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.
Gottschalk M, Sieme H, Martinsson G, Distl O.The objectives of the present study were to evaluate genetic parameters for semen quality traits of 241 fertile German Warmblood stallions regularly employed in artificial insemination (AI). Stallions were owned by the National Studs Celle and Warendorf in Germany. Semen traits analyzed were gel-free volume, sperm concentration, total number of sperm, progressive motility and total number of progressively motile sperm. Semen protocols from a total of 63,972 ejaculates were collected between the years 2001 and 2014 for the present analysis. A multivariate linear animal model was employed for es...
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...
Joonè CJ, Bertschinger HJ, Gupta SK, Fosgate GT, Arukha AP, Minhas V, Dieterman E, Schulman ML.Few studies have investigated ovarian function in the mare undergoing porcine zona pellucida (pZP) immunocontraception despite reported ovarian dysfunction in other species. Objective: This study aimed to describe ovarian function and oestrous cyclicity in pony mares following treatment with either the conventional pZP vaccine or a novel recombinant form of the vaccine derived from porcine ZP3 and ZP4 (reZP). In addition, the contraceptive efficacy of pZP vs. reZP was assessed. Methods: Blinded, randomised, prospective clinical trial. Methods: Mares (n = 21) were randomised into 3 groups of 7:...
Reghini MF, Ramires Neto C, Segabinazzi LG, Castro Chaves MM, Dell'Aqua Cde P, Bussiere MC, Dell'Aqua JA, Papa FO, Alvarenga MA.Degenerative changes of the endometrium are directly related to age and fertility in mares. Chronic degenerative endometritis (CDE) is correlated with uterine fluid retention and reduced ability to clear uterine inflammation. Recent research in the areas of equine surgery and sports medicine has shown that platelet-rich plasma (PRP) treatment acts as an immunomodulator of the inflammatory response. Therefore, the aim of this study was to determine if the uterine infusion of PRP could modulate the local inflammatory response and modify the intrauterine NO concentrations after artificial insemin...
Gibb Z, Aitken RJ.In vitro sperm storage is a necessary part of many artificial insemination or in vitro fertilization regimes for many species, including the human and the horse. In many situations spermatozoa are chilled to temperatures between 4 and 10°C for the purpose of restricting the metabolic rate during storage, in turn, reducing the depletion of ATP and the production of detrimental by-products such as reactive oxygen species (ROS). Another result of lowering the temperature is that spermatozoa may be "cold shocked" due to lipid membrane phase separation, resulting in reduced fertility. To overcome ...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Guignot F, Reigner F, Perreau C, Tartarin P, Babilliot JM, Bed'hom B, Vidament M, Mermillod P, Duchamp G.Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryo...
Riera FL, Roldán JE, Gomez J, Hinrichs K.Transfer of donor oocytes to the oviducts of inseminated recipient mares (oocyte transfer, OT) presents a valuable method for production of foals from otherwise infertile mares. Little information is available, however, on factors affecting success of OT in a clinical setting. We report the findings over three breeding seasons in a commercial OT program developed at an equine embryo transfer center in Argentina. Overall, 25 mares were enrolled, and 197 follicle aspiration procedures were performed. The average mare age was 23 years. Follicle aspiration was performed with a needle placed throug...
Haadem CS, Nødtvedt A, Farstad W, Thomassen R.Pregnancy rates with cooled equine semen can be unsatisfactory and show great variation. Information about first cycle pregnancy rates and pregnancy rates per cycle are often lacking from publicly available records. This retrospective cohort study was performed to evaluate the fertility of the Norwegian Coldblooded trotter. The aim of the study was to compare the breeding results after insemination with fresh, extended with those of cooled, shipped semen among Norwegian Coldblooded trotter mares. First cycle pregnancy rate was the main parameter used to measure fertility. Stud-books were colle...
Reilas T, Rivera Del Alamo MM, Liepina E, Yeste M, Katila T.Cervical patency is considered to be important for uterine drainage after mating or artificial insemination (AI), and failure to relax or premature tightening of the cervix can lead to persistent endometritis. This study investigated the hypothesis that cervical occlusion after AI increases accumulation of fluid, polymorphonuclear leukocytes (PMNs), and cytokines in the uterine lumen. Endometrial swabs were obtained from 29 normal cyclic mares during the first, third, and fifth estrus and biopsies during the first and fifth estrus. All mares were inseminated during the second and fourth estrus...
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...
Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, Varner DD, Cavinder CA.The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Ferreira JC, Ignácio FS, Rocha NS, Thompson DL, Pinto CR, Meira C.The present experiment was divided into two studies to investigate the effect of age and endometrial degeneration on uterine blood flow of mares throughout the immediate post-breeding period. In study 1, uterine blood flow was characterized in mares (n = 7 mares/group) with minimal, moderate or severe endometrial degenerative changes (GI, GII and GIII, respectively). In study 2, the effect of age was investigated using young (≤ 6 years) and old (≥ 15 years) mares (n = 7 mares/group). Uterine vascular perfusion and mesometrial pulsatility index (PI) were evaluated every hour from H0 (moment...
Christoffersen M, Söderlind M, Rí»ºlk SR, Pedersen HG, Allen J, Krekeler N.Infectious endometritis is a major cause of infertility in the mare and inflicts major losses on the equine breeding industry. The ability of the mare to eliminate uterine infections has been studied intensively for decades; however, despite identification of several factors contributing to the multifactorial pathogenesis and improved treatment, infectious endometritis remains a significant problem in a subpopulation of broodmares. Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most commonly isolated pathogens from the uterus of mares, suffering from infectious endom...
Neuhauser S, Dörfel S, Handler J.Preservation of epididymal spermatozoa is important to save genetic material of endangered species and breeds, or in case of unexpected injury, which will end the breeding career of valuable sires. Seminal plasma (SP) influences sperm quality in a dose-dependent manner and its addition to preserved semen immediately before insemination may be beneficial for sperm fertility. Increased plasma membrane stability of epididymal spermatozoa reduces freezing injury of cells, and the addition of SP after freezing and thawing might have activating and protecting effects on spermatozoa within the female...
Alghamdi AS, Madill S, Foster DN, Troedsson MH.When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutro...
Martínez-Boví R, Cuervo-Arango J.Haemorrhagic anovulatory follicle is the most common pathological anovulatory condition in the mare, but its cause remains unknown. An experimental model to induce luteinised unruptured follicles (LUF) with flunixin-meglumine (FM) has been developed. Luteinised unruptured follicles share similar morphological and hormonal characteristics with haemorrhagic anovulatory follicles. Objective: To test the effect of intrafollicular administration of prostaglandins PGE2 and PGF2α during the periovulatory period on ovulation and pregnancy in FM-treated mares. Methods: In vivo experiment in a crossove...
Seidel GE.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...
Cuervo-Arango J, Mateu-Sánchez S, Aguilar JJ, Nielsen JM, Etcharren V, Vettorazzi ML, Newcombe JR.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)...
Macías-García B, González-Fernández L, Loux SC, Rocha AM, Guimarães T, Peña FJ, Varner DD, Hinrichs K.Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA...
Albrizio M, Moramarco AM, Nicassio M, Micera E, Zarrilli A, Lacalandra GM.It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level fo...
Coffman EA, Pinto CR, Snyder HK, Leisinger CA, Cole K, Whisnant CS.A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulat...
Morrell JM, Richter J, Martinsson G, Stuhtmann G, Hoogewijs M, Roels K, Dalin AM.A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from "normal" ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sp...
Avanzi BR, Ramos Rdos S, Araujo GH, Fioratti EG, Trinca LA, Dell'Aqua JA, Melo E Oña CM, Zahn FS, Martin I, Alvarenga MA, Papa FO.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...
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...
Woodward EM, Christoffersen M, Horohov D, Squires EL, Troedsson MH.Research has shown that 6 h after breeding is a critical time during the uterine innate immune response, and the failure to respond appropriately will result in persistent breeding-induced endometritis. This presents a potential opportunity to modulate the course of inflammation towards a timely resolution. Objective: To evaluate the effects of immune modulation on endometrial mRNA expression of inflammatory genes in susceptible mares 6 h after breeding. The hypothesis was that immune modulation alters endometrial cytokine expression in susceptible mares. Methods: A randomised controlled s...
Kino S, Osawa T, Mizuno S, Watanabe G, Taya K, Izaike Y.To clarify effect of postbreeding intramuscular administration of deslorelin on mares, plasma LH and progesterone concentrations and conception rate were evaluated. In Experiment 1, deslorelin, buserelin, or a placebo was administered on day 9 (day 0=day of last breeding or AI). Plasma samples were collected daily from days 6 to 15, and additional samples were taken after treatment. There was a significant increase in hormonal concentrations after deslorelin treatment. In Experiment 2, deslorelin or placebo was injected on day 9. There was no difference in conception rates at days 15 and 30 be...
Govaere JL, Hoogewijs MK, De Schauwer C, De Vliegher S, Van Soom A, Duchateau L, de Kruif A.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...
Sessions-Bresnahan DR, Graham JK, Carnevale EM.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 ...
Morrell JM, Georgakas A, Lundeheim N, Nash D, Davies Morel MC, Johannisson A.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...
de Oliveira RA, Scarlet D, Ille N, Aurich C.A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 106 and 100 × 106 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensat...
Palmer E, Duchamp G, Cribiu EP, Mahla R, Boyazoglu S, Bézard J.The aim of this study was to test the possibility that ovulation can occur from a preovulatory follicle emptied of its follicular fluid. Transport of the oocyte into the oviduct and fertilisation in 29% of cases demonstrated that ovulation can occur in the absence of follicular fluid but the higher fertility achieved in control mares (62.5%) suggested that follicular fluid does serve a role during ovulation, fertilisation and oviductal transport. Injection of horse oocytes into preovulatory follicles in mules after removal of the follicular fluid, followed by insemination of the mules with hor...
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...
Müller Z.From 341 stallions examined for sperm quality, 61% of warm-blooded stallions and 47% of cold-blooded stallions fulfilled the pre-existing criteria for their occasional use in insemination. From these stallions 51-71% of acceptable ejaculates were obtained. Altogether 959 mares were inseminated in an average of 1.36 oestrous cycles. For the insemination of one mare in one oestrous cycle on the average 2.2 insemination doses were used. These inseminations were carried out by 41 cattle insemination technicians trained in mare insemination. A pregnancy rate of 56% and a foaling rate of 48% were ac...
Greiser T, Sieme H, Martinsson G, Distl O.Objectives of the present study were to estimate genetic parameters for frozen-thawed semen traits of 271 fertile German Warmblood stallions and genetic correlations with raw semen quality traits. Semen samples were collected from stallions utilized for semen collection and artificial insemination (AI) on the Lower Saxon National Stud Celle and the North Rhine-Westphalian National Stud Warendorf. Semen quality variables were analyzed in 63,972 raw (gel-free volume, concentration, progressive motility, number of sperm) and 3681 frozen-thawed samples (motility, DNA fragmentation index (DFI), non...
Panzani D, Rota A, Pacini M, Vannozzi I, Camillo F.One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate an...
Pennington PM, Splan RK, Jacobs RD, Chen Y, Singh RP, Li Y, Gucek M, Wagner AL, Freeman EW, Pukazhenthi BS.Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embr...
Gutiérrez-Cepeda L, Fernández A, Crespo F, Ramírez MÁ, Gosálvez J, Serres C.Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of e...
Choi YH, Love CC, Varner DD, Hinrichs K.This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract...
Haadem CS, Nødtvedt A, Farstad W, Thomassen R.Pregnancy rates with cooled equine semen can be unsatisfactory and show great variation. Information about first cycle pregnancy rates and pregnancy rates per cycle are often lacking from publicly available records. This retrospective cohort study was performed to evaluate the fertility of the Norwegian Coldblooded trotter. The aim of the study was to compare the breeding results after insemination with fresh, extended with those of cooled, shipped semen among Norwegian Coldblooded trotter mares. First cycle pregnancy rate was the main parameter used to measure fertility. Stud-books were colle...
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...
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...
Ramírez-Agámez L, Hernández-Avilés C, Varner DD, Love CC.Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for f...
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 24 h pre- or 4 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 ...
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...
