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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.
Ultrastructure, protein phosphorylation and mRNA status of equine oocytes matured in vivo and in vitro. Equine oocytes were collected by follicle aspiration in vivo or by dissection of material obtained from an abattoir, and the ultrastructure, protein phosphorylation and mRNA status of the oocytes were evaluated. Electron microscopy studies indicated that the nucleus had a smooth membrane in oocytes with a compact cumulus, whereas the nuclear membrane was undulated in all other groups. Oocytes with compact cumuli had only a few microvilli, whereas those with expanded cumuli had more microvilli. There were only small numbers of cortical granules close to the oolemma in oocytes with compact cumul...
Effect of timing of follicle aspiration on pregnancy rate after oocyte transfer in mares. Mares with preovulatory follicles >33 mm in diameter were administered hCG and were randomly assigned for aspiration of the dominant follicle at 24 h or 35 h after hCG administration. Oocytes recovered at 24 h were cultured for 12 h before transfer and oocytes recovered at 35 h were cultured for 1 h. Oocytes were transferred by flank laparotomy to the oviduct of the same mare, or to the oviduct of another oocyte donor. Recipient mares were inseminated before and after transfer. The oocyte recovery rates at 24 h and 35 h after hCG administration were not significantly different (10/15 (66%) ...
Apoptosis in equine granulosa cells and its relationship to cumulus expansion and oocyte chromatin configuration in ovarian follicles. During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa ...
Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa. In Expt 1, compact cumulus oocyte complexes (COCs) were matured in: (i) control medium (Hepes-buffered TCM-199 with 10% oestrous cow serum (OCS) + oestradiol, LH and FSH); (ii) Hepes-buffered TCM-199 with 20% follicular fluid; or (iii) control medium containing 250 ng progesterone ml(-1). Mature oocytes were collected by transvaginal aspiration as a positive control for the in vitro maturation (IVM) treatments. Oocytes were fertilized by ICSI and cultured in Menezo's B2 + 5% fetal calf serum (FCS). There were no significant differences among IVM treatments. In Expt 2, oocytes with expanded COC...
Production of live foals from sperm-injected oocytes harvested from pregnant mares. In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilizat...
Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro. In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increas...
Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare. The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm w...
Morphology of the oocyte-follicular connection in the mare. The present study characterised the oocyte-follicular connection (i.e., oocyte fixation site) in Graafian follicles of the mare morphologically. Antral follicles were dissected in toto from ovaries obtained from oestrous, dioestrous and transitional mares after slaughter. The location of the cumulus oophorus complex in relation to the ovulation fossa, the width and density of the blood vessels surrounding the cumulus oophorus complex, the relative dimensions and histological aspects of the cumulus oophorus were investigated. For ultrastructural analysis of the junctional regions, cumulus-oocyt...
Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation. We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+ channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopus oocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to -10 mV on conditioning prepulse voltages, could be shifted in...
Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare. The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro matur...
Maturation-promoting factor (MPF) and mitogen activated protein kinase (MAPK) expression in relation to oocyte competence for in-vitro maturation in the mare. In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We...
Cumulus expansion, chromatin configuration and meiotic competence in horse oocytes: a new hypothesis. When recovered from the follicle, horse oocytes may be categorised as having either a compact or an expanded cumulus. Cumulus expansion is strongly associated with follicle atresia. Oocytes with expanded and compact cumuli have similar proportions in the germinal vesicle stage when recovered from the follicle. However, during in vitro culture, a higher proportion of oocytes with expanded cumuli mature, and they do so more quickly, than do oocytes with compact cumuli. Using Hoechst 33258 to label chromatin, in the germinal-vesicle stage horse oocytes can be divided into those in which the nucle...
Follicular fluid is not a compulsory carrier of the oocyte at ovulation in the mare. The aim of this study was to test the possibility that ovulation can occur from a preovulatory follicle emptied of its follicular fluid. Transport of the oocyte into the oviduct and fertilisation in 29% of cases demonstrated that ovulation can occur in the absence of follicular fluid but the higher fertility achieved in control mares (62.5%) suggested that follicular fluid does serve a role during ovulation, fertilisation and oviductal transport. Injection of horse oocytes into preovulatory follicles in mules after removal of the follicular fluid, followed by insemination of the mules with hor...
Treatment of equine oocytes with A23187 after intracytoplasmic sperm injection. In vitro matured horse oocytes with a first polar body (n = 68) were each injected with a single spermatozoon and divided into 2 groups: Group 1 oocytes were treated with 10 microM calcium ionophore A23187 for 5 min while Group 2 oocytes received no activation treatment. After culture in vitro for 2 days, significantly more oocytes treated with A23187 (5/24, 21%) cleaved than oocytes without activation treatment (2/44, 5%, P<0.05). All 7 cleaved zygotes from both treatment groups were transferred to recipient mares but no pregnancies resulted.
Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare? In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes ...
Comparison of different methods for the recovery of horse oocytes. The object of this study was to compare 4 different methods of oocyte recovery from mares; 1) transvaginal follicle aspiration in vivo; 2) follicle aspiration in vitro; 3) oocyte recovery by isolation of follicles in vitro and 4) follicle scraping in vitro. Oocyte recovery was highest after follicle scraping (71.1%) and follicle isolation and rupture (61.3%). Follicle aspiration in vitro and in vivo yielded oocytes on 31.2% and 19.3% of occasions, respectively. The output of different types of cumulus-oocyte-complexes was different among the methods; the portion of compact cumulus-oocyte-compl...
Timing of in vivo maturation of equine preovulatory oocytes and competence for in vitro maturation of immature oocytes collected simultaneously. The objects of this study were to monitor the development of the cumulus complex and nuclear maturation in oocytes recovered from preovulatory follicles following treatment to induce ovulation and to investigate the in vitro maturation competence of oocytes recovered from smaller nonpreovulatory follicles of varying size. All follicles > or =5 mm in pony mares were individually punctured at 0, 6, 12, 24 and 35 h after an injection of LH to induce ovulation. The recovery rates of oocytes were 64% from 55 preovulatory follicles, 22% from 32 subordinate follicles and 52% from 227 small follicl...
Spontaneous multiple ovulation and development of multiple embryonic vesicles in a mare. A Warmblood mare was observed to ovulate spontaneously 12 follicles within 2 days, none of which exceeded 22 mm in diameter. On Days 13 and 17 after ovulation, 6 embryonic vesicles were identified in the uterus by ultrasonography but by Day 26, 5 of the vesicles had disappeared. Development of the surviving conceptus was monitored until Day 42. Plasma progesterone concentrations rose to 14 ng/ml on Day 7, decreased over the next 8 days and then plateaued to around 4-6 ng/ml until Day 70. The occurrence of multiple spontaneous ovulations was diagnosed repeatedly in this mare. However, the devel...
Equine oocyte-cumulus morphology as affected by follicular size. From the ovaries of 256 slaughtered mares a total of 1713 follicles were isolated from which 1641 (95.8%) oocytes were recovered (6.4/mare). A total of 564 follicles and oocytes were evaluated for the degree of vascularisation of the follicle wall, the appearance of the follicular fluid and the location and morphology of the cumulus-oocyte-complex. Follicles with a diameter of >10 mm displayed more numerous, well branched and more pronounced blood vessels than the smaller ones (4-10 mm diameter) and most of them contained clear, yellowish fluid with few granulosa cells. The percentage of oo...
Oocyte transfer in mares. Oocytes were collected from dominant preovulatory follicles of donor mares 24 hours after administration of human chorionic gonadotropin. Oocytes were incubated in vitro for 12 or 18 hours before transfer to recipient mares, representing maturation times after human chorionic gonadotropin administration of 36 and 42 hours, respectively. Pregnancy rates after transfer were 4 of 5 in the 36-hour group and 2 of 3 in the 42-hour group. The overall pregnancy rate achieved (6/8 mares) indicated that oocyte transfer may be useful clinically.
Chorionic gonadotropin secretion is associated with an inhibition of follicular growth and an improvement in oocyte competence for in vitro maturation in the mare. This study reports the follicular growth and oocyte competence for in vitro maturation and fertilization under the influence of circulating eCG. Three to 7 successive ultrasound-guided follicular punctures were performed on 4 pregnant mares from Day 23 until Day 75 of pregnancy and on 5 control mares whose embryonic vesicle was crushed on Day 22. All follicles larger than 5 mm were punctured 24 h after the largest follicle reached 18 mm. Expanded cumulus oocyte complexes (COCs) were stained at recovery to analyze the nuclear stage. Compact COCs were cultured in vitro for 46 h and either staine...
Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in-vitro fertilization or intracytoplasmic sperm injection. The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm i...
Intracytoplasmic sperm injection of in vitro-matured equine oocytes. Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and process...
Equine oocyte competence for nuclear and cytoplasmic in vitro maturation: effect of follicle size and hormonal environment. Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal ph...
Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse. Horse oocytes with expanded (EX) cumuli appear to have greater meiotic competence than do horse oocytes with compact (CP) cumuli but are thought to come from atretic follicles. We evaluated the relationships among cumulus expansion, follicle viability, initial chromatin configuration, and meiotic competence of horse oocytes. Follicle walls were sectioned for histological examination, and the follicles were scraped to obtain the oocytes. Half of the oocytes were evaluated immediately and half were matured for 24 h in vitro. Cumulus expansion was significantly associated with follicle atresia. I...
Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes. Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment...
Cytogenetic analysis of horse oocytes matured in vitro for different periods of time. This paper presents the results of recovering horse oocytes by aspiration and maturation in vitro for 24, 30, 36 or 42 h. A total of 522 oocytes were recovered from 221 ovaries (2.4 per ovary) and 271 oocytes (51.9%) were selected for in vitro maturation (IVM). Oocytes were cultured in maturation medium (TCM 199 + estrus cow serum [ECS] + follicle-stimulating hormone [FSH] + 17 beta-estradiol + gentamycin). One hundred and seventy oocytes were cytogenetically analysed (68.3%). Cytogenetic analysis showed that the stage of maturation (first telophase-TI or second metaphase-MII) for fertilizatio...
Effect of cycloheximide on nuclear maturation of horse oocytes and its relation to initial cumulus morphology. The period of protein synthesis necessary for meiotic maturation in horse oocytes initially having compact or expanded cumulus cells was studied. Oocytes incubated in the presence of cycloheximide after 0, 4, 8, 12 or 16 h maturation in vitro (total incubation time 24 h) were matured for 24 h, or were incubated with cycloheximide for 24 h and then matured for 24 h. Incubation with cycloheximide from 0 h was effective in suppressing maturation (no significant increase in maturing oocytes compared with controls fixed directly after removal from the follicle) in both expanded and compact groups a...
In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification. The study was designed to examine the suitability of immature horse oocytes for vitrification. Immature oocytes derived from slaughtered horse ovaries were transferred to a vitrification solution (EFS; 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in modified phosphate-buffered saline) directly (Groups 1 and 4) or were first exposed to 20% ethylene glycol solution for 10 min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20 degrees C (Groups 1, 2, and 3) or 30 degrees C (Groups 4, 5, and 6). After vitrification and warming, their viability was assessed by maturation ...
Transvaginal aspiration. This article describes in detail the procedures for collection of equine oocytes using a transvaginal ultrasound probe. Success in obtaining oocytes from humans, bovines, and horses are presented. The effect of repeated follicular aspiration of both cattle and horses is reviewed.
