Reproductive technology in horses encompasses a range of scientific techniques and procedures aimed at assisting and enhancing equine reproduction. These technologies include artificial insemination, embryo transfer, and cryopreservation of gametes and embryos. They are employed to improve breeding efficiency, manage genetic diversity, and preserve valuable genetic material. Artificial insemination involves the collection and introduction of semen into the mare's reproductive tract, while embryo transfer allows for the harvesting and implantation of embryos from donor to recipient mares. Cryopreservation involves freezing and storing sperm, oocytes, or embryos for future use. This page compiles peer-reviewed research studies and scholarly articles that investigate the methodologies, applications, and outcomes of reproductive technologies in equine breeding and management.
Gastal EL, Aguiar FLN, Gastal GDA, Alves KA, Alves BG, Figueiredo JR.The mammalian ovary is responsible for essential stages of folliculogenesis and hormonal production, regulating the female physiological functions during the menstrual/estrous cycles. The mare has been considered an attractive model for comparative studies due to the striking similarities shared with women regarding in vivo and in vitro folliculogenesis. The ovarian follicular population in horses contains a large number of oocytes enclosed in preantral follicles that are yet to be explored. Therefore, the in vitro manipulation of equine preantral follicles aims to avoid the process of atre...
Panzani D, Fanelli D, Camillo F, Rota A.In industrialized countries, the donkey population had a dramatic decrease during the last century and this has brought almost all European donkey breeds to risk. Embryo technologies have already been employed as a tool for the conservation of endangered equid species (e.g. Equus Przewalskii). Today it is possible to obtain pregnancies after the transfer of donkey embryos in synchronized recipients, even though embryo transfer is not widely used as a reproductive technique in this species. So far, very few foals are born after transfer of cryopreserved embryos. To date, no pregnancies have bee...
Aguiar FLN, Gastal GDA, Alves KA, Alves BG, Figueiredo JR, Gastal EL.During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achievin...
Dos Reis WVA, de Braga D, Júnior MV, Gheller JM, de Deco-Souza T, da Costa E Silva EV, Sampaio BFB, Macedo GG.Dispersed ovulation at the breeding (BS) and anestrus at non-breeding season (NBS) are major impediments to embryo transfer and insemination programmes. The present study aimed to evaluate a hormonal P4/E2-based synchronisation protocol in mares during both the BS and the NBS on ovarian/follicle behaviour. Mares underwent a hormone protocol to synchronise their ovulation during the BS (n = 8) and NBS (n = 10), starting (D0) with the insertion of an intravaginal device containing 1 g of P4 and 7 mg Estradiol Benzoate IM. (EB). On D9, the device was removed and injected with 0.25 mg o...
Fernández-Hernández P, Sánchez-Calabuig MJ, García-Marín LJ, Bragado MJ, Gutiérrez-Adán A, Millet Ó, Bruzzone C, González-Fernández L....Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the ...
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...
Miró J, Marín H, Catalán J, Papas M, Gacem S, Yeste M.In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work inten...
Cuervo-Arango J, Martín-Peláez MS, Claes AN.The echogenicity of the early CL undergoes obvious changes during the first 5 days of luteal development. This phenomenon could potentially be used to estimate the age of the CL in mares which have not been checked frequently for the diagnosis of ovulation. The objective of this retrospective study was to determine the accuracy of estimating the age of the early CL (Day 0 to Day 4; Day 0 = Day of ovulation) in recipient mares examined for the first time in the breeding season to be used in a commercial embryo transfer (ET) program. The post-transfer pregnancy rate and embryo loss of 28 recip...
Palmer E, Chavatte-Palmer P.Reproductive technologies aim at improving fertility with the ultimate result of improving genetic selection. In equidae, the respective contribution of different methods of horse management and breeding to genetic progress remain difficult to evaluate as breeding strategies affect the number of offspring per mare or stallion whereas different selection methods (based on pedigree, performance, genomics or progeny's performance) will be applicable at different ages, leading to different accuracy in the estimation of the breeding value. Here, a mathematical model was applied to evaluate theoreti...
Canesin HS, Ortiz I, Rocha Filho AN, Salgado RM, Brom-de-Luna JG, Hinrichs K.Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38...
Claes A, Cuervo-Arango J, Colleoni S, Lazzari G, Galli C, Stout TA.The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient...
Squires E.Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12-24 hours and transport them to recipient stations. Embryo freezing has become more common us...
Prell MJ, McCue PM, Moffett PD, Graham JK.Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration ...
Souza JRM, Gonçalves PBD, Bertolin K, Ferreira R, Ribeiro ASS, Ribeiro DB, Rechsteiner SMDEF, Araújo RR, Pimentel CA, Pimentel AMH.The aim of the present study was to evaluate the correlation of age and heat cycle to determine reproductive efficiency in young and aged Thoroughbred mares bred on foal heat (FH) or on second heat (SH) after foaling. Embryo mortality (EmbM) was determined every time a mare was found open after a positive pregnancy diagnosis. Parturition to breeding interval, pregnancy rate (PregR) and EmbM rate were the dependent variables and the treatments were breeding on the FH or on SH. The cutoff age to obtain above-average probability for the EmbM was 10 years old. PregR in mares bred on FH was lower ...
Antczak DF.The development of placentation that coincided with the evolution of mammals presented new challenges to the transmission of life from one generation to the next, particularly with regard to the possibility of maternal immunological recognition and destruction of the developing conceptus. The balance between immunity and tolerance dominates the immunological relationship between mother and fetus during mammalian pregnancy, and the focal point of this relationship lies at the interface between the trophoblast cells that comprise the outermost layer of the placenta and the maternal endometrial t...
Dordas-Perpinyà M, Normandin L, Dhier T, Terris H, Cochard A, Frilley C, Huiban F, Bruyas JF.The aim of this study was to assess the efficacy of different doses of buserelin acetate and another GnRH agonist, triptorelin acetate, in saline solution in a single subcutaneous injection, to induce ovulation of growing pre-ovulatory follicle in mare and compare it with the classical treatment of a single injection of hCG. The study is split into 3 experiments over different breeding seasons in the same stud with a random distribution of treatment. The first one was to compare the injection of 6 mg of buserelin with 1,500 IU of hCG; the second one consisted of comparing different doses of ...
Miró J, Morató R, Vilagran I, Taberner E, Bonet S, Yeste M.Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze...
Angel D, Canesin HS, Brom-de-Luna JG, Morado S, Dalvit G, Gomez D, Posada N, Pascottini OB, Urrego R, Hinrichs K, Velez IC.Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated...
Nieto-Olmedo P, Martín-Cano FE, Gaitskell-Phillips G, Ortiz-Rodríguez JM, Peña FJ, Ortega-Ferrusola C.In order to determine whether differences in uterine blood flow between pregnant and non-pregnant mares can be used to predict the presence of the equine embryo prior to flushing in an embryo transfer program, power Doppler ultrasonography was used on a total of 52 mares on days 7 or 8 post-ovulation. Computer analysis of Doppler images was subsequently performed using ImageJ v1.48 software. Vascular perfusion of the endometrium was analyzed using spot meter techniques, measuring mean pixel intensity and area of blood flow. Mares with positive flushings presented a higher uterine blood flow ar...
Goncalves GR, Morotti F, Colombo AHB, Bonato DV, Bizarro-Silva C, Rosa CO, Cavalieri FLB, Seneda MM.Age and antral follicle count (AFC) are related to fertility in cattle, but this information remains limited in mares. Methods: To verify the influence of age and AFC on the reproductive characteristics of mares, 15 Quarter Horse donors, with 5-15 antral follicles, ranging from three to 17 years old, healthy and in good nutritional status, were divided into groups with low AFC (≤9 follicles) or high AFC (≥10 follicles) and mares considered young (≤9 years) or old (≥10 years). Mares were submitted to ultrasonography to determine the dominant follicle diameter, follicular growth rate, de...
Immonen I, Cuervo-Arango J.The reproductive management of mares for frozen semen artificial insemination (AI) can be costly and labor intensive. Predicting the exact time of ovulation can be challenging even when ovulation-inducing drugs are used. The main objective of this retrospective study was to determine whether there was an effect of interval between examinations to detect ovulation on likelihood of pregnancy and early embryonic loss in mares after postovulatory breeding with a single straw of frozen/thawed semen. The second objective was to determine the efficacy of two different drugs (human chorionic gonadotro...
Ducheyne KD, Rizzo M, Cuervo-Arango J, Claes A, Daels PF, Stout TAE, de Ruijter-Villani M.Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts ex...
Rodríguez MB, Gambini A, Clérico G, Ynsaurralde-Rivolta AE, Briski O, Largel H, Sansinena M, Salamone DF.Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ...
Carnevale EM, Metcalf ES.Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning...
Len J, Beehan D, Eilts B, Ebrahimie E, Lyle S.The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 106 sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator fo...
Riera FL, Roldán JE, Espinosa JM, Fernandez JE, Ortiz I, Hinrichs K.Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a po...
Alonso MA, Silva LA, Affonso FJ, Lemes KM, Celeghini ECC, Lançoni R, Carvalho HF, de Arruda RP.Establishment of pregnancy after embryo transfer is the ultimate goal of an embryo transfer program and increasing pregnancy rates and reducing pregnancy loss are mandatory. The utilization of treatments to improve conception rates in recipient mares has been the focus of several research groups over the last years and the results are controversial. Some studies using human chorionic gonadotrophin (hCG) found promising results. Our hypothesis was that hCG administration would cause an additional stimulation on luteal function, uterine and luteal vascularization and progesterone concentration, ...
Ortiz I, Dorado J, Morrell JM, Diaz-Jimenez MA, Pereira B, Consuegra C, Hidalgo M.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
Frank BL, Doddman CD, Stokes JE, Carnevale EM.In this retrospective study the morphological characteristics of oocytes and cleavage stage embryos were associated with pregnancy results from clinical intracytoplasmic sperm injection (ICSI) in mares. Oocytes were collected from preovulatory follicles, and images (×200; n=401) were captured for measurements of ooplasm, the perivitelline space and zona pellucida. After ICSI and before transfer into recipients' oviducts, cleavage stage embryos were imaged (n=178). Oocyte donor ages (3-13, 14-19, 20-23, 24-27 years) were compared, as were mares aged 3-13 years without versus with recent histor...
Cuervo-Arango J, Claes AN, Stout TAE.In recent years, the number of equine invitro-produced embryos (IVP) has increased markedly; as yet, there are few reports on what constitutes an 'ideal' recipient for an IVP embryo. This study retrospectively investigated the effects of recipient mare oestrous cycle characteristics on the likelihood of pregnancy after transfer of IVP (n=264) and invivo-derived embryos (n=262). IVP embryos tolerated only a narrow window of recipient mare 'synchrony', with transfer on Day 4 after ovulation resulting in a higher likelihood of ongoing pregnancy (69%) than transfer on Days 3, 5 or 6 (53.2%, 41.3% ...
Peña FJ, Martín-Cano FE, Becerro-Rey L, Ortega-Ferrusola C, Gaitskell-Phillips G, da Silva-Álvarez E, Gil MC.The mammalian ejaculate is very well suited to proteomics studies. As such, research concerning sperm proteomics is offering a huge amount of new information on the biology of spermatozoa. Among domestic animals, horses represent a species of special interest, in which reproductive technologies and a sizeable market of genetic material have grown exponentially in the last decade. Studies using proteomic approaches have been conducted in recent years, showing that proteomics is a potent tool to dig into the biology of the stallion spermatozoa. The aim of this review is to present an overview of...
Love CC.Stallion semen evaluation is an important part of the breeding soundness evaluation. The results of the semen evaluation cannot be interpreted without a thorough knowledge of the mare and management effects that may have played a role or may affect the potential fertility of the stallion evaluated. There are considerations and limitations that the clinician should understand about each test. Any sperm quality test must be interpreted with a clear understanding of how it relates to fertility.
Traversari J, Aepli H, Knutti B, Lüttgenau J, Bruckmaier RM, Bollwein H.The anti-Müllerian hormone (AMH) plays an inhibitory role during folliculogenesis by regulating the number of follicles entering the growing pool. Antral follicle counts (AFC) are highly correlated with serum AMH concentrations and both appear to be related to the ovarian reserve in several species. Few data on AMH and AFC in mares exist, especially with regard to fertility. Therefore, the objective of the current study was to investigate the interrelationship between antral follicle count, serum AMH concentrations and fertility outcome in mares. One hundred and twenty-seven mares were enroll...
Bedford SJ, Kurokawa M, Hinrichs K, Fissore RA.In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single mo...
Perkins NR.This article reviews therapies and strategies commonly used to treat diseases of the mare's genital tract and modulate the reproductive cycle of the mare. Many reproductive treatments are based on historical use and empirical evidence rather than well controlled clinical studies. This article attempts to present practical information in a summary form while highlighting the need for continued research documenting the efficacy and safety of reproductive therapies.
Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, Varner DD, Cavinder CA.The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Summers PM, Shephard AM, Hodges JK, Kydd J, Boyle MS, Allen WR.Blastocysts were collected non-surgically from 2 Przewalski's horse and 2 Grant's zebra mares and transferred extra-specifically to domestic horse and donkey recipients. Nine Przewalski's horse embryos were transferred surgically, and 2 non-surgically, to domestic Welsh-type pony mares. After surgical transfer, 7 (77.8%) pregnancies were established and 4 foals were born. Twelve Grant's zebra embryos were transferred surgically to 5 pony and 7 domestic donkey recipients respectively and 1 non-surgically to a donkey; 3 (60%) zebra-in-horse pregnancies were established and 2 went to term. Only 2...
Consuegra C, Crespo F, Bottrel M, Ortiz I, Dorado J, Diaz-Jimenez M, Pereira B, Hidalgo M.The aim of this study was to assess different concentrations of sucrose-based extenders combined with bovine serum albumin (BSA) as an alternative to stallion sperm cryopreservation with permeable cryoprotectants. Semen samples (n = 16) were collected from six stallions. Sperm was cooled, filled in 0.5 mL straws and frozen in nitrogen vapor. Post-thaw sperm kinetic parameters, plasma and acrosome membrane integrity were statistically compared among treatments. In Experiment 1, extenders containing 1% of BSA and different concentrations of sucrose (mmol/L, M): 0, 50, 100, 250, 350 and 450...
Troedsson MH, Ababneh MM, Ohlgren AF, Madill S, Vetscher N, Gregas M.The objective of this study was to determine whether periovulatory treatments with PGF2alpha affects the development of the CL, and whether the treatment was detrimental to the establishment of pregnancy. Reproductively sound mares were assigned randomly to one of the following treatment groups during consecutive estrus cycles: 1. 3,000 IU hCG within 24 hours before artificial insemination and 500 microg cloprostenol (PGF2alpha analogue) on Days 0, 1, and 2 after ovulation (n=8), 2. 2 mL sterile water injection within 24 hours before artificial insemination and 500 microg cloprostenol on Days ...
Betteridge KJ, Eaglesome MD, Flood PF.Two experiments were conducted using 14 mares. In Exp. 1, mares were inseminated with semen treated with TEPA, which, in other species, has been shown to lead to an arrest in ovum cleavage at 2--4 cells. The oviducts and/or uterus were then flushed 7--10 days after ovulation in 6 mares (Group A) or 2--6 days after ovulation in 5 mares (Group B). Fresh eggs were found in the oviduct flushes of 5 Group A and 5 Group B mares: 9 of the 10 eggs appeared to have cleaved, but none had developed beyond 16-cells. Seven eggs contained spermatozoa and 3 of 4 eggs from each group showed evidence of fertil...
Lagares MA, Castanheira PN, Amaral DC, Vasconcelos AB, Veado JC, Arantes RM, Stahlberg R.The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
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...
Stout TA.During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the followin...
Wu Z, Zheng X, Luo Y, Huo F, Dong H, Zhang G, Yu W, Tian F, He L, Chen J.The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess w...
Morrell JM, Garcia BM, Pena FJ, Johannisson A.The purpose of this study was to determine if the quality of stored stallion semen doses could be enhanced by the scaled-up version of Single Layer Centrifugation using Androcoll-E-Large. Three semen doses from each of fifteen stallions were transported overnight to the Swedish University of Agricultural Sciences (SLU) for processing 24 h after semen collection. Sperm quality in the resulting SLC-selected samples was significantly improved compared to the uncentrifuged samples: mean progressive motility was increased by 8% on the day of processing (P < 0.001) and by 13% after 24 h cold storage...
Alonso A, Baca Castex C, Ferrante A, Pinto M, Castañeira C, Trasorras V, Gambarotta MC, Losinno L, Miragaya M.The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P 0.05). Blastocysts were obtained with cooled spermatozoa, and ...
Cuervo-Arango J, Claes AN, Stout TAE.In recent years, the number of equine invitro-produced embryos (IVP) has increased markedly; as yet, there are few reports on what constitutes an 'ideal' recipient for an IVP embryo. This study retrospectively investigated the effects of recipient mare oestrous cycle characteristics on the likelihood of pregnancy after transfer of IVP (n=264) and invivo-derived embryos (n=262). IVP embryos tolerated only a narrow window of recipient mare 'synchrony', with transfer on Day 4 after ovulation resulting in a higher likelihood of ongoing pregnancy (69%) than transfer on Days 3, 5 or 6 (53.2%, 41.3% ...
Frank BL, Doddman CD, Stokes JE, Carnevale EM.In this retrospective study the morphological characteristics of oocytes and cleavage stage embryos were associated with pregnancy results from clinical intracytoplasmic sperm injection (ICSI) in mares. Oocytes were collected from preovulatory follicles, and images (×200; n=401) were captured for measurements of ooplasm, the perivitelline space and zona pellucida. After ICSI and before transfer into recipients' oviducts, cleavage stage embryos were imaged (n=178). Oocyte donor ages (3-13, 14-19, 20-23, 24-27 years) were compared, as were mares aged 3-13 years without versus with recent histor...
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...
Morrell JM, Pihl J, Dalin AM, Johannisson A.There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
Sinowatz F, Wessa E, Neumüller C, Palma G.Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species-specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly ...
Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF.In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cool...
Aguiar FLN, Gastal GDA, Alves KA, Alves BG, Figueiredo JR, Gastal EL.During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achievin...
Marinone AI, Losinno L, Fumuso E, Rodríguez EM, Redolatti C, Cantatore S, Cuervo-Arango J.Advanced maternal age is an important predisposing factor on the reduction of reproductive efficiency. The aim of this study was to evaluate the effect of donor's age on several reproductive parameters in a commercial equine embryo transfer program. Donors were classified into 3 age groups: Group 1=fillies (3 and 4 years old), Group 2=middle age mares (aged 5-10) and Group 3=old mares (aged 13-25). Embryo recovery, multiple ovulation and pregnancy rates and interovulatory intervals were compared amongst age groups. Group 1 (171/244, 70.1%) and Group 2 (774/1081, 71.6%) had a higher (P<0.005) e...
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
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...
Alonso MA, Silva LA, Affonso FJ, Lemes KM, Celeghini ECC, Lançoni R, Carvalho HF, de Arruda RP.Establishment of pregnancy after embryo transfer is the ultimate goal of an embryo transfer program and increasing pregnancy rates and reducing pregnancy loss are mandatory. The utilization of treatments to improve conception rates in recipient mares has been the focus of several research groups over the last years and the results are controversial. Some studies using human chorionic gonadotrophin (hCG) found promising results. Our hypothesis was that hCG administration would cause an additional stimulation on luteal function, uterine and luteal vascularization and progesterone concentration, ...
Brinsko SP, Varner DD.Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable preg...
Sánchez-Calabuig MJ, Fernández-González R, Hamdi M, Smits K, López-Cardona AP, Serres C, Macías-García B, Gutiérrez-Adán A.Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Objective: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Methods: Experiments comparing in vitro and in vivo produced embryos. Methods: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10...
