Equid semen refers to the reproductive fluid produced by male horses, which contains sperm cells necessary for fertilization. The quality and viability of equid semen are important for successful breeding programs and can be influenced by various factors, including genetics, age, diet, and environmental conditions. Key parameters used to assess semen quality include volume, concentration, motility, and morphology of sperm cells. Techniques for collecting, evaluating, and preserving equid semen, such as cryopreservation, are critical for artificial insemination practices. This page compiles peer-reviewed research studies and scholarly articles that explore the physiology, assessment, and management of equid semen in the context of equine reproduction.
Loomis PR.Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major imped...
Rigby SL, Brinsko SP, Cochran M, Blanchard TL, Love CC, Varner DD.This study evaluated motility and fertility of uncentrifuged and centrifuged equine semen following dilution in a skim milk-glucose extender with or without supplemental Tyrode's medium. In addition, the effect of seminal plasma addition to each extender was evaluated. For Experiment 1, motility of 48h cooled, stored spermatozoa was evaluated following eight dilution treatments: uncentrifuged and diluted 1:4 (v/v) in skim milk-glucose extender (EZ Mixin CSTJ; CST-1:4) or in CST supplemented 65:35 (v/v) with modified Tyrode's medium (KMT-1:4); uncentrifuged and diluted to 25x10(6) spermatozoa/m...
Graham JK.For many years, scientists have sought to develop laboratory assays that accurately predict the fertilizing capacity of a semen sample. This goal, however, has proven elusive and will most likely be very difficult to achieve, due to the complex nature of the problem. Part of the problem results from the many attributes that a spermatozoon must possess to fertilize an egg, and how laboratory assays can evaluate all of these attributes simultaneously. The percentage of motile sperm in a sample is most commonly used to evaluate semen quality. This assay, however, is not highly correlated with the...
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...
McDonnell SM.This study is part of ongoing work toward developing pharmacological methods for enhancing and inducing ejaculation in stallions with ejaculatory dysfunction or disabilities that interfere with normal breeding behavior. The objective was to evaluate a treatment regimen involving oral imipramine followed by intravenous xylazine that, in uncontrolled field clinical trials, had shown promise for a higher rate of ejaculation and fewer side effects using a more easily obtained and administered form of imipramine. Eight stallions each underwent eight trials in which treatment consisted of imipramine...
Katila T.The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h a...
Carvalho AL, Dias JM, Sanz L, Romero A, Calvete JJ, Romão MJ.The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.
Ball BA, Vo AT, Baumber J.To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
Nie GJ, Wenzel JG.Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay...
Arienti G, Polci A, De Cosmo A, Saccardi C, Carlini E, Palmerini CA.The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein p...
Janett F, Thun R, Ryhiner A, Burger D, Hassig M, Hertzberg H.The objective of this study was to evaluate the effect of Eqvalan (ivermectin) on stallion semen quality and freezability. Experiments were performed using 22 Freiberger stallions, randomly divided into a control and test group. Semen was collected once a week for 17 weeks from October 1997 to February 1998. Eqvalan was given orally to all stallions of the test group at a therapeutic dose of 0.2 mg ivermectin/kg. Besides measuring the scrotal width, ejaculates were collected to determine the volume, concentration, and the motility and morphology (normal sperm, major defects, vacuoles and acros...
Merkies K, Chenier T, Plante C, Buhr MM.Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of ea...
Blottner S, Warnke C, Tuchscherer A, Heinen V, Torner H.The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were eva...
Neild DM, Chaves MG, Flores M, Miragaya MH, Gonzalez E, Agüero A.The hypo-osmotic test has been used successfully on equine semen and was considered to be a simple and accessible method which could be a useful addition to routine equine semen analysis. It was therefore of interest to determine whether the hypo-osmotic test is significantly correlated to proposed criteria of fertility. The stallions were divided into two groups on the basis of threshold levels of fertility. A significant difference (P<0.05) was found between the two groups for the following parameters: progressive motility, morphologically normal spermatozoa, percentage of swelling with t...
Stradaioli G, Sylla L, Zelli R, Verini Supplizi A, Chiodi P, Arduini A, Monaci M.The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas ...
Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E.The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addit...
Alvarenga MA, Landim-Alvarenga FC, Moreira RM, Cesarino MM.The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E1...
Multigner L, Magistrini M, Ducot B, Spira A.Several reports have suggested that human semen quality has declined throughout the world over the last few decades. Chemicals in the environment acting as endocrine disruptors have been implicated as a possible cause. If this is indeed the case, then similar effects may be observed in animals. We report data on secular trends in semen quality of stallions collected during the last two decades by French National Studs. Methods: We analyzed 1489 ejaculates collected from 390 Breton draught stallions between 1981 and 1996 and 341 ejaculates from 86 anglo-arab thoroughbred stallions from 1985 and...
Brinsko SP, Crockett EC, Squires EL.The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Brinsko SP, Rowan KR, Varner DD, Blanchard TL.This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loa...
Lagares MA, Petzoldt R, Sieme H, Klug E.The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane...
Malmgren L.The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two dif...
Trimeche A, Yvon JM, Vidament M, Palmer E, Magistrini M.The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA.The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known ...
Troedsson MH, Liu IK, Crabo BG.After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa...
Shore MD, Macpherson ML, Combes GB, Varner DD, Blanchard TL.It has become a common practice in the equine breeding industry to send 2 insemination doses for breeding with transported cooled semen, one to be used for the initial insemination upon arrival, and the other to be held a second insemination the next day. One fertile stallion and 36 fertile mares were used to determine if breeding once with 1 dose of semen cooled for 24 h would improve fertility compared with breeding twice, 1 d apart, with half the dose of semen cooled for 24 h on the first day of breeding and half cooled for 48 h on the second day of breeding. Mares were given two intramuscu...
Liberda J, Tichá M, Zralý Z, Svecová D, Vezník Z.The interaction of seminal plasma proteins, sperms and detergent-released sperm proteins of three species with different types of acidic polysaccharides was studied. Heparin-binding activity of boar, bull and stallion seminal plasma proteins, sperms and sperm proteins was compared with their ability to interact with polysaccharides differing in the presence of the sulfate groups or in their saccharide moiety (chondroitin sulfate, dextran sulfate, fucoidan, hyaluronic acid). Bull seminal plasma proteins were characterized by higher affinity to heparin, fucoidan and dextran sulfate, while signif...
Squires EL, Brubaker JK, McCue PM, Pickett BW.In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit t...
Leipold SD, Graham JK, Squires EL, McCue PM, Brinsko SP, Vanderwall DK.Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy r...
Yang F, Wu S, Yang W, Yu J, Liu B, Zeng S.To study the effects of exercise on donkey semen parameters, Dezhou donkey (Equus asinus) jackasses (n = 18) were assigned to three groups: control (CN, n = 6), 1-hour exercise (1 h EX, n = 6) and 2-hour exercise (2 h EX, n = 6) groups. They were exercised with a walking machine for 3 days/week for 8 weeks. Semen parameters and reproductive hormones were evaluated weekly. The heart rate, rectal temperature, blood haematology indexes, and the body condition score (BCS) were also measured. Results showed that the ejaculation volume increased while the sperm concentration decreased after ...
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...
Byers SW, Dowsett KF, Glover TD.Three stallions were bled each hour for 25 h at 28-day intervals throughout 1 year. Testosterone levels were pulsatile. Pulse frequency and pulse amplitude were higher in the summer months than at other times (P less than 0.01). The number of testosterone pulses also varied throughout the day, with the greatest frequency occurring in the afternoon (14.00-17.00 h) and at night (22.00-01.00 h). Mean testosterone levels were highest in the summer (P less than 0.01) but showed a secondary, smaller increase in the autumn. Semen characteristics were assessed from measurements of 222 stallions. Semen...
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 ...
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...
Pérez-Marín CC, Vizuete G, Vazquez-Martinez R, Galisteo JJ.Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. Objective: To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. Methods: Randomised controlled experiment. Methods: Horse (n=19) and donkey (n=16) embryos (≤300 μm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L...
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 ...
Mari G, Bucci D, Love CC, Mislei B, Rizzato G, Giaretta E, Merlo B, Spinaci M.The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (...
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...
Casey PJ, Robertson KR, Liu IK, Espinoza SB, Drobnis EZ.Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
Sieme H, Bonk A, Hamann H, Klug E, Katila T.The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto...
Assumpção TI, Lançoni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Malmgren L.The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two dif...
Squires EL.There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on t...
López ML, Olea N, Retamal CA.Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific d...
Amann RP, Cristanelli MJ, Squires EL.Motility and fertility of frozen-thawed semen differs greatly amongst stallions. Differences in seminal plasma might be one cause of this variation. For 8 ejaculates from each of 17 stallions, seminal plasma was saved at -20 degrees C and spermatozoa were cryopreserved. Based on post-thaw sperm motility, seminal plasma samples from 7 stallions (2 good, 3 variable, 2 poor sperm motility) were selected for measurement of electrolytes, protein content and analysis by sodium dodecylsulphate gel electrophoresis (10% gel, Coomassie blue stain). Variation in seminal plasma was significant (P less tha...
Consuegra C, Crespo F, Dorado J, Ortiz I, Diaz-Jimenez M, Pereira B, Hidalgo M.Vitrification of sperm is based on high-speed freezing by direct exposure to liquid nitrogen using non-permeable cryoprotectants, mainly disaccharides; yet, the concentration of cryoprotectants has a species-specific effect on the sperm cell. The aim of this study was to assess different sucrose concentrations for stallion sperm vitrification. Semen samples (n = 9) were collected from three stallions, centrifuged and resuspended to a concentration of 50 × 10 sperm/ml in a base extender (INRA96 + 1% of bovine serum albumin) with three different sucrose concentrations (Molar): 20 mM (S...
Guimarães T, Lopes G, Ferreira P, Leal I, Rocha A.Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the exte...
Bugno-Poniewierska M, Kij-Mitka B, Podstawski Z, Długosz B, Lasek O, Mrowiec P, Tischner M.The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, u...
Fazeli AR, Steenweg W, Bevers MM, van den Broek J, Bracher V, Parlevliet J, Colenbrander B.The hemizona assay (HZA) has been developed as a diagnostic test to predict the fertilisation potential of human spermatozoa. The aim of this study was to develop an HZA for stallion spermatozoa and to investigate a possible relationship between fertility and the outcome of the HZA in this species. Equine oocytes were obtained from ovaries collected at a slaughterhouse and by transvaginal, ultrasound-guided follicle aspiration. They were then denuded from cumulus cells and stored in salt solution at 4 degrees C until use. On the day of the experiments the oocytes were bisected, thus providing ...
Loomis PR, Squires EL.Success with frozen semen requires attention to detail and a basic understanding of the techniques for properly handling and thawing and inseminating frozen semen. Practitioners should also be familiar with strategies used for managing mares for insemination with thawed semen. This manuscript will review those techniques and also present fertility data collected in a commercial setting. Factors that affect pregnancy rates for mares inseminated with frozen-thawed semen such as timing and frequency of insemination were examined for two separate data sets consisting of 332 and 536 mare cycles col...
Jung H, Kim N, Yoon M.The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell po...
Thomson CH, Thompson DL, Kincaid LA, Nadal MR.To test the hypothesis that prolactin mediates the increase in seminal volumes induced by sexual stimulation in stallions, semen was collected from six stallions every other day for 26 d. The last eight collection days were treatment days. For each stallion, four treatments were randomly assigned to the first four of the eight treatment collection days, and then repeated in reverse order on the last four collection days; 1) CONTROL: semen collected per normal procedures; 2) Sexually stimulated: stallions were presented to mares in a chute for 10 min before collection; 3) Bromocriptine (dopamin...
Usuga A, Gutiérrez V, López ME, Pérez LF, Jaramillo L, Rojano B, Restrepo G.Microbial growth in semen may cause a decline of sperm quality and fertility; however, the addition of antifungals to semen extender has been shown to impair the overall fertility of the sperm. The aim of this study was to evaluate the antifungal activity of conventional and natural compounds, and their effect on the motility and kinetics of cooled stallion semen. A total of 15 ejaculates from five stallions were collected using the artificial vagina. Each ejaculate was supplemented with: fluconazole at 12.5 (F1), 25 (F2) and 50 (F3) mg/ml; amphotericin-B at 6.5 (A1), 12.5 (A2) and 25 (A3) mg/...
Rodriguez H, Bustos Obregon E.The fertilizing ability of stallion semen was analyzed using fresh and frozen samples, obtained before (June-July) or during (October-November) the breeding season. Thirty ejaculates obtained from 4 stallions were used. The analysis comprises routine seminogram; ATP concentration (Comhaire et al., 1983); subjective and objective motility and sperm velocity (Makler, 1980). Freezing was done following the technique of Martin et al. (1979). Sperm velocity, ATP content and objective motility in ejaculates of subjective motility >50% show values of 14.0 + or - 0.84 mu m s(-1); 4.8 + or - 2.7x10(...
Orsolini MF, Verstraete MH, van Heule M, Orellana D, Ortega A, Meyers S, Dini P.Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there ...
Brito LFC, Loomis PR, Klohonatz KM, Althouse GC.Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, ...
Backman T, Bruemmer JE, Graham JK, Squires EL.The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on t...
Campbell ML, England GC.Uterine contractions may play an important role in the transportation of spermatozoa towards the site of fertilisation in the oviduct of mares. M-mode ultrasound was used to measure the number, amplitude and duration of uterine contractions in each uterine horn and the uterine body of oestrous mares for four minutes before and four minutes after either coitus, or the artificial insemination of either 80.0 ml of fresh semen or 10.0 ml of fresh semen. The direction of the uterine contractions in each uterine horn and the uterine body was measured before and after coitus. Coitus and the inseminat...
