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.
Grady ST, Watts AE, Thompson JA, Penedo MCT, Konganti K, Hinrichs K.This study aims to determine if intra-ovarian injection of bone marrow-derived mesenchymal stem cells (MSCs) improves or restores ovarian function in aged females. Methods: Prospective randomized study of eight aged mares and six young mares receiving intra-ovarian injection of MSCs or vehicle. Main outcome measures were antral follicle count and serum anti-Müllerian hormone (AMH) (aged and young mares), and for aged mares, oocyte meiotic and developmental competence; gross and histological ovarian assessment; evaluation of presence of chimerism in recovered granulosa cells and in ovarian tis...
Hendriks WK, Colleoni S, Galli C, Paris DBBP, Colenbrander B, Stout TAE.Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine emb...
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...
Merlo B, Mari G, Iacono E.The increase in demand for in vitro produced horse embryos is fostering the development of commercial laboratories for this purpose. Nevertheless, blastocyst production after intracytoplasmic sperm injection (ICSI) is still not as great as desired in most of these laboratories. In relation to horse oocyte classification, both expanded and compact cumulus-oocyte-complexes (COCs) are used for in vitro embryo production. The aim of this study was to compare in vitro embryo developmental capacity of COCs from horses including those with only the corona radiata, frequently collected after aspiratio...
Hinrichs K.A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm num...
Rizzo M, Ducheyne KD, Deelen C, Beitsma M, Cristarella S, Quartuccio M, Stout TAE, de Ruijter-Villani M.Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. Objective: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro-matured (IVM) oocytes. Methods: Morphometric and morphological analysis. M...
Spacek SG, Carnevale EM.Equine follicular fluid (FF) provides autocrine and paracrine factors from theca, granulosa, and cumulus cells, both reflecting and impacting oocyte and follicle maturation. We hypothesized that maturation of oocytes in FF from old versus young mares has a deleterious effect on oocyte maturation and their subsequent developmental potential. Follicular fluid was collected from the large, dominant follicle from young mares (4-13 years) or old mares (21-26 years) and classified as: (1) Noninduced follicular fluid (NFF), FF from noninduced follicle 33 ± 3 mm, or (2) Induced follicular fluid (I...
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...
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...
Davis KA, Klohonatz KM, Mora DSO, Twenter HM, Graham PE, Pinedo P, Eckery DC, Bruemmer JE.Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n = 10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a ...
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...
Diaw M, Salgado RM, Canesin HS, Gridley N, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at col...
Canesin HS, Brom-de-Luna JG, Choi YH, Pereira AM, Macedo GG, Hinrichs K.Previous studies have found low rates of blastocyst development (0-11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volu...
Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Stokes JE, Carnevale EM.Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's e...
Ortiz-Escribano N, Bogado Pascottini O, Woelders H, Vandenberghe L, De Schauwer C, Govaere J, Van den Abbeel E, Vullers T, Ververs C, Roels K....The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objective: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Methods: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to l...
Douet C, Parodi O, Martino NA, Lacalandra GM, Nicassio M, Reigner F, Deleuze S, Dell'Aquila ME, Goudet G.Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) techn...
Cuervo-Arango J, Claes AN, Ruijter-Villani M, Stout TA.Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus. Objective: To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET). Methods: R...
González-Fernández L, Sánchez-Calabuig MJ, Alves MG, Oliveira PF, Macedo S, Gutiérrez-Adán A, Rocha A, Macías-García B.Equine cumulus-oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This difference could be related to divergent glucose metabolism. To test this hypothesis in the present study, eCOCs, cCOCs and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30h, at which time maturation rate, glucose metabolism and the expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. There were significant differences between eCOCs and cCOCs in maturation ...
Paulini F, Chaves SB, Rôlo JLJP, Azevedo RB, Lucci CM.Visualization and clear understanding of the ovarian structures are important in determining the stage of oestrus, helping to diagnose several pathologies and supporting advances in reproductive technologies. In this research, computerized microtomography (microCT) was used to explore and characterize the ovarian structure of seven mammalian species. Ovaries of rats, female dog, queens, cows, mares, sows and a female donkey were used. After microCT scanning, the same samples were prepared for histologic evaluation, used here as a validation criterion. It was possible to distinguish regions of ...
Franciosi F, Tessaro I, Dalbies-Tran R, Douet C, Reigner F, Deleuze S, Papillier P, Miclea I, Lodde V, Luciano AM, Goudet G.The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previous...
Bertero A, Ritrovato F, Evangelista F, Stabile V, Fortina R, Ricci A, Revelli A, Vincenti L, Nervo T.The purpose of this study was to observe -matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, = 922) were subjected to different maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation perce...
Canesin HS, Brom-de-Luna JG, Choi YH, Ortiz I, Diaw M, Hinrichs K.We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured wer...
Scarlet D, Ille N, Ertl R, Alves BG, Gastal GDA, Paiva SO, Gastal MO, Gastal EL, Aurich C.The objective of this study was to determine whether (1) systemic and intrafollicular cortisol concentrations in horses are directly related and (2) supraphysiological levels of glucocorticoids affect in vitro maturation (IVM) rates of oocytes. Specifically, we studied the (1) changes in the intrafollicular cortisol and progesterone in context with granulosa cell gene expression during maturation of equine follicles (from 5-9 mm, 10-14 mm, 15-19 mm, 20-24 mm, and ≥25 mm in diameter) and (2) effects of cortisol supplementation on IVM rates and gene expression of equine cumulus-oocyte comple...
Carnevale EM.Assisted reproductive techniques that are based on oocyte manipulations have gained acceptance in the equine industry. Methods to collect and handle immature or maturing oocytes have been developed, and systems to ship oocytes now allow for collection in one location and intracytoplasmic sperm injection (ICSI) in another. Subsequently, ICSI-produced embryos can be transferred onsite, shipped to another location, or cryopreserved. Methods for the collection, identification, culture, maturation, and shipment of equine oocytes are reviewed, with an emphasis on procedures from laboratories providi...
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...
Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A.In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm-oviduct interaction. Equine sperm-oviduct binding appears to be...
Ellinger I.Active placental transport of maternal serum calcium (Ca(2+)) to the offspring is pivotal for proper development of the fetal skeleton as well as various organ systems. Moreover, extracellular Ca(2+) levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR) translates changes in extracellular Ca(2+)-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca(2+)-transport such as the murine int...
Bucci D, Giaretta E, Merlo B, Iacono E, Spinaci M, Gadani B, Mari G, Tamanini C, Galeati G.Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0, and 9.0 for 4 hours at 38 °C, 5% CO with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group); ...
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...
Dini P, Bogado Pascottini O, Ducheyne K, Hostens M, Daels P.In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hou...
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...
Choi YH, Ritthaler J, Hinrichs K.Cloned animals possess mitochondria derived from the host ooplast, which typically differ genetically from those of the donor. This is of special concern to horse breeders, as maternal lines are prized and athletic performance is a key factor in genetic value. To evaluate the feasibility of producing mitochondrial-identical cloned foals, we collected oocytes from immature follicles of two mares, BL and SM, maternally related to the donor stallion. In vitro matured, enucleated oocytes were treated with roscovitine-synchronized donor cells and blastocysts were transferred transcervically to reci...
Dini P, Bogado Pascottini O, Ducheyne K, Hostens M, Daels P.In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hou...
Martoriati A, Lalmanach AC, Goudet G, Gérard N.A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate foll...
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...
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...
Deleuze S, Goudet G, Caillaud M, Lahuec C, Duchamp G.In vivo techniques, such as intraoviductal oocyte transfer (OT) and intrafollicular oocyte transfer (IFOT), can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production after transfer of in vivo recovered oocytes (1) into a recipient's oviduct or (2) into her preovulatory follicle either immediately after ovum pick-up or (3) after in vitro maturation (IVM). Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten da...
Moros-Nicolás C, Douet C, Reigner F, Goudet G.In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre-incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre-incubation with progesterone. In experiments 1 and 2, IVF was performed in the...
Bøgh IB, Bézard J, Duchamp G, Baltsen M, Gérard N, Daels P, Greve T.In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those...
Morel MC, Newcombe JR, Hayward K.The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Th...
Martoriati A, Caillaud M, Goudet G, Gérard N.Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured...
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...
Choi YH, Love LB, Westhusin ME, Hinrichs K.Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h a...
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...
Dell'Aquila ME, Albrizio M, Guaricci AC, De Santis T, Maritato F, Tremoleda JL, Colenbrander B, Guerra L, Casavola V, Minoia P.The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cy...
Leemans B, Gadella BM, Stout TA, Nelis H, Hoogewijs M, Van Soom A.Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat in...
Scott TJ, Carnevale EM, Maclellan LJ, Scoggin CF, Squires EL.Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory folli...
Clulow JR, Evans G, Maxwell WM, Morris LH.The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-t...
Li X, Morris LH, Allen WR.The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modi...
Hinrichs K.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...
Wuri L, Agca C, Agca Y.This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compa...
Lorenzo PL, Liu IK, Carneiro GF, Conley AJ, Enders AC.Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group ...
Choi YH, Landim-Alvarenga FC, Seidel GE, Squires EL.Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine...
Bedford SJ, Kurokawa M, Hinrichs K, Fissore RA.In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain i...
Agnieszka N, Joanna K, Wojciech W, Adam O.The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solu...
De Coster T, Velez DA, Van Soom A, Woelders H, Smits K.Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matur...
Hinrichs K, Choi YH, Norris JD, Love LB, Bedford-Guaus SJ, Hartman DL, Velez IC.To evaluate the efficiency of foal production following intracytoplasmic sperm injection (ICSI) and blastocyst culture of oocytes from mares that died or were euthanized under field conditions. Methods: Prospective case series. Methods: 16 mares (age, 3 to 19 years) that died or were euthanized for various causes. Methods: Ovaries were collected immediately before euthanasia (n = 10) or after death (6). Ovaries were transported to the laboratory for oocyte recovery (15 mares), or oocytes were recovered at a remote location and shipped to the laboratory (1). Oocytes underwent ICSI, and presumpt...
Altermatt JL, Marolf AJ, Wrigley RH, Carnevale EM.Objectives of the experiment were to determine the effects of mare age and gonadotropin treatments on dominant follicle vascularity, ovarian blood flow and dominant follicle growth and to associate follicular vascularity with oocyte developmental capacity. Growing follicles >30 mm from young (4-9 years) and old (>20 years) mares were assessed for blood flow using color Doppler ultrasonography before maturation induction with recombinant equine LH (eLH) and immediately prior to oocyte collection at 20-24 h after eLH. Pulsed Doppler was used to obtain resistance indices of ovarian arteries...
Zhang JJ, Muzs LZ, Boyle MS.In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 ...
Pereira BC, Ortiz I, Dorado JM, Diaz-Jimenez MA, Consuegra C, Gosalvez J, Hidalgo M.DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post-mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus-oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO ) before (control group) or after a permeable cryoprotectant-free vitrification method using 1 M sucrose (vitrification group). Af...
