Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
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...
Neuhauser S, Bollwein H, Siuda M, Handler J.Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze...
Boye JK, Katzman SA, Kass PH, Dujovne GA.In equids, it is common to inject lidocaine into the testicles at the time of routine castration to provide analgesia. The effects of lidocaine on equine sperm have not been evaluated in vitro or on epididymal sperm collected following castration. The aims of this study were to determine effects of clinically relevant doses of lidocaine on equine spermatozoa in vitro using freshly collected semen and to compare the characteristics of epididymal spermatozoa after routine castration with or without intra-testicular lidocaine administration. We hypothesized that increasing concentrations of lid...
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...
Ghallab ARM, Abou-Ahmed MM, Fad AM, El-Badry DA, Shahat AM, Moawad AR.Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed. Objective: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa. Methods: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divid...
Ortiz-Rodriguez JM, Martín-Cano FE, Ortega-Ferrusola C, Masot J, Redondo E, Gázquez A, Gil MC, Aparicio IM, Rojo-Domínguez P, Tapia JA....Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact ...
Brasileiro LS, Segabinazzi LGTM, Menezes E, Salgueiro CC, Novello G, Scheeren VFDC, Alvarenga MA, Nunes JF.The aim of this study was to evaluate the effect of coconut water as a component of extender in different formulations for cooling equine sperm. One ejaculate of fourteen stallions was collected. Sperm was diluted to 50 × 10 sperm/mL using five different extenders: ACP-105: powdered coconut water extender (ACP-105, ACP Biotecnologia, Brazil); ACP-Milk: ACP-105 + 20 g/L of skimmed milk; ACP-EY 2.5%: ACP-105 + 2.5% of egg yolk; ACP-EY 5%: ACP-105 + 5% of egg yolk; and BotuSêmen (Botupharma, Botucatu, Brazil) and cooled in passive cooling device (BotuFlex, Botupharma, Botucatu, Brazil) at 5...
Sales F, Ferreira-Silva J, Vieira J, Basto S, Filho P, Rocha J, Moura MT, Oliveira M.Post-diluents could potentially increase semen cryotolerance, but remain poorly explored in horses. The aim was to evaluate the efficiency of post-diluents on frozen-thawed semen viability of two stallions (S1-S2). The cryopreserved semen was thawed at 50°C for 40 seconds. Semen motility and acrosin activity (AA) were determined during the thermo-resistance test (TRT). Progressive motility of S2 semen decreased after 60 and 90 minutes of TRT (TRT60 and TRT90) on the control compared to both post-diluents. The total motility of both S1 and S2 decreased on TRT60 and TRT90 semen control versus b...
Martins HS, Martins-Filho OA, Araujo MS, Martins NR, Lagares MA.Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. Objective: This work aimed...
Delerue M, Breuil MF, Duquesne F, Bayon-Auboyer MH, Amenna-Bernard N, Petry S.Taylorella equigenitalis can be transmitted during artificial insemination. This report describes clinical T. equigenitalis transmission by cryopreserved stallion semen. T. equigenitalis isolates from a mare's vaginal discharge and semen from the same batch of the cryopreserved semen used for the insemination gave identical API ZYM, antibiotic susceptibility, and multilocus sequence typing results (ST-46); furthermore, the multilocus sequence typing lineage ST-46 is known to circulate in the country of semen collection. These results support the need for strict contagious equine metritis scre...
Yasine A, Ashenafi H, Geldhof P, Bekana M, Tola A, Van Brantegem L, Van Soom A, Duchateau L, Goddeeris BM, Govaere J.Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 10 to >5 × 10 trypanosomes, were added to semen samples. Subsequently, SLC was performed ...
Blommaert D, Sergeant N, Delehedde M, Jouy N, Mitchell V, Franck T, Donnay I, Lejeune JP, Serteyn D.A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentra...
Diaz-Jimenez M, Dorado J, Consuegra C, Ortiz I, Pereira B, Carrasco JJ, Gomez-Arrones V, Domingo A, Hidalgo M.The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 μL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma memb...
Huber D, Amsler E, Vidondo B, Kaeser R, Wespi B, Sieme H, Burger D.There exist differences in the reproductive behavior of stallions and mares under free-running and domestic in-hand breeding conditions. Contrary to artificial insemination programs, a stallion mates a mare multiple times per estrus under natural conditions. The objective of this study was to determine if multiple periovulatory artificial inseminations (MI), four times in two different time intervals instead of two, would result in increased pregnancy outcome or higher incidences of breeding induced endometritis. Methods: Eighty-two estrous mares were allocated randomly to one of three experim...
Álvarez C, Luño V, González N, Guerra P, Gil L.This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility...
Vafaei F, Kohram H, Zareh-Shahne A, Ahmad E, Seifi-Jamadi A.This study was aimed to evaluate the effect of permeable cryoprotectants in combination with trehalose or sucrose on the freezing capacity of stallion sperm. For this purpose, the ejaculates (n = 24) were collected from four healthy mature Turkmen stallions. The ejaculates were pooled and diluted with one of the extenders containing a combination of 5% of permeating (dimethylacetamide [DMA]; dimethylformamide [DMF] or glycerol) and 50 mM of nonpermeating cryoprotectant agents (CPAs) (sucrose or trehalose) to a final concentration of 200 × 10 spermatozoa/mL. The extended samples were cryopr...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Arenas R, Morrell JM, Hidalgo M.Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN) and conventional freezing using 0.5 mL straws frozen in LN vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Signif...
Rota A, Sabatini C, Przybył A, Ciaramelli A, Panzani D, Camillo F.To increase sperm motility, several molecules have been tested in mammals. Methylxanthines have shown effects on sperm motility, capacitation, and on in vitro fertilization processes. The aim of the study was to evaluate if the post-thaw addition of caffeine and/or pentoxifylline changes motility parameters of cryopreserved stallion and donkey spermatozoa. Straws derived from 14 horses and 7 donkeys were thawed and diluted in a milk-based extender to obtain the following final concentrations: CTR (control, no additives), CAF 5 (5 mM caffeine), CAF 10 (10 mM caffeine), PTX 5 (5 mM pentoxify...
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...
Duguma A, Lemma A, Hibste A.Equine reproduction is unique by having long behavioral estrus and differences in time of breeding between breeds and individuals of mares. An experimental study was conducted at the Balderas Sport Horses and Recreational Center, Addis Ababa, Ethiopia, from January to June, 2018, to evaluate conception rate to frozen semen in local and exotic crossbreed mares. Mares were teased to characterize estrus behavior and examined by ultrasound in determining imminent ovulation. Inseminations were done post ovulation within an average of 6-9 h using frozen-thawed semen. The overall conception rate to ...
Alamaary MS, Haron AW, Ali M, Hiew MWH, Adamu L, Peter ID.Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equa...
Gonzalez-Castro RA, Carnevale EM.We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF ...
Kareskoski M, Venhoranta H, Virtala AM, Katila T.Artificial insemination (AI) with cooled stallion semen has increased markedly during the last decades in all countries, but fertility is often lower than when fresh semen or natural mating is used. The objective of this study was to examine field data (1634 ,cycles 523 Standardbred (SB) mares, 575 Finnhorse (FH) mares, and 90 stallions) using multivariable logistic regression for factors influencing the pregnancy rate (PR) after AI with cooled transported semen from SB and FH stallions. The PR per cycle for the material was 47%: Finnhorses 42% and Standardbreds 53%. When assessed with multiva...
Hannan MA, Haneda S, Itami Y, Wachi S, Saitoh T, Cheong SH, Nambo Y.There has been no report of equine embryo transfer in Japan for the last 24 years. Our objective was to establish an effective protocol for embryo transfer in domestic horse. A Hokkaido native pony was bred by deep-horn artificial insemination with frozen semen from a Connemara pony. Embryo collection was performed using a non-surgical method on day 7. Two embryos were obtained from three flushes (67% recovery) and were transferred fresh into crossbred recipient mares. Both recipient mares were diagnosed pregnant (100% pregnancy rate) 5 days after embryo transfer and had normal progesterone le...
Treulen F, Aguila L, Arias ME, Jofré I, Felmer R.During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-tha...
Soni Y, Talluri TR, Kumar A, Ravi SK, Mehta JS, Tripathi BN.Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acro...
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Morrell JM.Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC witho...
Neuhauser S, Gösele P, Handler J.During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen sim...
Ruiz AJ, Tibary A, Heaton RA, Hargreaves IP, Leadon DP, Bayly WM.Although coenzyme Q10 (CoQ10) serves as an antioxidant and energy source for spermatozoa when added to stallion semen before cooling or freezing, the effects of feeding CoQ10 on semen quality have not been studied. We assessed the effects of daily oral ingestion of CoQ10-ubiquinol by stallions on their plasma CoQ10 concentrations and semen quality. Seven mature Andalusian stallions ate 1g ubiquinol/day for 4 weeks followed by a 4-week washout period. Four horses initially completed an additional 4-week control period without ubiquinol. Blood was sampled weekly for determination of plasma CoQ1...
Soni Y, Talluri TR, Kumar A, Ravi SK, Mehta JS, Tripathi BN.Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acro...
Pasing S, von Lewinski M, Wulf M, Erber R, Aurich C.The aim of this study was to evaluate the stress response of stallions (n = 16) aged 3-13 years with previous sexual experience to semen collection by determination of heart rate, heart rate variability, and cortisol in saliva. Recordings were done on two consecutive days. The time intervals from leaving the box until arrival in the collection barn and from first exposure to the teaser mare until ejaculation as well as the number of mounts until ejaculation were neither affected by collection day nor by age, sexual experience (i.e., the number of breeding seasons the stallion experienced), or ...
Griggers S, Paccamonti DL, Thompson RA, Eilts BE.Urospermia has been reported as a cause of infertility in numerous species. The detrimental effects of urine on spermatozoa are due, at least in part, to changes in pH and osmolarity. Semen was collected and subjected to conditions of varying pH (Experiment 1), of varying osmolarity (Experiment 2), and various quantities and concentrations of urine (Experiment 3) and effects on motility were recorded. Finally, semen was contaminated with urine and then either of 2 semen extenders was added, with or without centrifugation, in an attempt to alleviate the detrimental effect of urine on motility (...
Khlifaoui M, Battut I, Bruyas JF, Chatagnon G, Trimeche A, Tainturier D.The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at diff...
Schmidt K, Deichsel K, de Oliveira RA, Aurich J, Ille N, Aurich C.We hypothesized that housing of stallions in a thermoneutral temperature zone during autumn and winter does not only influence metabolism and hair shedding but also improves the characteristics of raw and processed semen. Fertile Shetland pony stallions were followed from October to June. This time coincided with the seasons autumn, winter and spring. Ponies were kept in outside paddocks (group CON, n = 8) or in indoor stables (group ST, n = 8) from October to March when ST stallions returned to outdoor paddocks, but ponies remained in the same groups. The rectal temperature was measured o...
Miller CD.This manuscript is a review of current protocols, advantages, and disadvantages of breeding mares with frozen-thawed equine semen. Issues affecting pregnancy rates are discussed, including proper mare selection, induction of ovulation, insemination dose, timing of insemination (single-dose versus multiple-dose insemination), methods of insemination (transrectal-guided deep-horn versus hysteroscopic insemination), and post-insemination mare management procedures. In a retrospective analysis of breeding records, a single-dose of frozen-thawed semen was inseminated within 6h post-ovulation; the p...
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...
Brum AM, Sabeur K, Ball BA.The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P<0.05) in the percentage of live, caspase-activated spermatozoa (2.9+/-0.7% vs 14.2+/-6.4%; mean+/-S.E.M.), low mitochondrial membrane potential (MMP; 6.8+/-1.1 vs 23.8+/-3.7), altered plasma membrane permeability (1.3+/-0.2 vs 3.0+/-0.5), DNA fragmentation (2.0+/-1.3 vs 14.3+/-3.6), total motility (81.8+/-3.3 vs 35.1+/-5.4), and progressive motility (66.3+/-4.3 vs 24...
Morrell JM, Mari G, Kútvölgyi G, Meurling S, Mislei B, Iacono E, Rodriguez-Martinez H.Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the ...
Cochran JD, Amann RP, Froman DP, Pickett BW.Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm mo...
Battut IB, Kempfer A, Lemasson N, Chevrier L, Camugli S.Spermatozoa from some stallions do not maintain an acceptable fertility after freezing and thawing. The selection of frozen ejaculates that would be suitable for insemination is mainly based on post-thaw motility, but the prediction of fertility remains limited. A recent study in our laboratory has enabled the determination of a new protocol for the evaluation of fresh stallion semen, combining microscopical observation, computer-assisted motility analysis and flow cytometry, and providing a high level of fertility prediction. The purpose of the present experiment was to perform similar invest...
Nikitkina EV, Dementieva NV, Shcherbakov YS, Atroshchenko MM, Kudinov AA, Samoylov OI, Pozovnikova MV, Dysin AP, Krutikova AA, Musidray AA....The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), p...
de Andrade AF, Zaffalon FG, Celeghini EC, Nascimento J, Tarragó OF, Martins SM, Alonso MA, Arruda RP.Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autol...
Brogan PT, Beitsma M, Henning H, Gadella BM, Stout TA.Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm reco...
Albrizio M, Moramarco AM, Nicassio M, Micera E, Zarrilli A, Lacalandra GM.It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level fo...
Ebel F, Vallejos A, Gajardo G, Ulloa O, Clavel E, Rodríguez-Gil JE, Ramírez-Reveco A.The aim of this study was to evaluate seasonal changes in basic parameters of sperm quality and freezability behaviour of ejaculates from 10 fertile heavy draft stallions. A total of 140 ejaculates were collected, processed and evaluated during both the breeding (September-November) and non-breeding seasons (April-June). Fresh semen was evaluated for volume, concentration, total spermatozoa per ejaculate, plasma membrane integrity and total sperm motility. Cryopreserved samples were evaluated for plasma membrane integrity and sperm motility by the CASA system, and for the freezability index (F...
Thomas AD, Meyers SA, Ball BA.The primary objective of this study was to assess plasma membrane characteristics and activation of signal transduction pathways in equine spermatozoa during both in vitro capacitation and cryopreservation. Significant plasma membrane restructuring, as assessed by measurement of plasma membrane lipid disorder and phospholipid scrambling, was not observed until after cryopreservation and subsequent thawing (P < 0.05). Although in vitro capacitated cells also displayed increased plasma membrane lipid disorder and phospholipid scrambling (P < 0.05), it appeared that regulation of these even...
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...
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...
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...
Turner RM, McDonnell SM, Hawkins JF.Ejaculation was pharmacologically induced in a 13-year-old Quarter Horse stallion with a spiral fracture of the radius. The owners desired to have semen from the stallion frozen prior to euthanatizing the horse, but because of the debilitating injury, standard methods of semen collection could not be used. With the stallion standing quietly in a stall, a plastic collection bag was positioned over the stallion's penis, and clomipramine hydrochloride (2.2 mg/kg of body weight, IV) was administered. Fifty-five minutes later, xylazine hydrochloride (0.5 mg/kg, IV) was administered. The stallion ej...
Rota A, Sgorbini M, Panzani D, Bonelli F, Baragli P, Ille N, Gatta D, Sighieri C, Casini L, Maggiorelli MM, Aurich C, Camillo F.Reproductive management of male donkeys employed for artificial breeding has been poorly studied. The aim of this study was to evaluate the effect of housing system, with the animals grouped together in a paddock or kept in individual boxes, on sexual behaviour, cortisol and testosterone concentration and seminal characteristics of adult male donkeys. The study included four Amiata donkey jacks (stallions) from which ejaculates, saliva and blood were collected during two distinct 3 weeks periods, one in the group and one in the box housing system. Time needed for semen collection was shorter ...
Hartwig FP, Lisboa FP, Hartwig FP, Monteiro GA, Maziero RR, Freitas-Dell'Aqua CP, Alvarenga MA, Papa FO, Dell'Aqua JA.During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of...
Ortiz I, Dorado J, Ramírez L, Morrell JM, Acha D, Urbano M, Gálvez MJ, Carrasco JJ, Gómez-Arrones V, Calero-Carretero R, Hidalgo M.The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Si...
Bliss SB, Voge JL, Hayden SS, Teague SR, Brinsko SP, Love CC, Blanchard TL, Varner DD.The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/...
Kareskoski AM, Reilas T, Andersson M, Katila T.With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with exten...
Quiñones-Pérez C, Martínez A, Ortiz I, Crespo F, Vega-Pla JL.Despite the advances in reproductive technology, there is still a considerable number of low sperm quality cases in stallions. Recent studies in humans have detected several seminal microflora-spermatozoa associations behind some idiopathic infertility cases. However, no studies are available on horses, and there is limited information on the microflora present in stallion ejaculates. Accordingly, the objective of this study was to examine associations to the presence of bacteria families with five sperm quality parameters: concentration, total number of spermatozoa, total and progressive moti...
Greiser T, Sieme H, Martinsson G, Distl O.The objectives of the present study were to analyze systematic effects on semen quality traits from fresh and frozen-thawed semen collected in and outside season. A total of 4,681 reports on semen traits of 121 stallions representing Arabian, Thoroughbred, Quarter Horse and four warmblood breeds used for artificial insemination at the Lower Saxon National stud Celle were edited for analysis of gel-free volume, sperm concentration, total number of sperm and total number of motile sperm in fresh semen and in frozen-thawed semen progressive motility, DNA fragmentation index and non-viable sperm. ...
Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specifici...
