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.
Feugang JM, Ishak GM, Eggert MW, Arnold RD, Rivers OS, Willard ST, Ryan PL, Gastal EL.Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid wer...
Zeitoun MM, Ateah MA, Almaiman AT, Mansour MM.The present study attempted to investigate the effect of various concentrations of spirulina platensis additions to the semen extender on Arabian stallion spermatozoa quality. Semen samples were collected with artificial vagina from five fertile stallions and diluted with an extender containing spirulina (2, 4, 6, and 8 mg/100 mL) or without spirulina (control). Aliquots of diluted semen were cooled (5°C, 90 minutes) and frozen (-196°C, 7 days), then physical traits of thawed spermatozoa were examined. Furthermore, antioxidant activities of superoxide dismutase (SOD), glutathione reductase (...
Alpoim-Moreira J, Fernandes C, Pimenta J, Bliebernicht M, Rebordão MR, Castelo-Branco P, Szóstek-Mioduchowska A, Skarzynski DJ, Ferreira-Dias G.Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA...
Yang F, Wu S, Yang W, Yu J, Liu B, Zeng S.To study the effects of exercise on donkey semen parameters, Dezhou donkey (Equus asinus) jackasses (n = 18) were assigned to three groups: control (CN, n = 6), 1-hour exercise (1 h EX, n = 6) and 2-hour exercise (2 h EX, n = 6) groups. They were exercised with a walking machine for 3 days/week for 8 weeks. Semen parameters and reproductive hormones were evaluated weekly. The heart rate, rectal temperature, blood haematology indexes, and the body condition score (BCS) were also measured. Results showed that the ejaculation volume increased while the sperm concentration decreased after ...
Amorim GB, Segabinazzi LG, Oliveira OM, Perecmanis S, Arruda R, Canisso IF.Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulatio...
Pastorello M, Gastal MO, Godoi DB, Gastal EL.Among female livestock, the mare has the shortest interval from parturition to first ovulation. Due to the scarcity of research on postpartum mares, little progress has been made on the characterization of the resumption of ovarian cyclicity after parturition. This study compared follicular and gonadotropin dynamics during follicle emergence and deviation in postpartum lactating (PP Lactating) versus non-postpartum cycling (N-PP Cycling) mares. On the day of parturition, every PP Lactating mare was paired with a N-PP Cycling mare. Comparisons were made by considering the partum-ovulation inter...
Aurich C, Kaps M.The behaviour of mares is often detrimental to their performance resulting in frequent demand for methods to suppress gonadal function. In addition, prevention of unintended reproduction especially in feral horse populations may require methods for suppression of gonadal function. Surgical ovariectomy is a safe method but not an acceptable approach in feral mares and undesired in mares where future breeding is considered. There are different approaches for artificial prolongation of the luteal phase resulting in transient inhibition of oestrus and ovulation. Among those, treatment with natural...
Doyle JL, Carroll CJ, Corbally AF, Fahey AG.The breeding of sport horses to compete in the Olympic disciplines of show jumping, eventing, and dressage is fast becoming a global industry with the increased use of reproductive technologies, including artificial insemination and embryo transfer. Reproductive technologies have facilitated the dissemination of genetics from elite horses across multiple countries and breeds as breeders are no longer limited by location. Due to this increased level of crossbreeding, there is an increased need for estimated breeding values (EBVs) for sport horse performance that can be compared across breeds an...
Claes A, Stout TAE.In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yield...
Gupta SK.Mammalian zona pellucida (ZP) is composed of three to four glycoproteins, which plays an important role during fertilization. Mutations in the genes encoding zona proteins are reported in women with empty follicle syndrome, degenerated oocytes and those with an abnormal or no ZP further emphasizing their relevance during fertility. Immunization with either native or recombinant ZP glycoproteins/proteins leads to curtailment of fertility in various animal species. Observed infertility is frequently associated with ovarian pathology characterized by follicular atresia and degenerative changes in...
Fanelli D, Tesi M, Monaco D, Diaz-Jimenez M, Camillo F, Rota A, Panzani D.In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, ...
Derisoud E, Auclair-Ronzaud J, Palmer E, Robles M, Chavatte-Palmer P.Although puberty can occur as early as 14-15months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects c...
Ávila ACA, Diniz NC, Serpa RT, Chaves MMBC, Viu MAO, de Oliveira RA.Endometritis is defined as inflammation of the endometrium that may be acute or chronic, infectious, or non-infectious. Endometritis is an important cause of subfertility in mares. Considering the antimicrobial characteristics, immune-stimulating ability, and low cost of ozone (O3) therapy, we aimed to evaluate the effectiveness of intrauterine O3 therapy as an alternative treatment for endometritis in mares. Twenty-five mares with a known reproductive history of uterine infection and inflammation were allocated into three groups: Group 1 (control; n = 7), uterine lavage with Ringer's lactat...
Andrade AFC, Knox RV, Torres MA, Pavaneli APP.When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. Depending on the species or how semen is collected, volume and concentration of SP components varies considerably. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (e.g., BSP - bull; HSP-1 - stallion; Major seminal plasma protein P...
Monteiro RA, Cunha RM, Guerra MMP, de Almeida VM, Peña-Alfaro CE, Silva SV.The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. Af...
Zuidema D, Kerns K, Sutovsky P.Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use ...
Khanam A, Swelum AA, Khan FA.Pharmacologically induced ex copula ejaculation is a method used for collection of semen when the traditional methods of semen collection are not feasible. Common indications for this method include health issues that either preclude the physical act of mating or result in impaired erection and ejaculation. The method also offers an alternative when there is a lack of equipment and facilities required for semen collection using the conventional artificial vagina method. A variety of pharmacological protocols for ex copula ejaculation in stallions have been evaluated in both research and clinic...
Zhao Q, Tao C, Pan J, Wei Q, Zhu Z, Wang L, Liu M, Huang J, Yu F, Chen X, Zhang L, Li J.Fixed-time artificial insemination (FTAI) technology uses exogenous reproductive hormones to regulate the sexual cycle and ovulation of sows without oestrus identification, which improves the sow breeding utilisation rate, reduces the number of non-productive days, and elevates the efficiency of pig farm management. In this study, we aimed to optimise FTAI procedures. Healthy 190-day-old and about 90 kg Large White × Landrace crossing breed replacement gilts (n = 166) which were of unknown reproductive status were randomly selected and divided into three groups: a control group (n = 62...
Angel-Velez D, De Coster T, Azari-Dolatabad N, Fernandez-Montoro A, Benedetti C, Bogado Pascottini O, Woelders H, Van Soom A, Smits K.Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using th...
Bresnahan DR, Lupole RE, Stilz CR, Carnevale EM.Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment...
Ginther OJ.Equine embryo mobility and cessation of mobility (fixation) provide explanations to several enigmas in reproductive biology of the pregnant mare and provide an efficient solution to the twinning problem, the bane of brood-mare owners. Embryo mobility is maximum on Days 12 to 15 (Day 0 = ovulation) while the spherical embryo is growing from 9 to 23 mm in diameter. During mobility, the embryo can be anywhere in the uterine lumen regardless of side of ovulation. Mobility solved the enigmas of how a small embryo can block luteolysis in a relatively massive uterus and why the side of ovulation do...
Rodrigues LT, Segabinazzi LG, Oliveira SN, Frasson M, Papa FO.Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4-6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smear...
Ghallab AM, Shahat AM, Fadl AM, Ayoub MM, Moawad AR.The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooli...
Claes A, Cuervo-Arango J, van den Broek J, Galli C, Colleoni S, Lazzari G, Deelen C, Beitsma M, Stout TA.In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce. Objective: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen-thawed IVP embryos and identify factors influencing success. Methods: Retrospective clinical study. Methods: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The ...
Catalán J, Llavanera M, Bonilla-Correal S, Papas M, Gacem S, Rodríguez-Gil JE, Yeste M, Miró J.The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, De Schauwer C, De Kruif A, De Vliegher S.The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E s...
Contreras MJ, Arias ME, Fuentes F, Muñoz E, Bernecic N, Fair S, Felmer R.Cryopreservation of stallion semen does not achieve the post-thaw quality or fertility results observed in other species like cattle. There are many reasons for this, but the membrane composition and intracellular changes in stallion sperm predispose them to low resistance to the cooling, freezing, and subsequent thawing process. Damage to the sperm results from different processes activated during cryopreservation, including oxidative stress, apoptosis, and structural modifications in the sperm membrane that increase the deleterious effect on sperm. In addition, significant individual variabi...
Varner DD.The conventional approach to evaluation of stallion semen dates back several decades, and includes evaluation of spermatozoal concentration, semen volume, spermatozoon morphological characteristics, and spermatozoal motility patterns initially and following in-vitro storage. While an analysis performed in this manner does have predictive value, incorporation of some more newly developed techniques may improve the predictive value of the examination. This communication addresses some newer tests that can be applied today for evaluation of semen, as well some tests that may be available in the c...
Campbell ML.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...
Ducheyne KD, Rizzo M, Daels PF, Stout TAE, de Ruijter-Villani M.Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed usin...
Philpott M.This review summarizes the major infectious diseases of the three major agricultural species (cattle, sheep and pigs) and horses, and presents the evidence for and against the possibility of infectious agents being transmitted between animals via the venereal route or by the use of semen or early embryos in commercial artificial insemination (AI) or embryo transfer (ET). Cattle feature most prominently in the widespread distribution of frozen semen, and national and international organizations have set out guidelines to work towards disease-free bull studs with semen free from potential pathog...
de Leon PM, Campos VF, Corcini CD, Santos EC, Rambo G, Lucia T, Deschamps JC, Collares T.The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in t...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh ...
Peña FJ, Ortega Ferrusola C, Martín Muñoz P.Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simul...
Zeitoun MM, Ateah MA, Almaiman AT, Mansour MM.The present study attempted to investigate the effect of various concentrations of spirulina platensis additions to the semen extender on Arabian stallion spermatozoa quality. Semen samples were collected with artificial vagina from five fertile stallions and diluted with an extender containing spirulina (2, 4, 6, and 8 mg/100 mL) or without spirulina (control). Aliquots of diluted semen were cooled (5°C, 90 minutes) and frozen (-196°C, 7 days), then physical traits of thawed spermatozoa were examined. Furthermore, antioxidant activities of superoxide dismutase (SOD), glutathione reductase (...
Marzano G, Mastrorocco A, Zianni R, Mangiacotti M, Chiaravalle AE, Lacalandra GM, Minervini F, Cardinali A, Macciocca M, Vicenti R, Fabbri R....Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidativ...
Chandley AC, Fletcher J, Rossdale PD, Peace CK, Ricketts SW, McEnery RJ, Thorne JP, Short RV, Allen WR.Chromosomal abnormalities have been detected in seven mares isolated by their poor reproductive performance. All had small or rudimentary gonads and absent or irregular oestrous cycles. Two mares had an XO genotype, one was a 65,XXX female and another a 64,XY sex-reversed female. Two other mares were sex chromosome mosaics of the 63,X/64,XX type. The seventh mare showed a normal female karyotype but a small extra autosomal fragment was found in a few cells.
Smits K, Govaere J, Peelman LJ, Goossens K, de Graaf DC, Vercauteren D, Vandaele L, Hoogewijs M, Wydooghe E, Stout T, Van Soom A.The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subse...
Thompson DL, Pickett BW, Squires EL, Amann RP.Factors affecting testicular measurements in situ and the relationships among the measurements and various reproductive characteristics were studied using data from 48 stallions. Mean values during the breeding season are provided for scrotal width, widths and lengths of individual testes, combined weight of testicular parenchyma, daily sperm production and daily sperm output. Testicular measurements were highly repeatable from day to day and for repeated measurements on a given day; technician provided the largest source of variation in the measurements of a given stallion. Age significantly ...
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...
Love CC.Semen collection techniques in the stallion have evolved considerably over the last 70 to 80 years and are used today primarily for artificial insemination. Semen can be collected from stallions that are otherwise unable to breed, allowing continued use of valuable animals. There are many options for collection of semen from stallions that present with ejaculatory dysfunction (see the article by McDonnell elsewhere in this issue.) Although there are many advantages to the use of artificial breeding, the collector must understand each step of the collection procedure as well as stallion prefere...
Samper JC, Hellander JC, Crabo BG.Studies were designed to investigate whether sperm motility determined with a Hamilton-Thorn HTM-2000 motility analyzer (HTM), or the percentage of spermatozoa that passed through glass wool (GW), Sephadex (S), or glass wool/Sephadex (GWS) filters could be used to predict the fertilizing potential of fresh or frozen semen. In the fresh semen study, 10 randomly selected ejaculates from 4 stallions exclusively used for A.I. breeding were assayed during the season. The 521 mares used were inseminated with 500 x 10(6) motile spermatozoa after gynaecological examination every 2 days. In the frozen ...
Galli C, Lagutina I, Duchi R, Colleoni S, Lazzari G.The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids an...
Woods J, Bergfelt DR, Ginther OJ.The effects of pre-ovulatory and post ovulatory insemination on pregnancy rate and embryonic-loss rate were studied in 268 mares in two experiments. Within each experiment mares were randomised within replicates as follows: to be inseminated on the day the pre-ovulatory follicle reached 35 mm (pre-ovulatory group), to be inseminated on the day of ovulation (Day 0 group), and to be inseminated on the day after ovulation (Day 1 group). Ultrasonic pregnancy diagnoses were performed on Days 11, 12, 13 and 14 (Experiment 1) and Days 11, 12, 13, 14, 15, 20 and 40 (Experiment 2). Combined for the two...
Fernandes CB, Devito LG, Martins LR, Blanco ID, de Lima Neto JF, Tsuribe PM, Gonçalves CG, da Cruz Landim-Alvarenga F.Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the ...
Vidament M.Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ...
Miller CD.This manuscript is a review of current protocols, advantages, and disadvantages of breeding mares with frozen-thawed equine semen. Issues affecting pregnancy rates are discussed, including proper mare selection, induction of ovulation, insemination dose, timing of insemination (single-dose versus multiple-dose insemination), methods of insemination (transrectal-guided deep-horn versus hysteroscopic insemination), and post-insemination mare management procedures. In a retrospective analysis of breeding records, a single-dose of frozen-thawed semen was inseminated within 6h post-ovulation; the p...
Hinrichs K, Choi YH, Walckenaer BE, Varner DD, Hartman DL.Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vi...
Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment...
Morrell JM, Mari G, Kútvölgyi G, Meurling S, Mislei B, Iacono E, Rodriguez-Martinez H.Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the ...
Hanlon DW, Stevenson M, Evans MJ, Firth EC.The objective of this study was to utilise multivariable statistical methods appropriate for clustered data to identify mare-related explanatory variables that significantly affected the reproductive performance of Thoroughbred mares in the Waikato region of New Zealand. In addition, we aimed to determine the relative contribution of the mare, stallion and stud farm to reproductive performance. Methods: A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during three consecutive breeding seasons (2006-2008). A total of 1,482 individual mares ...
Choi YH, Roasa LM, Love CC, Varner DD, Brinsko SP, Hinrichs K.This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
