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.
Sieme H, Martinsson G, Rauterberg H, Walter K, Aurich C, Petzoldt R, Klug E.The objective of this research was to improve the techniques in processing chilled and frozen-thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim-up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 ...
Tremoleda JL, Van Haeften T, Stout TA, Colenbrander B, Bevers MM.Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and exami...
Morris LH, Tiplady C, Allen WR.To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are...
Quintero-Moreno A, Miró J, Teresa Rigau A, Rodríguez-Gil JE.The aim of this study was to test the presence of separate sperm subpopulations, with specific motility characteristics, in stallion ejaculates by using a computer-assisted semen motility analysis (CASA) system. Motility data were analyzed with a hierarchical clustering of variables based on a correlation or covariance matrix to select like parameters of sperm motility descriptors that better explain the kinetics of spermatozoa. The statistical analyses clustered the whole motile sperm population in both fresh and 24 h stored ejaculates into four separate groups. There were significant differe...
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Agüero A.Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 3325...
Li GP, Seidel GE, Squires EL.Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the IC...
Brinsko SP, Rigby SL, Lindsey AC, Blanchard TL, Love CC, Varner DD.This study was conducted to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, and to determine whether prebreeding intrauterine treatment with prostaglandin E(2) would improve pregnancy rates. Estrus was synchronized in 36 fertile Quarter Horse and Thoroughbred broodmares. When a dominant follicle >or=33 mm diameter was present, mares were treated with 2500 units hCG intravenously and were assigned to one of four treatment groups for insemination with five million total sperm in 200 microl extender the next day as follows: (1) Group PGE-HYS (n=9): 0...
Ball BA, Gravance CG, Wessel MT, Sabeur K.A testis-specific isoform of angiotensin-converting enzyme (ACE) has been identified in a number of mammalian species. The purpose of this study was to characterize the activity of ACE in equine spermatozoa, seminal plasma, and testis. Activity of ACE was determined in seminal plasma, ejaculated and epididymal spermatozoa from mature stallions as well as from pre- and postpubertal testis. The effect of addition of angiotensin II on equine sperm motility was also evaluated. The activity of ACE in detergent extracted sperm plasma membrane was approximately 13-fold higher than that detected in se...
Brinsko SP, Blanchard TL, Rigby SL, Love CC, Varner DD.The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculat...
Rosati I, Berlinguer F, Bogliolo L, Leoni G, Ledda S, Naitana S.It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS ...
Rathi R, Colenbrander B, Stout TA, Bevers MM, Gadella BM.Progesterone (P(4)) is a physiological inducer of the acrosome reaction (AR) in stallion spermatozoa. However, the capacitation-dependent changes that enable progesterone binding, and the nature of the signaling cascade that is triggered by progesterone and results in induction of the AR, are poorly understood. The aim of the current study was, therefore, to investigate the protein kinase dependent signaling cascades involved in progesterone-mediated induction of the AR in stallion spermatozoa. In addition, we aimed to determine whether bicarbonate, an inducer of sperm capacitation, acted via ...
Devireddy RV, Swanlund DJ, Alghamdi AS, Duoos LA, Troedsson MH, Bischof JC, Roberts KP.The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, wh...
Pommer AC, Rutllant J, Meyers SA.Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar ph...
Mantovani R, Rora A, Falomo ME, Bailoni L, Vincenti L.The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprote...
Pommer AC, Rutllant J, Meyers SA.Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditio...
Carver DA, Ball BA.Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during...
Schembri MA, Major DA, Suttie JJ, Maxwell WM, Evans G.Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P&...
Macpherson ML, Simmen RC, Simmen FA, Hernandez J, Sheerin BR, Varner DD, Loomis P, Cadario ME, Miller CD, Brinsko SP, Rigby S, Blanchard TL.The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected fr...
Hinrichs K, Love CC, Brinsko SP, Choi YH, Varner DD.Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P <...
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...
Lo CC, Thompson JA, Lowry VK, Varner DD.We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as sub...
Champion ZJ, Vickers MH, Gravance CG, Breier BH, Casey PJ.Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are both present in blood plasma and IGF-I has been measured in epididymal fluid and seminal plasma. This study was designed to investigate the direct effects of GH or IGF-I on the motility of mature equine spermatozoa in vitro. We compared the effects of one concentration (100 ng/ml) of recombinant bovine GH (rbGH) and recombinant human IGF-I (rhIGF-I) on motility and motion characteristics of equine spermatozoa over a 24 h period. Motility was maintained longer in spermatozoa treated with either rbGH or rhIGF-I during a 24 h period...
Baumber J, Vo A, Sabeur K, Ball BA.Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallion...
Lindsey AC, Morris LH, Allen WR, Schenk JL, Squires EL, Bruemmer JE.The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Al...
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...
Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment...
Ball BA, Vo A.The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescenc...
Kosiniak K.The use of an 'open' artificial vagina made it possible to separate the individual jets in the ejaculate of the stallion. A total of 132 jets were collected in this way from twenty-one ejaculates. After macro- and microscopic examination and sperm counting, glycerylphosphorylcholine (GPC), ergothioneine and fructose levels were determined for every jet. The values obtained for the first three jets were similar and accounted for about 76% of all spermatozoa, and 70% of GPC. These jets also contained the highest concentration of ergothioneine and fructose.
Farlin ME, Jasko DJ, Graham JK, Squires EL.The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (in...
Boyle MS, Cran DG, Allen WR, Hunter RH.The morphology of the uterotubal junction (UTJ) and caudal isthmus during the peri-ovulatory period, and the distribution of spermatozoa within the region, were studied in 10 Pony mares. The proximal tip of the uterine horn and caudal 1-2 cm of the isthmus were removed during oestrus or shortly after ovulation from animals mated or artificially inseminated within the previous 24 h. The tissues were incised longitudinally and fixed for scanning electron microscopy. Analysis of micrographs showed deep longitudinal and oedematous folds in the preovulatory samples. After ovulation, much of the fol...
Kjöllerström HJ, do Mar Oom M, Chowdhary BP, Raudsepp T.The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm-FISH and molecular analysis of FKBP6 - a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 6...
Zevallos Valenzuela GE, Ferrante A, Verón GL, Miragaya M, Marín-Briggiler CI, Vazquez-Levin MH.Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), o...
Brinsko SP, Rigby SL, Lindsey AC, Blanchard TL, Love CC, Varner DD.This study was conducted to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, and to determine whether prebreeding intrauterine treatment with prostaglandin E(2) would improve pregnancy rates. Estrus was synchronized in 36 fertile Quarter Horse and Thoroughbred broodmares. When a dominant follicle >or=33 mm diameter was present, mares were treated with 2500 units hCG intravenously and were assigned to one of four treatment groups for insemination with five million total sperm in 200 microl extender the next day as follows: (1) Group PGE-HYS (n=9): 0...
Liu DY, Lopata A, Pantke P, Baker HW.The present study demonstrates that horse and marmoset monkey sperm can bind to the human zona of salt-stored oocytes that failed to fertilize in vitro. Marmoset monkey sperm are also able to penetrate the salt-stored human zona. In contrast, human sperm do not bind to the zona of either horse or marmoset monkey oocytes. These results suggest that human sperm binding to the zona pellucida is more strictly species-specific than it is for horse and marmoset monkey sperm. In contrast, horse and marmoset monkey sperm contain receptors recognized by the human zona.
Abraham-Peskir JV, Chantler E, Uggerhøj E.We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their inciden...
de la Torre J, Crespo F, Arroyo F, Zabal-Aguirre M, Abdoon AS, Gosálvez J.Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after inc...
Alghamdi AS, Fedorka CE, Scoggin KE, Esteller-Vico A, Beatty K, Davolli G, Ball BA, Troedsson MHT.Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To eval...
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...
Eterpi M, Magistrini M, Couty I, Gavin-Plagne L, Aguirre-Lavin T, Schmitt E, Carion O.Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes pr...
Kato H, Seidel GE, Squires EL, Wilson JM.In vitro matured horse oocytes with a first polar body (n = 68) were each injected with a single spermatozoon and divided into 2 groups: Group 1 oocytes were treated with 10 microM calcium ionophore A23187 for 5 min while Group 2 oocytes received no activation treatment. After culture in vitro for 2 days, significantly more oocytes treated with A23187 (5/24, 21%) cleaved than oocytes without activation treatment (2/44, 5%, P<0.05). All 7 cleaved zygotes from both treatment groups were transferred to recipient mares but no pregnancies resulted.
González-Fernández L, Macedo S, Lopes JS, Rocha A, Macías-García B.Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%...
Turner RM, Love CC, McDonnell SM, Sweeney RW, Twitchell ED, Habecker PL, Reilly LK, Pozor MA, Kenney RM.An 8-year-old stallion was evaluated because of recurrent urinary tract infections and chronic intermittent urospermia. After extensive diagnostic testing, it was hypothesized that the stallion had a reflex dyssynergia of the bladder and urethral sphincter. Initial attempts to manage the urospermia included semen fractionation, semen collection after voluntary urination, and use of semen extenders. None of these efforts reliably yielded a quality ejaculate. Administration of imipramine hydrochloride (1.2 mg/kg of body weight, PO, 4 hours prior to semen collection) was initiated in an attempt t...
Silva DM, Holden SA, Lyons A, Souza JC, Fair S.The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100×106 spermatozoa mL-1 with 0.02mM vitamin E (VE) and 0, 1, 10 or 20ng mL-1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stall...
Senra RL, Ramírez-López CJ, Magalhães-Júnior MJ, Neves JGDS, Barros E, Waddington B, Guimarães SEF, Guimarães JD, Baracat-Pereira MC.Horses are seasonal polyoestrous animals, and the photoperiod is the main factor modulating their reproductive activity. There is no consensus on the andrological and biochemical factors that influence breeding seasonality. To assess the involvement of climate in reproduction, Mangalarga Marchador stallions were monitored over 1 year regarding semen quality and seminal plasma proteome. Here, we show that kallikrein (KLKs) proteoforms in seminal plasma are involved in climate conditioning of reproduction. During the breeding season, greater abundance and different types of KLKs occurred simult...
McCue PM, Fleury JJ, Denniston DJ, Graham JK, Squires EL.A technique was developed for oviductal insemination of mares, in which a small number of motile spermatozoa are deposited directly into the oviduct. Pregnancy rates in mares inseminated by traditional intrauterine artificial insemination were compared with rates in mares inseminated by oviductal insemination. Fifteen mares were inseminated with 5 x 10(8) progressively motile spermatozoa by intrauterine artificial insemination, and 14 mares were inseminated with 5 x 10(4) progressively motile spermatozoa by oviductal insemination. Pregnancy rates in mares inseminated by intrauterine artificial...
Pruß D, Oldenhof H, Wolkers WF, Sieme H.The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calc...
Okólski A, Babusik P, Tischner M, Lietz W.Comparisons were made between 2 methods of oocyte recovery from the ovarian follicles of slaughtered mares: 500 oocytes (3 per mare) were obtained by aspiration of follicular fluid from ovaries of 162 mares, and 120 oocytes (8 per mare) by isolation and rupture of follicles from ovaries of 14 mares. In the oocytes recovered after rupture of follicles, 89.2% were morphologically unchanged, in comparison to 29.3% obtained by aspiration of follicular fluid. Stallion spermatozoa capacitated in vitro were tested on zona-free hamster oocytes. The stallion spermatozoa were washed in TCM-199 and prein...
Lançoni R, Celeghini ECC, Gonella-Diaza AM, Júnior VG, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very l...
Kareskoski AM, Palviainen M, Johannisson A, Katila T.For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expressio...
Hernández-Avilés C, Ramírez-Agámez L, Love CC, Friedrich M, Pearson M, Kelley DE, Beckham AMN, Teague SR, LaCaze KA, Brinsko SP, Varner DD.Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were ...
Toscano WA, Toscano JS, Toscano DG, Gross MK.The soluble calmodulin-sensitive isoform of adenylyl cyclase isolated from equine sperm is unique because it requires Mn(2+) rather than Mg(2+) for activity. To gain insight into the molecular action of metals on sperm adenylyl cyclase, the kinetics of Mn(2+) and ATP effect was examined. A biphasic response to increases in ATP concentration was observed when metal was held constant. When [Mn(2+)] exceeded [ATP], however, greatly enhanced enzyme activity was observed. The kinetic profiles were consistent with allosteric activation of adenylyl cyclase by Mn(2+). Linear transformation of the data...
Kareskoski M, Vakkamäki J, Laukkanen K, Palviainen M, Johannisson A, Katila T.Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of...
Runcan EE, Pozor MA, Zambrano GL, Benson S, Macpherson ML.The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. Objective: To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal...
Engle CE, Foley CW, Witherspoon DM, Scarth RD, Goetsch DD.Three experiments were conducted on the metabolism of stallion sperm. In experiment 1, whole and washed sperm were incubated under aerobic and anaerobic enviroments and analyzed before and after controlled incubation for motility, pH, lactic acid, glucose, fructose, and O2 comsumption. In experiment 2, whole and washed sperm were incubated aerobically and anaerobically with and without uterine tubal fluids. Experiment 3 was the same as experiment 2, except added substrates of glucose and lactic acid were studied. The same examinations were made in experiments 2 and 3 as for experiment 1. Motil...
Serafini R, Varner DD, Bissett W, Blanchard TL, Teague SR, Love CC.The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabil...
Pace MM, Sullivan JJ.Fertilization rate was highest in mares inseminated with frozen semen within 12 hr of ovulation. Foaling rate was improved (P less than 0-05) by increasing the number of motile spermatozoa inseminated from 40 X 10(6) to 80 X 10(6) but was not further improved by increasing the number to 160 X 10(6) or by increasing the frequency of insemination from once to twice daily. The fertilizing capacity of spermatozoa frozen in one of the hydrogen ion extenders studied was dependent upon relative osmotic pressure and method of freezing (ampoules or pellets). Adjusting glycerol concentration from 7% to ...
Álvarez C, Luño V, González N, Gil L.Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted...
