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.
Mlodawska W, Okólski A.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...
Hinrichs K, Matthews GL, Freeman DA, Torello EM.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.
Goudet G, Leclercq L, Bézard J, Duchamp G, Guillaume D, Palmer E.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...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F.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 inje...
Grøndahl C, Hansen TH, Hossaini A, Heinze I, Greve T, Hyttel P.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...
Goudet G, Bézard J, Duchamp G, Gérard N, Palmer E.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...
Hinrichs K, Williams KA.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...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato F.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...
Sosnowski J, Lechniak D, Brzozowska M, Switoński M.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...
Alm H, Hinrichs K.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...
Hochi S, Kozawa M, Fujimoto T, Hondo E, Yamada J, Oguri N.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 ...
Squires EL, Cook NL.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.
Squires EL.Equine oocytes obtained either by transvaginal ultrasound-guided follicular aspiration or from slaughterhouse ovaries can be matured in vitro. This generally requires culture in TCM-199 containing serum and hormones for 30 to 36 hours. With this protocol, approximately 50% to 60% of the oocytes are at metaphase-II at the end of the culture period. At least some of these oocytes appear viable based on production of fertilized eggs either through in vitro fertilization or fertilization in vivo of a recipient mare. The success of producing equine embryos in vitro is still extremely low. More than...
Dell'aquila ME, Fusco S, Lacalandra GM, Maritato F.The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with froz...
Dobrinski I, Thomas PG, Ball BA.Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3...
Grøndahl C, Hyttel P, Grøndahl ML, Eriksen T, Gotfredsen P, Greve T.The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscop...
Brinsko SP, Ball BA, Ellington JE.Oocytes were harvested from mare ovaries obtained at slaughter and were divided into 3 groups based on the age of the donor. The age groups consisted of young (2 to 7 yr), middle-aged (8 to 14 yr) and aged (>or=15 yr) mares. There were no differences between age groups in the proportions of follicles available for examination or the proportions of normal, abnormal or total oocytes collected. After 24 h of culture, the overall maturation rate to the second metaphase (MII) was 52.7%. Maturation rates for oocytes obtained from young and middle-aged mares were similar, but oocytes from aged mar...
Del Campo MR, Donoso X, Parrish JJ, Ginther OJ.Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages...
Hinrichs K, Martin MG, Schmidt AL, Friedman PP.Two experiments were conducted to evaluate the effect of follicular components on the maintenance of meiotic arrest in horse oocytes. In Expt 1, oocytes were incubated for 24 h with follicular fluid, or with granulosa cells suspended either in medium or in follicular fluid at 25 x 10(6) cells ml-1. None of the treatments resulted in significant maintenance of the germinal vesicle stage over that of non-suppressive control. Culture with follicular fluid plus granulosa cells resulted in a significantly higher proportion of oocytes at metaphase I compared with controls. In Expt 2, oocytes were di...
Del Campo MR, Del Campo CH, Mapletoft RJ, Ginther OJ.Morphology and location of the attached cumulus-oocyte complex (COC) were studied in slaughter-house ovaries in horses (49 follicles, 9 to 44 mm), cattle (68 follicles, 6 to 18 mm), and llamas (38 follicles, 3 to 14 mm). The expected point of ovulation was marked, using the ovulation fossa in mares and the center of the projecting follicular surface in cattle and llamas. A follicle was dissected from an ovary, and tissue was removed from the follicle until the COC became visible by transillumination. However, most llama follicles protruded prominently from the ovarian surface so that dissectio...
Azuma T, Choi YH, Hochi S, Oguri N.The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or ...
Braun J.Although foals born after embryo transfer are eligible for registration in the majority of horse breeds, application of embryo transfer is still rare. This is mainly due to the lack of a possibility for superovulation. Uterine stage embryos can be recovered by a non-surgical flushing technique. Transfer can be accomplished by non-surgical as well as surgical methods. In contrast to the situation in cattle, most related technologies are scarcely available. Methods of cryopreservation as well as bisection of embryos are hampered by the fact that suitable embryos (morula) can be collected from th...
Younis AI, Sehgal PK, Biggers JD.A single s.c. injection of 1000 IU of pregnant mare's serum gonadotrophin (PMSG) stimulates the growth of multiple antral follicles in cynomolgus monkeys (Macaca fascicularis). The number of cumulus-enclosed oocytes (CEO) from six non-stimulated controls was 36 (mean = 6). In contrast, a total of 95 CEO (mean = 31.7) were recovered from three animals stimulated and ovariectomized 3 days later, while 385 CEO (mean = 128.3) were obtained from three animals stimulated and ovariectomized 4 days later. A comparison of the effects of highly purified human follicle-stimulating hormone (FSH), human lu...
Redina OE, Amstislavsky SYa , Maksimovsky LF.This study examined the developmental capacity of oocytes in DD mice after they had been injected with pregnant mares' serum gonadotrophin at different stages of the oestrous cycle. The superovulation of mature DD mice at pro-oestrus, oestrus and metoestrus resulted in a large yield of viable embryos. The proportion of abnormal embryos was highest after injection of pregnant mares' serum gonadotrophin at dioestrus. The pool of viable oocytes was most synchronized with normal development after the hormone was injected at oestrus. The results demonstrate that oocytes of different morphology coul...
Choi YH, Okada Y, Hochi S, Braun J, Sato K, Oguri N.Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stai...
Willis P, Caudle AB, Fayrer-Hosken RA.Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microscopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 +/- 0.2 degree C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and...
Hochi S, Fujimoto T, Choi YH, Braun J, Oguri N.Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for...
Choi YH, Hochi S, Braun J, Sato K, Oguri N.The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semic...
Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG.The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-specific label. Oocytes were classified as having either compact (CP) or expanded (EX) cumuli at the time of collection. Centrifugation of oocytes was effective in allowing visualization of the germinal vesicle. Two main chromatin configurations were found in oocytes known to have a germinal vesicle: condensed chromatin (CC), in which the chromatin formed a dense mass surrounding the nucleolus; and fluorescing nucleus (FN), in which the entire nucleu...
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB, Dunbar BS.This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubiliz...
Desantis S, Ventriglia G, Zizza S, De Santis T, Di Summa A, De Metrio G, Dell'aquila ME.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...
Love LB, Choi YH, Love CC, Varner DD, Hinrichs K.Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocyte...
Choi YH, Love LB, Varner DD, Hinrichs K.Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not diff...
Benammar A, Derisoud E, Vialard F, Palmer E, Ayoubi JM, Poulain M, Chavatte-Palmer P.Although there are large differences between horses and humans for reproductive anatomy, follicular dynamics, mono-ovulation, and embryo development kinetics until the blastocyst stage are similar. In contrast to humans, however, horses are seasonal animals and do not have a menstrual cycle. Moreover, horse implantation takes place 30 days later than in humans. In terms of artificial reproduction techniques (ART), oocytes are generally matured in vitro in horses because ovarian stimulation remains inefficient. This allows the collection of oocytes without hormonal treatments. In humans, in viv...
Schmid RL, Kato H, Herickhoff LA, Schenk JL, McCue PM, Chung YG, Squires EL.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...
Kölle S, Dubois CS, Caillaud M, Lahuec C, Sinowatz F, Goudet G.In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. ...
Meuffels-Barkas J, Wilsher S, Allen WRT, Ververs C, Lueders I.Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we post...
Morris LHA.The development of techniques to produce equine embryos in vitro is reviewed with specific reference to intracytoplasmic sperm injection (ICSI). Unexplored 50 years ago, this technology has progressed rapidly in the last 20 years to become a commercial reality for the equine breeding industry. Improvements in our understanding of oocyte and embryo competence in the horse have been key factors in overcoming some of the initial problems associated with ICSI. It is now possible to obtain high nuclear maturation and cleavage rates in vitro and the most limiting factor, presently, is the low rate o...
Cook NL, Masterson KR, Battaglia D, Beck R, Metcalf ES.Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxid...
Dell'Aquila ME, De Felici M, Massari S, Maritato F, Minoia P.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...
Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G.Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo devel...
Salgado RM, Brom-de-Luna JG, Resende HL, Canesin HS, Hinrichs K.The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. Methods: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between inject...
Angel-Velez D, Meese T, Hedia M, Fernandez-Montoro A, De Coster T, Pascottini OB, Van Nieuwerburgh F, Govaere J, Van Soom A, Pavani K, Smits K.In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), a...
Catandi GD, LiPuma L, Obeidat YM, Maclellan LJ, Broeckling CD, Chen T, Chicco AJ, Carnevale EM.Dietary supplementation is the most feasible method to improve oocyte function and developmental potential . During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant s...
Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Carnevale EM.Intracytoplasmic sperm injection (ICSI) is an established method to fertilise equine oocytes, but not all oocytes cleave after ICSI. The aims of the present study were to examine cytoskeleton patterns in oocytes after aging in vitro for 0, 24 or 48h (Experiment 1) and in potential zygotes that failed to cleave after ICSI of oocytes from donors of different ages (Experiment 2). Cytoplasmic multiasters were observed after oocyte aging for 48h (P<0.01). A similar increase in multiasters was observed with an increased interval after ICSI for young mares (9-13 years) but not old (20-25 years) mares...
Sessions-Bresnahan DR, Graham JK, Carnevale EM.IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine ...
Squires EL, McCue PM.Embryo recovery from single ovulating mares is approximately 50 per cent per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A purified, ...
Mlodawska W, Okolski A.The effect of filly age on morphology of the ovaries, collected oocytes and their capacity for in vitro maturation (IVM) was examined. The ovaries of slaughtered fillies were classified into three groups, according to filly age: (I) <10 month old (<10MF); (II) approximately 1 year old (1YF); and (III) approximately 1.5 year old (1.5YF). The ovaries of mares were used as a control group. Ovarian morphology and collected oocytes were evaluated. Only oocytes with expanded (Ex) and compact (Cm) cumuli were used for IVM. In <10MF, 1YF, 1.5YF and mare groups, corpora lutea were found in the...
Martinez de Andino EV, Brom-de-Luna JG, Canesin HS, Rader K, Resende HL, Ripley AM, Love CC, Hinrichs K.To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). Methods: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IF...
Accogli G, Douet C, Ambruosi B, Martino NA, Uranio MF, Deleuze S, Dell'Aquila ME, Desantis S, Goudet G.Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern diffe...
Blanco ID, Devito LG, Ferreira HN, Araujo GH, Fernandes CB, Alvarenga MA, Landim-Alvarenga FC.This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25mg EPE and treated with 2500 IU hCG, (2) superovulation with 25mg EPE, and (3) cont...
Lewis N, Hinrichs K, Leese HJ, McGregor Argo C, Brison DR, Sturmey RG.The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturat...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF.Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be...
Clérico G, Taminelli G, Veronesi JC, Polola J, Pagura N, Pinto C, Sansinena M.Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato F.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...
Marchal R, Caillaud M, Martoriati A, Gérard N, Mermillod P, Goudet G.The aim of this study was to investigate the role of growth hormone (GH) on in vitro cumulus expansion and oocyte maturation in equine and porcine cumulus-oocyte complexes (COCs), and to approach its way of action. Equine COCs were cultured in a control medium (TCM199, 5 mg/ml BSA, 1 microg/ml estradiol, and antibiotics) supplemented with either 0.5 microg/ml equine GH or 5 microg/ml equine LH. Porcine COCs were cultured in a basal medium (TCM199 with 570 microM cysteamine) supplemented with 0, 0.1, 0.5, or 1 microg/ml porcine GH or in a control medium (basal medium with 10 ng/ml epidermal gro...
Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, Paccamonti D, Graff KJ, Godke RA.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...
Hinrichs K.Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
Roels K, Smits K, Ververs C, Govaere J, D'Herde K, Van Soom A.In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with det...
