Insemination in horses refers to the process of introducing sperm into the reproductive tract of a mare to achieve pregnancy. This can be accomplished through natural breeding or artificial insemination (AI). Artificial insemination involves collecting semen from a stallion and manually depositing it into the mare's uterus. This technique allows for greater control over breeding, including the selection of genetic traits and the management of breeding schedules. Various methods of artificial insemination exist, such as fresh, chilled, or frozen semen insemination, each with specific protocols and considerations. This page compiles peer-reviewed research studies and scholarly articles that explore the techniques, outcomes, and advancements in insemination practices in equine reproduction.
Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E.This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 ...
Woodward EM, Christoffersen M, Campos J, Squires EL, Troedsson MH.The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10(9) freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention ...
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...
Ruiz L, Echegaray A, Lafuente A.The aim of this study was the optimization of the sperm freezing protocols for the Pure Breed Andalusian Horse (AH) stallions. The study was performed in 84 ejaculates from 14 stallions (6 ejaculates per stallion). We examined the effect of individual stallion, centrifugal force and centrifugation extender on post-thaw sperm quality. Neither centrifugal force nor centrifugal extender had any significant effect on post-centrifugation or post-thawing sperm quality. Stallion was the principal source of variation in our experiments, showing individual significant differences (p < 0.05) in all para...
Mozūraitis R, Būda V, Kutra J, Borg-Karlson AK.Urine samples from 14 mares, belonging to five breeds, were collected at estrus and diestrus to search for estrous specific volatile compounds which could be used to determine ovulation time. Around 150 volatiles were collected from urine head-space samples by solid phase micro extraction technique, and analyses were conducted by gas chromatography-mass spectrometry methods. Comparison of chromatographic profiles of volatile substances revealed that concentrations of m- and p-cresols were significantly greater during estrus while diethylphthalate was more abundant at diestrus. Monitoring of m-...
Ferrer MS, Lyle SK, Paccamonti DL, Eilts BE, Hosgood G, Godke RA.Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in ran...
Clulow JR, Buss H, Evans G, Sieme H, Rath D, Morris LH, Maxwell WM.Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freez...
Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K.Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sper...
Monteiro GA, Papa FO, Zahn FS, Dellaqua JA, Melo CM, Maziero RR, Avanzi BR, Alvarenga MA, Guasti PN.The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides ...
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...
Canisso IF, Carvalho GR, Morel MD, Ker PG, Rodrigues AL, Silva EC, Coutinho Da Silva MA.As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Objective: To compare the seminal parameters of cryopreserved donkey semen in lactose-EDTA and lactose-yolk extenders and the fertility rates on horse mares. Methods: TRIAL 1: Sperm total and progressive motility, vigour (scale 0-5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was ...
Mari G, Castagnetti C, Rizzato G, Mislei B, Iacono E, Merlo B.Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simp...
Pojprasath T, Lohachit C, Techakumphu M, Stout T, Tharasanit T.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integ...
Broaddus CC, Balasuriya UB, Timoney PJ, White JL, Makloski C, Torrisi K, Payton M, Holyoak GR.The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infecte...
Heckenbichler S, Deichsel K, Peters P, Aurich C.Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm ...
de Andrade AF, Zaffalon FG, Celeghini EC, Nascimento J, Tarragó OF, Martins SM, Alonso MA, Arruda RP.Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autol...
Newcombe JR, Cuervo-Arango J.One hundred and fifty-four mares were inseminated with fresh semen either during the pre- or post-ovulatory periods at different intervals relative to ovulation: 36-24 h (n = 17) and 24-0 h (n = 30) before ovulation; 0-8 h (n = 21), 8-16 h (n = 24), 16-24 h (n = 48) and 24-32 h (n = 14) h after ovulation. All mares received the same routine post-mating treatment consisting of an intrauterine infusion with 1 litre of saline and antibiotics followed 8 h later by an intravenous administration of oxytocin. Artificial inseminations (AI) from 36 h before ovulation up to 16 h post-ovulation were perf...
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 ...
Newcombe JR, Paccamonti D, Cuervo-Arango J.Data were analysed retrospectively from fourteen breeding seasons at an Equine Fertility Clinic for the effect of interval between pre- and postovulatory examinations for immediate postovulatory insemination on pregnancy rate (PR) and embryo loss rate (ELR). Mares of various breeds and ages were examined at intervals which varied from 0.5 to 15h between the pre- and postovulatory period over 867 cycles. When ovulation was detected they were inseminated with a single dose of commercial frozen-thawed semen. All mares were treated in the post-insemination period with intrauterine antibiotics and ...
Pillet E, Duchamp G, Batellier F, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ul...
Pronost S, Pitel PH, Miszczak F, Legrand L, Marcillaud-Pitel C, Hamon M, Tapprest J, Balasuriya UB, Freymuth F, Fortier G.The vast majority of equine arteritis virus (EAV) infections are inapparent or relatively mild, but may occasionally cause outbreaks of equine viral arteritis. The event observed in France during the summer of 2007 was the most important seen in the country, with mortality and disruption of economic activity. Objective: To describe the different stages seen during the outbreak and to show how molecular tools were used for both the detection and management of the crisis. Methods: EAV detection was performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in blood, nasal sw...
Samper JC, Plough T.In the last decade, there has been a significant increase in the quality and commercial use of frozen equine semen. The emergence of new reproductive technologies, coupled with the high prices for an insemination dose from some stallions, the increasing costs of import and export and the marketing policies of stallion agents or owners in the sport horse industry has stimulated the fractionation of doses for insemination. Consequently, the sperm number and the volume of an insemination dose are significantly reduced. To deliver lower doses of sperm in lower volumes compared to the standard dose...
Hinrichs K.In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development ...
Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD, Chowdhary BP, Raudsepp T.Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200...
Katila T, Reilas T, Nivola K, Peltonen T, Virtala AM.The major horse breeds in Finland are the Finnhorse (FH) and the American Standardbred (SB). The foaling rates of the FH have consistently been lower than those of the SB. During the last years, a decreasing trend in foaling rates of both breeds has been observed. The purpose of this study was to describe and compare the structure of these two mare populations for age, reproductive history and mating type. In addition, changes over the years were studied that could explain the decline in foaling rates. Methods: In Finland, the mating statistics and foaling rates per stallion are published year...
Nath LC, Anderson GA, McKinnon AO.To evaluate the reproductive efficiency of horse farms in north-east Victoria and identify aspects of management to be targeted for improving reproductive efficiency. Methods: Retrospective study. Methods: Records from seven Thoroughbred (TB) and four Standardbred (STB) studs in north-east Victoria from 1990 to 2001 were reviewed; 8813 cycles in 4455 mares were analysed. TB mares were inseminated by natural mating, whereas STB mares (89%) were artificially inseminated. Results: The overall early pregnancy rate per cycle was 68.8% for TB mares and for STB mares, 68.3%. Multiple pregnancy per cy...
Jackson RE, Bormann CL, Hassun PA, Rocha AM, Motta EL, Serafini PC, Smith GD.To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Methods: Controlled clinical study. Methods: An assisted reproductive technology laboratory. Methods: Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Methods: One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24...
Clulow JR, Evans G, Maxwell WM, Morris LH.The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-t...
Hannan MA, Haneda S, Itami Y, Wachi S, Saitoh T, Cheong SH, Nambo Y.There has been no report of equine embryo transfer in Japan for the last 24 years. Our objective was to establish an effective protocol for embryo transfer in domestic horse. A Hokkaido native pony was bred by deep-horn artificial insemination with frozen semen from a Connemara pony. Embryo collection was performed using a non-surgical method on day 7. Two embryos were obtained from three flushes (67% recovery) and were transferred fresh into crossbred recipient mares. Both recipient mares were diagnosed pregnant (100% pregnancy rate) 5 days after embryo transfer and had normal progesterone le...
Li X, Morris LH, Allen WR.The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1)...
Ibrahim M, Ferrer MS, Ellerbrock RE, Rollin E.The cytobrush is considered the method of choice to obtain endometrial samples. Rigid brush fibers, however, may induce endometrial irritation and bleeding, or cell fragmentation, decreasing quality and diagnostic value of the samples. It was hypothesized that samples collected using a novel cytotape would provide sample smears of greater quality and less blood contamination than the cytobrush. Endometrial samples were collected with a cytotape and a cytobrush from ten mares without endometritis. Endometritis was then induced with artificial insemination, and samples were again collected 6 h a...
Cuervo-Arango J, Aguilar J, Newcombe JR.Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm...
Leisinger CA, Medina V, Markle ML, Paccamonti DL, Pinto CRF.A novel in vivo model utilizing serial administrations of PGF2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median ...
Pommer AC, Linfor JJ, Meyers SA.Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong spe...
Jhamb D, Talluri TR, Sharma S, Juneja R, Nirwan SS, Yadav D, Pargi KK, Tanwar A, Kumar P, Kumar R, Mehta SC, Parashar M, Gaur M.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing...
Colombo I, Mislei B, Mari G, Iacono E, Merlo B.Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24Â h pre- or 4Â h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine resp...
Gamboa S, Machado-Faria M, Ramalho-Santos J.The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyz...
Stawicki RJ, McDonnell SM, Giguère S, Turner RM.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...
Grøndahl C, Hansen TH, Hossaini A, Heinze I, Greve T, Hyttel P.Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and process...
Hartmann C, Gerner W, Walter I, Saalmüller A, Aurich C.In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 ...
Diaz-Jimenez M, Rota A, Dorado J, Consuegra C, Pereira B, Camillo F, Panzani D, Fanelli D, Tesi M, Monaco D, Hidalgo M.Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 Ã...
Maziero RRD, Guaitolini CRF, Guasti PN, Monteiro GA, Martin I, Silva JPMD, Crespilho AM, Papa FO.Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400. Seminal samples were evaluated after thawing for sperm motion parameters by...
Lange-Consiglio A, Capra E, Giuliani D, Canesi S, Funghi F, Bosi G, Cretich M, Frigerio R, Galbiati V, Cremonesi F.Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral ovid...
Hinrichs K, DiGiorgio LM.A technique was developed in which immature horse oocytes, obtained from slaughterhouse specimens, were transferred to the pre-ovulatory follicle of a mare in vivo, with resulting oocyte maturation, ovulation, fertilization and embryo development. Oocytes were collected from all follicles greater than 3 mm, and were classified as immature, maturing, expanded or denuded. The transfers were performed in the standing, tranquilized mare. The ovary containing the pre-ovulatory follicle was grasped per rectum. A trochar and cannula were placed through the abdominal wall in the flank area, ipsilatera...
Ponthier J, Rigaux G, Parrilla-Hernandez S, Egyptien S, Gatez C, Carrasco Leroy C, Deleuze S.This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 μg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation ...
Woods J, Rigby S, Brinsko S, Stephens R, Varner D, Blanchard T.Two trials were conducted to investigate the effects of intrauterine infusion of PGE2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 m...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Bader H.A total of 23 mares were inseminated once within 0-6 h after clinical detection of ovulation, 14 with fresh and 9 with deep-frozen semen containing 0.1 x 10(9) to 4.7 x 10(9) motile spermatozoa. Within these two groups, the mares were slaughtered 2, 4 or 6 h after insemination and their genital tracts removed. The utero-tubal junction, isthmus and ampulla ipsilateral to the ovary in which ovulation occurred were flushed separately for sperm recovery. In 1 or 2 mares of each group, the uterine horn and corpus uteri, the cervix and vagina were also flushed. Tissue samples were collected from the...
Varner DD, Blanchard TL, Meyers PJ, Meyers SA.A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, fr...
Johannisson A, Lundgren A, Humblot P, Morrell JM.The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
Heckenbichler S, Deichsel K, Peters P, Aurich C.Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm ...
Gonzalez-Castro RA, Carnevale EM.Limited clinical information is available regarding sperm population parameters that are important for use with equine intracytoplasmic sperm injection (ICSI). Therefore, the appropriateness of a sample of sperm is typically not known before ICSI. The aim of our study was to determine which sperm population characteristics were predictive of ICSI outcome. Frozen-thawed sperm samples (n = 114) from 37 stallions in a clinical program were analyzed after ICSI for percentages of normal morphology (MORPH+), live as assessed by eosin/nigrosin stain (LIVE+), membrane intact as assessed by hypoosmot...
de Vasconcelos Franco JS, Faheem M, Chaveiro A, Moreira da Silva F.The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C...
Walbornn SR, Felix M, Schnobrich MR, Bradecamp EA, Scoggin CF, Stefanovski D, Hinrichs K.To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. 14 healthy mares were used; 11 completed the study and were included for analysis. Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in ran...
Sevinga M, Hesselink JW, Barkema HW.Because the incidence of retained placenta in Friesian mares is estimated to be high, and no reports have been published on the reproductive performance of Friesian mares after retained placenta, we studied postpartum reproductive performance in Friesian brood mares with (n = 54) and without (n = 50) retained placenta. We defined a retained placenta as the failure to expel all fetal membranes within 3 h after the delivery of a foal. We subdivided the group of mares with retained placenta into mares in which the placenta had been removed manually (n = 30) and mares in which it had not (n = 24)....
Güvenc K, Reilas T, Katila T.Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen co...
