Equine sperm refers to the male reproductive cells produced by stallions, essential for the process of fertilization and successful breeding in horses. The study of equine sperm encompasses various aspects, including morphology, motility, viability, and genetic integrity. These parameters are critical for assessing stallion fertility and improving breeding outcomes. Research in this field often focuses on understanding the factors that influence sperm quality, such as age, nutrition, and environmental conditions. Additionally, advancements in assisted reproductive technologies, such as artificial insemination and cryopreservation, rely heavily on the detailed study of sperm characteristics. This page compiles peer-reviewed research studies and scholarly articles that explore the biology, evaluation, and technological applications related to equine sperm.
Gracia-Calvo LA, Ezquerra LJ, Martín-Cuervo M, Durán ME, Tapio H, Gallardo JM, Peña FJ, Ortega-Ferrusola C.Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this proce...
Yeste M, Estrada E, Rocha LG, Marín H, Rodríguez-Gil JE, Miró J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Guimarães T, Lopes G, Pinto M, Silva E, Miranda C, Correia MJ, Damásio L, Thompson G, Rocha A.Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorgan...
Coutinho da Silva MA, Seidel GE, Squires EL, Graham JK, Carnevale EM.Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 comp...
Morrell JM, Johannisson A.The effect on sperm motility and chromatin integrity of adding homologous or heterologous equine seminal plasma (SP) to fresh stallion spermatozoa selected by single layer centrifugation (SLC) was studied. No statistical difference in mean progressive motility was seen after adding SP at time 0 h, although there were differences for individual stallions. The proportion of spermatozoa with high velocity was increased compared to untreated SLC-selected spermatozoa (P < 0.05), with significant differences between individuals (P < 0.01). When the SLC samples were stored for 24 h before a...
Varner DD, Gibb Z, Aitken RJ.Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermato...
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...
Álvarez C, Gil L, González N, Olaciregui M, Luño V.The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservatio...
Gallardo Bolaños JM, Balao da Silva C, Martín Muñoz P, Plaza Dávila M, Ezquerra J, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h ...
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...
Gallardo Bolaños JM, Balao da Silva CM, Martín Muñoz P, Morillo Rodríguez A, Plaza Dávila M, Rodríguez-Martínez H, Aparicio IM, Tapia JA....AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, w...
Leemans B, Gadella BM, Sostaric E, Nelis H, Stout TA, Hoogewijs M, Van Soom A.Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during...
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 ...
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 ...
Arkoun B, Gautier C, Delalande C, Barrier-Battut I, Guénon I, Goux D, Bouraïma-Lelong H.Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysi...
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...
Najjar A, Ben Said S, Benaoun B, Chetoui C, Ezzaouia M, Ben Mrad M.The study was undertaken in order to evaluate sperm morphology features of post-thawed semen of Tunisian Arab stallions. Forty two ejaculates was collected and frozen, during years 2009 and 2010, from 9 stallions aged between 9 to 24 years. After thawing, sperm morphology was studied after eosin-nigrosin stain. The percentages of abnormal head, mid piece, flagella, sperm with droplets and the total abnormal sperm were determined. Analysis of variance was carried out using SAS software. The results showed that all sperm morphology features varied among ejaculates within stallion and among stall...
Murphy C, English AM, Holden SA, Fair S.An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
Ponthier J, Franck T, Parrilla-Hernandez S, Niesten A, de la Rebiere G, Serteyn D, Deleuze S.Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extr...
Schmid-Lausigk Y, Aurich C.Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled-stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO...
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...
Morrell JM, Klein C, Lundeheim N, Erol E, Troedsson MH.Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not...
Bolaños JM, Morán AM, da Silva CM, Dávila MP, Muñoz PM, Aparicio IM, Tapia JA, Ferrusola CO, Peña FJ.To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the ...
Olaciregui M, Gil L, Montón A, Luño V, Jerez RA, Martí JI.Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was test...
Dias GM, López ML, Ferreira AT, Chapeaurouge DA, Rodrigues A, Perales J, Retamal CA.Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal ma...
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...
Gil L, Galindo-Cardiel I, Malo C, González N, Alvarez C.Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125-150 mM of cholesterol from pig liver and 0.5-0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6-8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100 × 10(6...
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...
Fayrer-Hosken RA, Hill NS, Heusner GL, Traylor-Wiggins W, Turner K.Ergot alkaloids cause a range of pathological conditions in mares. There is no evaluation of the effects of ergot alkaloids from endophyte-infected tall fescue on the stallion breeding soundness examination spermiogram. Objective: The objective of this study was to investigate the effect of ergot alkaloids from endophyte-infected tall fescue on the stallion's reproductive functions. Methods: Crossover toxicology experiment. Methods: Six stallions were fed either toxic endophyte-infected tall fescue seed or a nontoxic endophyte tall fescue seed (Flecha AR-542, MaxQ). The fescue seed content was...
The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the f...
Power MM, Gustavsson I, Switoński M, Plöen L.Synaptonemal complex analysis by electron microscopy of a trisomy 28 in a male horse demonstrated a trivalent or a bivalent plus a univalent in primary spermatocytes. Two of the chromosomes making up the trivalent were, most often, completely paired with each other and only partially paired or associated with the third one. Half of the spermatocytes analysed demonstrated heterologous pairing or association between the free axis of the trivalent and the sex bivalent. The pairings remained, to a large extent, into diakinesis-metaphase I. In most pachytene cells one autosomal bivalent showed prox...
Davis RO, Gravance CG, Casey PJ.Tissue variation in microscope slides made for spermatozoon analysis and variation introduced by the subjective techniques used to analyze these slides reduce the statistical power of studies that seek to use spermatozoon morphology to predict fertility. A simple specimen preparation method was developed to standardize stallion spermatozoon morphologic smears, and a new, automated spermatozoa morphometry instrument was used to objectively analyze the efficacy of the specimen preparation technique. The method achieved a standard spermatozoon concentration and reduced field-to-field variation in...
Bedford SJ, Kurokawa M, Hinrichs K, Fissore RA.In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain i...
Valenzuela OA, Couturier-Tarrade A, Choi YH, Aubrière MC, Ritthaler J, Chavatte-Palmer P, Hinrichs K.Assisted reproductive technologies (ARTs) such as intracytoplasmic sperm injection (ICSI), in vitro embryo culture and embryo transfer (ET) may be associated with alterations in fetal and placental development. In horses, ET has been used for decades. More recently, in vitro embryo production by ICSI and in vitro culture, followed by embryo transfer (ICSI-C) has become an accepted method for clinical foal production. However, no information is available on the effects of ICSI-C or even of standard ET itself on placental and neonatal parameters in horses. We therefore evaluated placental and ne...
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...
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...
Crespo F, Gosalvez J, Gutiérrez-Cepeda L, Serres C, Johnston SD.Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ...
Hidalgo M, Ortiz I, Dorado J, Morrell JM, Gosálvez J, Consuegra C, Diaz-Jimenez M, Pereira B, Crespo F.The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (as...
Gracia-Calvo LA, Ezquerra LJ, Martín-Cuervo M, Durán ME, Tapio H, Gallardo JM, Peña FJ, Ortega-Ferrusola C.Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this proce...
Wilhelm KM, Graham JK, Squires EL.Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for sper...
Len JA, Jenkins JA, Eilts BE, Paccamonti DL, Lyle SK, Hosgood G.The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility ...
Gacem S, Catalán J, Valverde A, Soler C, Miró J.In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISASD4C depths 10 and 20 µm, ISASD4C Leja 20 and drop displacement chamber (DDC), Spermtrack (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity-VCL) was dependent on chamber type. The highest ...
Mogielnicka-Brzozowska M, Fraser L, Dziekońska A, Gackowska K, Sobiewska M, Kuzborska A, Majewska AM, Filipowicz K, Kordan W.The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrei...
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 ...
Phetudomsinsuk K, Sirinarumitr K, Laikul A, Pinyopummin A.One of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions. Methods: Semen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Stand...
Rossi M, Gonzalez-Castro R, Falomo ME.Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 ...
Catalán J, Yánez-Ortiz I, Gacem S, Papas M, Bonet S, Rodríguez-Gil JE, Yeste M, Miró J.Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored...
Orsztynowicz M, Pawlak P, Podstawski Z, Nizanski W, Partyka A, Gotowiecka M, Kosiniak-Kamysz K, Lechniak D.Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four ...
Bruemmert JE, Coy RC, Squires EL, Graham JK.Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination w...
Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A.Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Morrell JM, Geraghty RM.A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. Objective: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. Methods: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing tech...
Miró J, Catalán J, Marín H, Yánez-Ortiz I, Yeste M.While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. ...
Blach EL, Amann RP, Bowen RA, Frantz D.Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender w...
Thomas PG, Ignotz GG, Ball BA, Brinsko SP, Currie WB.Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein sec...
Kumar P, Mehta JS, Ravi SK, Dedar RK, Purohit GN, Legha RA, Tripathi BN, Talluri TR.The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5,...
Plante G, Lusignan MF, Lafleur M, Manjunath P.Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a ...
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 ...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Hidalgo M.Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 μl), sperm concentrations (50, 100, 200 × 10 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 20...
