Topic:Assisted Reproductive Techniques
Assisted Reproductive Techniques (ART) in horses encompass a range of technologies designed to aid in the breeding process. These techniques include artificial insemination, embryo transfer, intracytoplasmic sperm injection (ICSI), and oocyte transfer. ART is employed to enhance reproductive efficiency, manage genetic diversity, and support breeding programs for both commercial and conservation purposes. Artificial insemination involves the collection and deposition of semen into the mare's reproductive tract, while embryo transfer entails the collection of a fertilized embryo from a donor mare and its implantation into a recipient mare. ICSI involves the direct injection of a single sperm into an oocyte to achieve fertilization. Oocyte transfer involves the transfer of an oocyte from one mare to another for fertilization and gestation. This page compiles peer-reviewed research studies and scholarly articles that examine the methodologies, advancements, and applications of assisted reproductive techniques in equine reproduction.
Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer. The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% ...
Chemotactic selection of frozen-thawed stallion sperm improves sperm quality and heterologous binding to oocytes. The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of r...
Vitrification of Equine In Vivo-Derived Embryos After Blastocoel Aspiration. Embryo cryopreservation is normally performed with great success in species like humans and cattle. The large size of in vivo-derived equine embryos and the presence of a capsule-impermeable to cryoprotectants-have complicated the use of embryo cryopreservation in equine reproduction. A breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after collapsing the blastocoel cavity using a micromanipulation system. High pregnancy rates have been obtained when vitrification is used in combination with embryo collapse.
Puncture of the Equine Embryonic Capsule and Its Repair In Vivo and In Vitro. Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured ...
Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review. 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...
Embryo technologies in donkeys (Equus Asinus). 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...
Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review. 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...
High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa. The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our k...
Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares. The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two group...
Dataset of endometrial blood flow from pregnant and non-pregnant mares on day 7 and 8 post-ovulation. This article provides the dataset for the use of power Doppler ultrasound to assess the equine uterus from the recent research article titled "Power Doppler can detect the presence of 7-8 days conceptuses prior to flushing in an equine embryo transfer program"(1). The vascularization of the endometrium was objectively assessed in mares by quantification of pixels in bitmap format (BMP) using computer assisted analysis of images. Fifty-two mares were examined on days 7 (26 mares) and 8 (26 mares) post-ovulation prior to performing flushing procedures for embryo recovery. Receiver operating char...
Laboratory Production of Equine Embryos. Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without...
Clinical Application of in Vitro Embryo Production in the Horse. The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor m...
Cryopreservation of equine oocytes: looking into the crystal ball. 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...
Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes. 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...
Advances in Holding and Cryopreservation of Equine Oocytes and Embryos. Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or h...
Current Reproductive Technologies Impacting Equine Embryo Production. 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...
Historical Aspects of Equine Embryo Transfer. Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, England, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral to the side of ovulation but the rates improved markedly when the whole uterus was flushed on realization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%-90%...
Energy metabolism of the equine cumulus oocyte complex during in vitro maturation. Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate...
Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm. Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300 ×g for 5', 300 ×g for 10', 1500 ×g for 5' and 1500 ×g for 10' vs no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantl...
Birth of first foals through embryo transfer after artificial insemination using frozen semen in Japan. Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to ...
Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation. 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...
Effects of cryopreservation on stallion sperm protamine messenger RNAs. Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in sta...
Assessing the fertility of two mares cloned from the same founder animal. Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pr...
Stage-specific metabolomic changes in equine oviductal fluid: New insights into the equine fertilization environment. A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF sample...
Assessing equine embryo developmental competency by time-lapse image analysis. The timing of early mitotic events during preimplantation embryo development is important for subsequent embryogenesis in many mammalian species, including mouse and human, but, to date, no study has closely examined mitotic timing in equine embryos from oocytes obtained by ovum pick-up. Here, cumulus-oocyte complexes were collected by transvaginal follicular aspiration, matured invitro and fertilised via intracytoplasmic sperm injection. Each fertilised oocyte was cultured up to the blastocyst stage and monitored by time-lapse imaging for the measurement of cell cycle intervals and identifica...
Time of first polar body extrusion affects the developmental competence of equine oocytes after intracytoplasmic sperm injection. 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 ...
Perspectives on the development and incorporation of assisted reproduction in the equine industry. Marked changes in equine breeding technologies have occurred over the past 25 years. Although there have been numerous reviews on assisted reproduction techniques for horses, few publications include the acceptance and impact of these techniques on the horse industry. In this review, several techniques are discussed, with an emphasis on how they developed in the horse industry and altered equine reproductive medicine. Embryo transfer has become a widely used technology, allowing multiple foals to be produced per year. Embryos can be collected, cooled or frozen, and shipped to a distant facilit...
Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection. Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at ...
Key Aspects of Donkey and Mule Reproduction. Donkeys are nonseasonal, polyestrous, territorial, and nonharem breeders. Although there are many similarities between horses and donkeys, there are also reproductive features that differ, from the longer cervix in the jenny to spermatogenic efficiency in the jack. Mules display reproductive cyclic activity but are rarely fertile. Frozen donkey semen has high pregnancy rates in mares, but lower rates in jennies. This article reviews key aspects of donkey and mule reproductive physiology, reproductive medicine, and assisted reproductive techniques that are useful for practitioners offering assi...
Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH). The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July-August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n = 10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg ...