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.
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...
Alonso A, Baca Castex C, Ferrante A, Pinto M, Castañeira C, Trasorras V, Gambarotta MC, Losinno L, Miragaya M.The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P 0.05). Blastocysts were obtained with cooled spermatozoa, and ...
Ferris RA, Frisbie DD, McCue PM.Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were pe...
Tuppits U, Orro T, Einarsson S, Kask K, Kavak A.The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen-thawed semen. Standardbred mares (n=18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen-thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonicall...
Aurich C, Schneider J.In the equine species, sex determination of the conceptus is of growing interest for the breeding industry. In horses, the sex ratio of the offspring depends on changes in body condition of the mother at conception and under natural conditions may thus markedly deviate from an expected 1:1 ratio. Insemination with sex-sorted spermatozoa allows a pronounced shift of the sex ratio but at present pregnancy rates are low and vary considerably under field conditions. In equine embryo transfer programmes, sex determination in embryos before transfer via genetic methods is a promising approach with h...
Morrell JM, Stuhtmann G, Meurling S, Lundgren A, Winblad C, Macias Garcia B, Johannisson A.Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and...
Bromfield EG, Aitken RJ, Gibb Z, Lambourne SR, Nixon B.While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop a...
Pugliesi G, Fürst R, Carvalho GR.The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast-freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s(-1) or 75 °C 7 s(-1) ). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing a...
Foss R, Ortis H, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. Objective: This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. Methods: In vitro experiment. Methods: Oocytes were recovered by transvaginal ultrasound-guided folli...
Galli C, Duchi R, Colleoni S, Lagutina I, Lazzari G.Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the ...
Panarace M, Pellegrini RO, Basualdo MO, Belé M, Ursino DA, Cisterna R, Desimone G, Rodríguez E, Medina MJ.Flow cytometry sex-sorting technology was developed in 1989. However, it is only the bovine species in which offspring of the desired sex are obtained at a commercial level. The aim of the present work was to evaluate efficiency parameters when using fresh sexed semen in a large-scale equine commercial embryo transfer program. During the 2009, 2010 and 2011 breeding seasons, 938 synchronized cycles were artificially inseminated. One hundred (10.6%) mares failed to ovulate, and for the remaining 838 useable cycles, 887 doses of sexed semen were used, representing 1.06 doses per cycle. In genera...
Hartwig FP, Lisboa FP, Hartwig FP, Monteiro GA, Maziero RR, Freitas-Dell'Aqua CP, Alvarenga MA, Papa FO, Dell'Aqua JA.During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of...
Serafini R, Longobardi V, Spadetta M, Neri D, Ariota B, Gasparrini B, Di Palo R.Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inse...
Morató R, Soares JM, Orero G, Mogas T, Miró J.The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino...
Woodward EM, Christoffersen M, Campos J, Betancourt A, Horohov D, Scoggin KE, Squires EL, Troedsson MH.Transient endometritis after breeding is necessary for clearance of bacteria and spermatozoa; however, in a subpopulation of mares, the inflammation fails to resolve in a timely fashion. The objective of this study was to describe the uterine inflammatory response in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) during the first 24 h after induction of uterine inflammation.Twelve mares were classified as susceptible (nZ6) or resistant (nZ6) to PBIE. Mares were inseminated over five estrous cycles and endometrial biopsies were collected at one time point per ...
Pasing SS, Aurich C, von Lewinski M, Wulf M, Krüger M, Aurich JE.An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen mot...
Gibb Z, Morris LH, Maxwell WM, Grupen CG.Cryopreserved, sex-sorted stallion sperm has been shown to have poor fertility. During this study, the effects of cryoprotectant (glycerol [GLY] and dimethyl formamide [DMF]), cryoprotectant equilibration time (0, 30, 60, 90, or 120 minutes), and cryoprotectant concentration (2%, 3%, or 4% vol/vol) on stored sex-sorted and stored nonsorted stallion sperm were evaluated. Total motility, viability, and DNA integrity (determined using sperm chromatin structure assay) of sperm were assessed after thawing. Equilibration for 90 minutes improved total motility (33.8%) compared with 0 (28.5%) or 120 m...
Vilés K, Rabanal R, Rodríguez-Prado M, Miró J.Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced e...
Canisso IF, Gallacher K, Gilbert MA, Korn A, Schweizer CM, Bedford-Guaus SJ, Gilbert RO.The major objective of this study was to determine whether short-term preovulatory progestagen treatment of mares could effectively delay ovulation. Secondary objectives were to determine the effect such supplementation had on signs of estrus, follicular growth, postovulatory luteal function and pregnancy rate. Thirteen cyclic mares of different breeds were used in this study during the natural breeding season. Once mares were confirmed in estrus with a follicle of 35 mm in diameter, they were assigned in random order to receive no treatment (control), placement of a progesterone-impregnated c...
Mambelli LI, Winter GH, Kerkis A, Malschitzky E, Mattos RC, Kerkis I.Mesenchymal stem cells (MSCs), because of their immunomodulation and trophic activities, in addition to their capacity to regenerate damaged tissues, have potential for treatment of many diseases. The success of stem cell therapies depends, in part, on the method of cell delivery, which should provide wide cell distribution and homing in to injured sites. The objective of the present study was to developing a novel strategy for delivery of MSCs into the uterus of mares with endometrosis (degenerative alteration of uterine glands and surrounding stroma). Endometrosis was confirmed in all mares ...
Woodward EM, Christoffersen M, Campos J, Horohov DW, Scoggin KE, Squires E, Troedsson MH.The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) within 24 h after breeding. Mares susceptible (n = 6) or resistant (n = 6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24 h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexame...
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...
Morrell JM, Timoney P, Klein C, Shuck K, Campos J, Troedsson M.Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, ...
Vieira LA, Gadea J, García-Vázquez FA, Avilés-López K, Matás C.After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
Klein C, Donahue JM, Sells SF, Squires EL, Timoney PJ, Troedsson MH.To determine the impact of antimicrobial-containing semen extender on the growth of Taylorella equigenitalis in semen culture-positive for contagious equine metritis (CEM) and the development of CEM after artificial insemination with CEM-positive semen extended with antimicrobial-containing semen extender. Methods: Prospective study. Methods: 21 mature mares free of CEM, 1 mature stallion experimentally infected with CEM, and semen from a stallion naturally infected with CEM. Methods: CEM-positive semen was incubated in semen extender with and without antimicrobials (amikacin [final concentrat...
Katila T.This review attempts to summarize the current knowledge on uterine inflammatory response after mating in horses, pigs and cattle. Post-mating endometritis has been extensively studied in horses as it has been considered to cause infertility. The inflammation is known to occur also in cattle, but it has not been investigated to a similar extent. There are a number of publications about mechanisms of post-mating uterine inflammation in pigs, which seem to resemble those in horses. The major focus of this review is the horse, but relevant literature is presented also on swine and cattle. Spermato...
Lindahl J, Dalin AM, Stuhtmann G, Morrell JM.One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. Results: Sixteen mares were insemi...
Katila T, Sankari S, Mäkelä O.A scintigraphic method was developed to study sperm migration in the reproductive tracts of mares. Mares (n=5) and stallions (n=2) were used to test various steps of the procedure and three other mares and a stallion were used to study sperm transportation. A radiolabelling solution was prepared from 99mTc (Technetium-99m) and hexamethyl propylene amine oxime. The highest labelling of spermatozoa (57-72%) was obtained by incubation of the spermatozoa with the radiolabelling solution for 20 min at 20 degrees C. Radioactivity outside the spermatozoa was removed by centrifugation and by two subse...
Ball BA, Sabeur K, Allen WR.Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objective: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also...
Zhang JJ, Muzs LZ, Boyle MS.In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 ...
Mari G, Morganti M, Merlo B, Castagnetti C, Parmeggiani F, Govoni N, Galeati G, Tamanini C.Since results with using sulpiride and domperidone are conflicting and since both have not been tested at the same time, the aim of this study was to compare the efficacy of these substances for the induction of ovulation in deep anestrous mares in the same experimental conditions and to determine their fertility after artificial insemination (AI) at the induced estrus. Twenty-six non-pregnant, non-lactating standardbred anestrous mares were randomly assigned to three groups and treated daily for 25 days (from February 3rd to February 28th) with either sulpiride (1mg/kg of body weight im SID, ...
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)...
Caballeros JE, Camacho C, Cazales N, Estradé MJ, Fiala-Rechsteiner S, Jobim MIM, Mattos RC.Rapid endometrial adaptations occur with the embryo entering the uterus to create a receptive uterine environment, which is essential for the conceptus' development. The aim of this study was to demonstrate ultrastructural and histological changes of the endometrium at day 5 after ovulation in cyclic and inseminated mares. Mares were daily examined by transrectal palpation and ultrasonographic examination of the reproductive tract until ovulation was detected. In the first cycle, endometrial biopsies from 10 cyclic mares (Cyclic group) were collected on day 5 post-ovulation. In the second cycl...
Hellander JC, Samper JC, Crabo BG.At the beginning of the breeding season an eight-year-old standardbred stallion had semen with virtually zero sperm motility and an approximately 90 per cent incidence of midpiece and tail defects. The motility of the sperm improved to 7 per cent when semen was collected daily but its morphology did not improve. Electron microscopy revealed that the defects consisted mainly of a loss of microtubules in the axoneme and of disorganised midpieces. A pregnancy rate of 24 per cent per cycle and 44 per cent for the season was achieved in 32 mares after the insemination of whole ejaculates collected ...
Choi YH, Love LB, Varner DD, Hinrichs K.This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In...
Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL.The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sp...
Crowe CA, Ravenhill PJ, Hepburn RJ, Shepherd CH.Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. Objective: Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. Methods: Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 200...
Panarace M, Pellegrini RO, Basualdo MO, Belé M, Ursino DA, Cisterna R, Desimone G, Rodríguez E, Medina MJ.Flow cytometry sex-sorting technology was developed in 1989. However, it is only the bovine species in which offspring of the desired sex are obtained at a commercial level. The aim of the present work was to evaluate efficiency parameters when using fresh sexed semen in a large-scale equine commercial embryo transfer program. During the 2009, 2010 and 2011 breeding seasons, 938 synchronized cycles were artificially inseminated. One hundred (10.6%) mares failed to ovulate, and for the remaining 838 useable cycles, 887 doses of sexed semen were used, representing 1.06 doses per cycle. In genera...
Morrell JM, Stuhtmann G, Meurling S, Lundgren A, Winblad C, Macias Garcia B, Johannisson A.Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and...
Meuffels-Barkas J, Wilsher S, Allen WRT, Ververs C, Lueders I.Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we post...
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H.Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was we...
Tongu EAO, Segabinazzi LGTM, Alvarenga ML, Monteiro A, Papa FO, Alvarenga MA.This study aimed to evaluate the effects of mesenchymal stem cell-conditioned medium (MSC-CM) on sperm parameters, intrauterine polymorphonuclear neutrophils (PMN), intrauterine fluid accumulation (IUF), and fertility in mares. In experiment 1, two ejaculates from ten stallions were extended to 50 million sperm/mL using a milk-based extender. Thereafter, 20 mL of extended semen was added of MSC-CM as follows: 0, 5, 10, 15, and 20 mL. Sperm kinetics and plasma membrane integrity were evaluated immediately after dilution (T0) and 2 h post-incubation at 37 °C (T2). In experiment 2, mares cha...
Brinsko SP.This manuscript presents a brief historical review of investigations related to equine artificial insemination. The origin of recommended insemination doses for use fresh, cooled and frozen semen will be reviewed. Over 30 years ago, an insemination dose of 500 x 10(6) progressively motile sperm (PMS) was recommended to maximize pregnancy rates when mares were bred with fresh semen under less than ideal conditions. Since that time, 500 x 10(6) progressively motile sperm has been almost universally accepted as a standard insemination dose, regardless of a stallion's fertility or the refinements ...
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...
Nouri H, Shojaeian K, Jalilvand G, Kohram H.The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation wer...
Dobrinski I, Thomas PG, Ball BA.Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3...
Güvenc K, Reilas T, Katila T.It is unclear whether AI of mares deep into the uterine horn causes more or less inflammation of the endometrium than conventional AI. Thus, we compared uterine inflammatory reactions of mares inseminated with two different doses of frozen-thawed semen into the tip of the uterine horn (UH) ipsilateral to the preovulatory follicle with those of mares inseminated into the uterine body (UB). Thirty-two mares were assigned to one of four groups (eight mares/group): UB20=AI into UB, 20 x 10(6)sperm/0.5 mL; UB200=AI into UB, 200 x 10(6)sperm/0.5 mL; UH20=AI into UH, 20 x 10(6)sperm/0.5 mL; UH200=AI ...
Samper JC.Semen quality, mare status and mare management during estrus will have the greatest impact on pregnancy rates when breeding mares with frozen semen. If semen quality is not optimal, mare selection and reproductive management are crucial in determining the outcome. In addition to mare selection, client communication is a key factor in a frozen semen program. Old maiden mares and problem mares should be monitored for normal cyclicity and all, except young maidens, should have at least a uterine culture and cytology performed. Mares with positive bacterial cultures and cytologies should be treate...
Malaluang P, Wagner LH, Cojkic A, Spergser J, Aurich C, Morrell JM.The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this st...
Al-Kass Z, Morrell JM.Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics ...
Carnevale EM, Maclellan LJ, Coutinho da Silva MA, Scott TJ, Squires EL.This study was designed to test 3 approaches for insemination and transfer of oocytes to recipient mares. Oocytes were recovered transvaginally from naturally cycling donor mares 24 to 26 h after an intravenous injection of 2500 IU of hCG when follicles reached 35 mm in diameter. Multiple oocytes (1 to 4) were transferred surgically into the oviducts of 4 or 5 recipient mares per group. Three groups of transfers were compared: 1) transfer of oocytes cultured in vitro for 12 to 14 h postcollection with insemination of the recipient 2 h postsurgery; 2) transfer of oocytes into the oviduct within...
Oddi S, Carluccio A, Ciaramellano F, Mascini M, Bucci R, Maccarrone M, Robbe D, Dainese E.Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight mai...
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...
