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Topic:Reproductive Technology
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.
Pregnancy outcomes using stallion epididymal sperm stored at 5 °C for 24 or 48 hours before harvest. The cryopreservation of epididymal sperm can be useful in a variety of circumstances for ensuring genetic preservation of a valued stallion. Although early studies have reported pregnancy rates significantly lower than those achieved with cryopreserved ejaculated sperm, two recent studies report over 60% one-cycle pregnancy rates with epididymal sperm stored for 24 hours at 5 °C before harvest and cryopreservation. The aims of this study were to: (1) attempt to replicate the one-cycle pregnancy rate of over 60% using epididymal sperm cooled and stored within the epididymis for 24 hours before...
Equine endometrial gene expression changes during and after maternal recognition of pregnancy. The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific colle...
Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. 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...
The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses. 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...
Morphometric Study of the Mare Oviductal Mucosa at Different Reproductive Stages. The objectives of this work were to describe some morphometric characteristics and to establish quantitative parameters of different regions of the equine oviductal mucosa from the isthmus, ampullary-isthmic junction (AIJ), and ampulla. Twenty-one mixed-bred mares were used for this study. Mares were selected in the following reproductive phases: anestrus, estrus, and diestrus. The left oviducts were examined with light microscopy, and rights ones were studied through the intraoviductal molds. The isthmus showed the smallest luminal area, mucosal area, epithelial perimeter, and luminal diamete...
Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants. The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1%...
The Impact of Reproductive Technologies on Stallion Mitochondrial Function. The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this p...
Embryo transfer in competition horses: Managing mares and expectations. Embryo transfer (ET) is an accepted and successful technique for obtaining foals from mares without interrupting their competition careers. Recent research, however, suggests that the potential of factors including heat, exercise, repeated embryo flushing and repeated manipulation of the reproductive cycle using exogenous hormones to have a negative impact on fertility may have been underestimated. This paper reviews the evidence base for involvement of these factors in repeated failures to recover embryos from nongeriatric competition mares without obvious clinical or pathological indications...
The effect of mare’s age on multiple ovulation rate, embryo recovery, post-transfer pregnancy rate, and interovulatory interval in a commercial embryo transfer program in Argentina. 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...
Assisted reproduction with gametes and embryos: what research is needed and fundable? Principles for selecting future research projects include interests of investigators, fundability, potential applications, ethical considerations, being able to formulate testable hypotheses and choosing the best models, including selection of the most appropriate species. The following 10 areas of assisted reproduction seem especially appropriate for further research: efficacious capacitation of bovine spermatozoa in vitro; improved in vitro bovine oocyte maturation; decreasing variability and increasing efficacy of bovine superovulation; improved fertility of sexed semen; improving equine IV...
Season does not influence embryo recovery rate and conceptus size until day 14 after ovulation in the horse. Although the horse is a seasonal breeding species, a considerable number of mares continue to cycle throughout autumn and winter. Slower equine embryo growth during the non-breeding season has been hypothesized, and because smaller embryo size is beneficial for cryopreservation, embryo collection outside the breeding season could be an interesting approach for the production of frozen horse embryos. In the present retrospective study, we have therefore analysed embryo recovery rates and conceptus size in mares (n = 30) throughout the year. Conceptus diameter was either size determined after co...
Asynchronous ovulation in mares: seasonal variations in frequency. The mare's reproductive system is rarely capable of sustaining multiple pregnancies to term. Multiple pregnancies largely derive from multiple ovulations, most commonly double ovulations, hence, double ovulations are of significant concern to breeders/veterinarians. Double ovulations may be synchronous or asynchronous. Asynchrony of up to 96 hours may result in two embryonic vesicles of very different sizes, which are challenging to detect at early ultrasonic pregnancy detection. This study aims to (1) document the incidence of synchronous and asynchronous double ovulation and (2) determine ...
The effect of the interval from PGF treatment to ovulation on embryo recovery and pregnancy rate in the mare. The objective of this study was to determine the effect of the interval from induced luteolysis to ovulation on fertility of mares from two different farms. At farm 1, 215 mares were inseminated with frozen/thawed semen during 513 estrous cycles over seven consecutive breeding seasons. Estrus was induced with analogues of PGF2α in 179 cycles. At farm 2, 375 embryo flushings were performed in 65 donor mares inseminated with fresh semen; of which, 327 were performed following artificial insemination after PGF-induced luteolysis. In both farms, the intervals from PGF treatment to ovulation (ITO)...
Microscopic examination of endometrial biopsies of retired sports mares: an explanation for the clinically observed subfertility? After their retirement from sports, performance mares often show a poor breeding success. The objective of this study was the microscopic evaluation of endometrial biopsies of retired sports mares (n = 189) to search for alterations that may explain subfertility. Mares of this study aged 3-23 years showed endometritis (30%) and endometrosis (77%); mild forms predominated. In regard to those mares biopsied during the breeding season (n = 99), 50% had glandular differentiation disorders, i.e. glandular inactivity (8%) or irregular glandular differentiation (42%). Compared to literature d...
Effect of cushioned or single layer semen centrifugation before sex sorting on frozen stallion semen quality. The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (...
Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential. 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...
Sex reversal syndrome in the horse: four new cases of feminization in individuals carrying a 64,XY SRY negative chromosomal complement. Horses are characterized as having a greater rate of chromosomal abnormalities than other species, which are mainly related to the sex chromosome pair and produce a series of different anomalies known as disorders in sexual development (DSD). In the present study, three Pura Raza Española (PRE) and one Menorquín (MEN) horses were studied and an incompatibility in their genetic and phenotypic sex were detected. Animals were karyotyped by conventional and molecular cytogenetic analyses and characterized using genomic techniques. Although all individuals, were totally unrelated, these animals h...
Current status of freeze-drying technology to preserve domestic animals sperm. In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
The interrelationship between anti-Müllerian hormone, ovarian follicular populations and age in mares. Anti-Müllerian hormone (AMH) is a granulosa-cell-derived glycoprotein, which plays an important inhibitory role during folliculogenesis. Concentrations of AMH are highly correlated with antral follicle counts (AFCs) in other species, which in turn are related to follicular reserve. Relatively little is known about AMH and AFC in the mare. Objective: To determine plasma AMH concentrations and AFCs in mares of different ages, to measure the repeatability of AMH concentrations and AFCs within and across oestrous cycles and to assess the relationship between plasma AMH concentrations and AFCs wit...
Stallion fertility: a focus on the spermatozoon. Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermato...
Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions? The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n = 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were in...
Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa. Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during...
A comparison of the histological structure of the placenta in experimental animals. The primary function of the placenta is to act as an interface between the dam and fetus. The anatomic structure of the chorioallantoic placenta in eutherian mammals varies between different animal species. The placental types in eutherian mammals are classified from various standpoints based on the gross shape, the histological structure of the materno-fetal interface, the type of materno-fetal interdigitation, etc. Particularly, the histological structure is generally considered one of the most useful and instructive classifications for functionally describing placental type. In this system,...
Effect of artificial insemination protocol and dose of frozen/thawed stallion semen on pregnancy results in mares. Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen-thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly de...
Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections. IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine ...
Potential differences between Leptospira serovars, host-adapted (Bratislava) and incidental (Copenhageni), in determining reproductive disorders in embryo transfer recipient mares in Brazil. The objective was to investigate potential differences between two Leptospira serovars, host-adapted (Bratislava) and incidental (Copenhageni), in causing reproductive disorders in mares. From August 2009 to March 2011, 608 adult recipient mares from eight studs located in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. These mares were 3-8 years of age, of various breeds, and were managed in a semiextensive system (embryo transfer centres). According to the reproductive history of these mares, the studs were categorised as Group A (357 mares: high prevalence of reprodu...
Effect of heterologous and homologous seminal plasma on stallion sperm quality. Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to inves...
