guided aspiration of immature follicles, or from ovaries postmortem, and can be effectively matured in vitro. Notably, the in vivo-matured oocyte may be easily recovered from the stimulated preovulatory follicle. Standard IVF is still not repeatable in the horse; however, embryos and foals can be produced by surgical transfer of mature oocytes to th...
Evaluation of foal production following intracytoplasmic sperm injection and blastocyst culture of oocytes from ovaries collected immediately before euthanasia or after death of mares under field conditions. To evaluate the efficiency of foal production following intracytoplasmic sperm injection (ICSI) and blastocyst culture of oocytes from mares that died or were euthanized under field conditions. Methods: Prospective case series. Methods: 16 mares (age, 3 to 19 years) that died or were euthanized for various causes. Methods: Ovaries were collected immediately before euthanasia (n = 10) or after death (6). Ovaries were transported to the laboratory for oocyte recovery (15 mares), or oocytes were recovered at a remote location and shipped to the laboratory (1). Oocytes underwent ICSI, and presumpt...
Follicular fluid leptin concentrations and expression of leptin and leptin receptor in the equine ovary and in vitro-matured oocyte with reference to pubertal development and breeds. There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, ...
Effects of FSH and LH on ovarian and follicular blood flow, follicular growth and oocyte developmental competence in young and old mares. Objectives of the experiment were to determine the effects of mare age and gonadotropin treatments on dominant follicle vascularity, ovarian blood flow and dominant follicle growth and to associate follicular vascularity with oocyte developmental capacity. Growing follicles >30 mm from young (4-9 years) and old (>20 years) mares were assessed for blood flow using color Doppler ultrasonography before maturation induction with recombinant equine LH (eLH) and immediately prior to oocyte collection at 20-24 h after eLH. Pulsed Doppler was used to obtain resistance indices of ovarian arteries...
Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes. Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro ma...
Fertilisation in the horse and paracrine signalling in the oviduct. The mammalian oviduct plays a crucial role in the preparation of gametes for fertilisation (transport and final maturation) and fertilisation itself. An increasing number of studies offers a comprehensive overview of the functions of the oviduct and its secretions, but this topic has had limited investigation in the horse. Limited data are available on the final oocyte maturation in the equine oviduct. However, in vitro and in vivo systems have been established to analyse the influence of equine oviduct epithelial cells (OEC) during maturation on the potential of oocytes for fertilisation and ...
In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model. Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of...
A pilot comparison of laser-assisted vs piezo drill ICSI for the in vitro production of horse embryos. Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells. The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P &...
Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker. The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in t...
G6PDH-activity in equine oocytes correlates with morphology, expression of candidate genes for viability, and preimplantative in vitro development. Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) matur...
The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes. The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained wi...
In vitro maturation and artificial activation of donkey oocytes. Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glut...
Efficacy of tuohy needle in oocytes collection from excised mare ovaries. TWO METHODS HAVE BEEN DESCRIBED TO RECOVER OOCYTES FROM EQUINE FOLLICLES IN EXCISED OVARIES: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (P < .05) than in the 16 g needle aspiration and ...
The equine oocyte: factors affecting meiotic and developmental competence. There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli...
In vitro production of equine embryos: state of the art. In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development ...
Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation). The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Th...
Enhancing reproductive performance in mares. Reproductive performance in mares can be enhanced by various techniques. Protocols hastening the onset of follicular development help establish pregnancy in mares and ensure that foals are born early in the year. The time spent breeding mares can be reduced by synchronizing estrus and inducing ovulation. After successful fertilization of the oocyte, the developing embryo can survive in the uterus only if postbreeding endometritis, if present, is treated.
Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares. Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in result...
Evaluation of the function of fresh and frozen-thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay. The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-t...
Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved? Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Results: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophor...
Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse. The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity ...
Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer. We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
The extracellular calcium-sensing receptor is expressed in the cumulus-oocyte complex in mammals and modulates oocyte meiotic maturation. The extracellular calcium-sensing receptor (CASR) plays an important role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in the extracellular Ca2+ ion concentration. We previously reported the localization and quantitative expression of CASR protein in human oocytes. In this study, we examined the expression and the functional role of CASR during oocyte meiotic maturation in a large mammal animal model, the horse. As in humans, CASR protein was found to be expressed in equine oocytes and cumulus cells. Western-blot analysis revealed a single 130 kDa band in denuded...
Optimalization of fluorescence in situ hybridization conditions in mare oocytes and mouse embryos. The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes an...
Lectin-binding sites in isolated equine cumulus-oocyte complexes: differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus. Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA(120), and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of bot...
Effects of age and equine follicle-stimulating hormone (eFSH) on collection and viability of equine oocytes assessed by morphology and developmental competency after intracytoplasmic sperm injection (ICSI). Young (4 to 9 yr) and old (>or=20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo d...
Efficiency of embryonic development after intrafollicular and intraoviductal transfer of in vitro and in vivo matured horse oocytes. In vivo techniques, such as intraoviductal oocyte transfer (OT) and intrafollicular oocyte transfer (IFOT), can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production after transfer of in vivo recovered oocytes (1) into a recipient's oviduct or (2) into her preovulatory follicle either immediately after ovum pick-up or (3) after in vitro maturation (IVM). Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten da...
