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.
Ginther OJ, Gastal EL, Gastal MO, Siddiqui MA, Beg MA.The effects of ultrasound morphology, vascularity, and follicular-fluid hormones of the preovulatory follicle on oocyte recovery rate and on follicle and oocyte maturity rates were studied for 60 spontaneous and solitary preovulatory follicles in mares. An ovulation-inducing dose of hCG was given when the follicle was >or=32 mm (Hour 0), and a procedure for oocyte recovery was done 30 h later (Hour 30). Between Hours 0 and 30, diameter of the follicle increased less and circulating estradiol (E2) concentrations decreased more in groups with successful versus nonsuccessful oocyte recovery an...
Lindbloom SM, Farmerie TA, Clay CM, Seidel GE, Carnevale EM.Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when...
Torner H, Alm H, Kanitz W, Goellnitz K, Becker F, Poehland R, Bruessow KP, Tuchscherer A.The aim of this investigation was to examine the chromatin configuration of the nucleus, pattern of mitochondrial aggregation and mitochondrial activity in parallel studies in the same horse oocytes. Horse oocytes recovered by ultrasound-guided follicle aspiration in vivo were classified according to two main initial cumulus morphologies as having compact or expanded cumulus. The percentage of oocytes with a diplotene meiotic configuration at the time of recovery from the follicles was highest in compact oocytes. Oocytes with expanded cumulus layers at the time of recovery matured more rapidly...
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. ...
Carnevale EM, Maclellan LJ.Assisted reproductive techniques have been developed to obtain pregnancies from subfertile mares and stallions and to salvage gametes after death. In recent years, these procedures have been used for clinical cases with repeated success. Although new developments occur, the basis for the success and future development of assisted reproductive techniques is our ability to collect and handle the equine oocyte successfully. This article focuses on important clinical aspects of oocyte collection and evaluation and briefly discusses the clinical use of assisted reproductive procedures in the horse....
Tharasanit T, Colleoni S, Lazzari G, Colenbrander B, Galli C, Stout TA.Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls....
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...
Choi YH, Love LB, Varner DD, Hinrichs K.The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured...
Purcell SH, Seidel GE, McCue PM, Squires EL.The aim of this study was to compare the efficacy of three approaches for recovering equine oocytes via transvaginal ultrasound-guided follicular aspiration. Fourteen mares were used as oocyte donors during the spring transition period and physiologic breeding season, and 11 mares were bred for use as oocyte donors during early gestation. In all mares, large (>20 mm) and small (10-20 mm) follicles were aspirated in eight rounds every 10-11 days. In each of the four rounds during the transition period, half the mares received 12.5 mg eFSH once daily for 4 days prior to aspiration. For each of t...
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, ...
Choi YH, Love LB, Varner DD, Hinrichs K.This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In...
Li X, Qin Y, Wilsher S, Allen WR.Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII s...
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...
Luciano AM, Goudet G, Perazzoli F, Lahuec C, Gérard N.The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte c...
Tharasanit T, Colenbrander B, Stout TA.Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as contr...
Vanderwall DK, Hyde KJ, Woods GL.To determine whether performance of transvaginal ultrasound-guided follicle aspiration (TVUFA) repeatedly in mares adversely affects their fertility. Methods: Historical prospective study. Methods: 23 mares that had never undergone TVUFA and 59 mares that had undergone TVUFA on 1 to 11 occasions. Methods: Mares were classified into 4 groups according to the number of TVUFA procedures previously performed on the ovary in which ovulation occurred at the time of insemination as follows: group 1, 0 TVUFAs (control group, n = 23 mares); group 2, 1 or 2 TVUFAs (40 mare-cycles); group 3, 3 or 4 TVUFA...
Conforti VA, Vanderwall DK, Woods GL.The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n ...
Mari G, Barbara M, Eleonora I, Stefano B.Ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) is a procedure applied in equine-assisted reproduction programs such as oocyte transfer and in vitro embryo production. Despite a large number of studies reporting that it is a repeatable and safe technique, little information is available about the effect of repeated punctures on fertility of mares. Moreover, even if flushing follicles improves the oocyte recovery rate, to our knowledge the efficiency of flushing estrous and diestrous follicles has not been evaluated. The aims of the present study were (1) evaluate if repe...
Hartt LS, Carling SJ, Joyce MM, Johnson GA, Vanderwall DK, Ott TL.Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected duri...
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 ...
Caillaud M, Duchamp G, Gérard N.A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. In...
Gil L, Saura S, Echegaray A, Martinez F, de Blas I, Akourki A, Gonzalez N, Espinosa E, Josa A.The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (...
Carnevale EM, Coutinho da Silva MA, Panzani D, Stokes JE, Squires EL.Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected i...
Vanderwall DK, Woods GL, Aston KI, Bunch TD, Li G, Meerdo LN, White KL.The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior...
Hinrichs K, Choi YH, Love LB, Varner DD, Love CC, Walckenaer BE.We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the ...
Carnevale EM, Coutinho da Silva MA, Maclellan LJ, Seidel GE, Squires EL.Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation. Recipients were inseminate...
Carnevale EM.Methods for the collection and transfer of equine oocytes have been developed, and uses of these techniques have resulted in new clinical and research possibilities. Because oocyte transfer avoids reproductive problems associated with the oviduct, uterus, and cervix, pregnancies can be produced from many mares that cannot carry a pregnancy or produce embryos. Oocytes for clinical transfers are usually collected from preovulatory follicles and cultured for a short interval or transferred directly into a recipient's oviduct. For oocyte transfer, the recipient is inseminated within the uterus. A ...
Pedersen HG, Watson ED, Telfer EE.The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ...
Dell'Aquila ME, Caillaud M, Maritato F, Martoriati A, Gérard N, Aiudi G, Minoia P, Goudet G.The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was incr...
Colleoni S, Luciano AM, Gandolfi F.Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes b...
Flores Bragulat AP, Ortiz I, Catalán J, Dorado J, Hidalgo M, Losinno L, Dordas-Perpinyà M, Yánez-Ortiz I, Martínez-Rodero I, Miró J, Gambini A.Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study...
Brück I, Greve T, Hyttel P.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...
Li X, Morris LH, Allen WR.This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old ...
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...
Goudet G, Belin F, Młodawska W, Bézard J.The effect of epidermal growth factor (EGF) on the in vitro maturation rate of equine oocytes was examined. Oocytes were collected from an abattoir (Expt 1) or using ultrasound-guided follicular puncture in vivo (Expt 2). All oocytes with a compact or expanded cumulus at recovery were cultured for 30 h in: medium 1 (TCM199 + fetal calf serum (FCS) + crude equine gonadotrophin (CEG) + oestradiol + antibiotics); medium 2 (TCM199 + EGF); medium 3 (medium 1 without FCS + EGF); or medium 4 (medium 1 without CEG + EGF). In Expt 1, 84% (37/44) and 87% (40/46) cumulus expansion (P > 0.05), and 39% (22...
Pereira BC, Ortiz I, Dorado J, Consuegra C, Diaz-Jimenez M, Demyda-Peyras S, Gosalvez J, Hidalgo M.DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liqui...
Aguilar JJ, Woods GL, Miragaya MH, Olsen LM, Vanderwall DK.The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would...
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...
Choi YH, Love LB, Varner DD, Hinrichs K.This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-2...
Pereira GR, Lorenzo PL, Carneiro GF, Bilodeau-Goeseels S, Kastelic J, Liu IK.The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein ...
Choi YH, Chung YG, Walker SC, Westhusin ME, Hinrichs K.This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei...
Deleuze S, Dubois CS, Caillaud M, Bruneau B, Goudet G, Duchamp G.Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectiv...
Vanderwall DK, Hyde KJ, Woods GL.To determine whether performance of transvaginal ultrasound-guided follicle aspiration (TVUFA) repeatedly in mares adversely affects their fertility. Methods: Historical prospective study. Methods: 23 mares that had never undergone TVUFA and 59 mares that had undergone TVUFA on 1 to 11 occasions. Methods: Mares were classified into 4 groups according to the number of TVUFA procedures previously performed on the ovary in which ovulation occurred at the time of insemination as follows: group 1, 0 TVUFAs (control group, n = 23 mares); group 2, 1 or 2 TVUFAs (40 mare-cycles); group 3, 3 or 4 TVUFA...
Choi YH, Gibbons JR, Canesin HS, Hinrichs K.Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant di...
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...
Hochi S.Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both i...
Ginther OJ, Gastal EL, Gastal MO, Siddiqui MA, Beg MA.The effects of ultrasound morphology, vascularity, and follicular-fluid hormones of the preovulatory follicle on oocyte recovery rate and on follicle and oocyte maturity rates were studied for 60 spontaneous and solitary preovulatory follicles in mares. An ovulation-inducing dose of hCG was given when the follicle was >or=32 mm (Hour 0), and a procedure for oocyte recovery was done 30 h later (Hour 30). Between Hours 0 and 30, diameter of the follicle increased less and circulating estradiol (E2) concentrations decreased more in groups with successful versus nonsuccessful oocyte recovery an...
Derisoud E, Auclair-Ronzaud J, Palmer E, Robles M, Chavatte-Palmer P.Although puberty can occur as early as 14-15months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects c...
Meyers SA.The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was us...
Love CC, Love LB, Varner DD, Hinrichs K.The relationship of holding time in media at room temperature (approximately 22 degrees C) to initial chromatin configuration and rate of in vitro maturation (IVM) of equine oocytes was determined. Only oocytes having a complete, compact cumulus were used in this study. Oocytes were removed from ovaries 3.5-8 h after slaughter and were put into one of four treatment groups: (1) immediate/fix (IF) = immediate fixation following removal from the ovary; (2) delay/fix (DF) = fixation after oocytes were held 1-4 h in medium at room temperature; (3) immediate/mature (IM) = immediate placement into m...
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...
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. ...
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...
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...
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...
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...
Gable TL, Woods GL.The objective was to test the hypothesis that increasing equine oocyte culture time from 48 to 96 or 144 h increases nucleus maturation of equine oocytes. The hypothesis was not supported because condensed chromatin-stage oocytes decreased (P<0.01) from 33/126 (26.2%) at 48 h or 34/95 (35.8%) at 96 h to 11/117 (9.4%) at 144 h, and polar body-stage oocytes decreased (P<0.01) from 65/126 (51.6%) at 48 h to 25/95 (26.3%) at 96 h and (P<0.01) to 1/117 (0.9%) at 144 h. Negative (non-staining) oocytes increased (P<0.01) from 16/126 (12.7%) at 48 h or 15/95 (15.8%) at 96 h to 39/117 (33.3%) at 144 h....
Merlo B, Iacono E, Bucci D, Spinaci M, Galeati G, Mari G.In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
Leal Reis I, Lopes B, Sousa P, Sousa AC, Branquinho MV, Caseiro AR, Rêma A, Briote I, Mendonça CM, Santos JM, Atayde LM, Alvites RD, Maurício AC.Horses are high-performance athletes prone to sportive injuries such as tendonitis and desmitis. The formation of fibrous tissue in tendon repair remains a challenge to overcome. This impels regenerative medicine to develop innovative therapies that enhance regeneration, retrieving original tissue properties. Multipotent Mesenchymal Stem/Stromal Cells (MSCs) have been successfully used to develop therapeutic products, as they secrete a variety of bioactive molecules that play a pivotal role in tissue regeneration. These factors are released in culture media for producing a conditioned medium (...
Alkhadrawy JMH, Aboelmaaty AM, Abou-Ahmed MM, Ghallab AM.Mares are the only companion animals simulating women in the large diameter of their follicles. Horses start reproduction at the age of three years, and some of them live for >30 years, so aging influences their reproductive capacity. Mares are sensitive to summer heat stress as they can sweat like humans. Unassigned: The current work aimed to study the effects of age (young versus senile), season (cold versus hot), and the hormonal treatments during embryo collection on the dominant and subordinate follicular dynamics and hemodynamics and circulating ovarian hormones in embryo donor mares ...
