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.
Serafini R, Ghosh S, Love CC, Medrano JMR, Teague SR, LaCaze KA, Varner DD.Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate a...
The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the f...
Kowalczyk A, Czerniawska-Piątkowska E, Kuczaj M.The purpose of this review was to analyze factors affecting the popularity of artificial insemination of mares in Europe in the context of sperm quality. Taking into account the prices of stallion semen on the world market, efficiency is important for the profitability of its use in artificial insemination programs in Europe. To increase the efficiency of a semen insemination facility, it is necessary to correctly and objectively assess the quality of semen. The available range of tools allows an effective evaluation of the potential fertility of a stallion. For several years, artificial insem...
Tsunoda RH, Serrano-Recalde EC, Arruda RP, Oliveira BMM, Pinto SCC, Celeghini ECC.The aim was to evaluate effects of addition of pentoxifylline to skimmed milk semen extender on uterine inflammatory response. Thirty-six estrous cycles of 15 mares were randomly divided into five groups for artificial insemination (AI): Control: mimicking the AI procedure (n = 7); Extender: deposition of skimmed milk based extender (n = 7); Extender + PTX: skimmed milk based extender plus pentoxifylline (7.18 mM; n = 8); Semen: semen diluted with extender without pentoxifylline (n = 7), and Semen + PTX: semen diluted with extender containing pentoxifylline (n = 7). M...
van Dorland A, Janett F, Bruckmaier R, Wach-Gygax L, Jeannerat E, Bollwein H, Sieme H, Burger D.Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ± standard deviation = 9.5 ± 4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were eva...
Sales FA, Ferreira-Silva JC, Vieira JI, Chaves MS, Caldas EL, Filho JP, Freitas VJ, Oliveira MA.Addition of extenders to thawed semen could improve fertility. Objective: To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). Methods: Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). Results: Progressive motility decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility decreased (P<0.05) after 60 min in thawed semen...
Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ.The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Thawed sperm were washed and resuspended in Ty...
Hernández-Avilés C, Love CC, Serafini R, Teague SR, Varner DD.Commonly marketed semen extenders contain various antibiotic types and concentrations to control bacterial growth from stallion's external genitalia. An experiment was conducted to test the effects of supplemental amikacin disulfate (1,000 μg/mL) + potassium penicillin G (1,000 IU/ML: INRA-AP), or ticarcillin-clavulanate (1,000 μg/mL: INRA-TIM) in the INRA 96 extender, on sperm function and antimicrobial activity, compared with extender without supplemental antibiotics (INRA-C). In freshly extended semen (Time 30m), no differences were observed among the three treatment groups for sperm m...
Dini P, Troch L, Lemahieu I, Deblende P, Daels P.The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm Equine (Aidmics Biotechnology), Androvision (Minitube) and NucleoCounter SP-100™ (ChemoMetec). The total motility and progressive motility were determined by the iSperm Equine and the Androvision using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above-mentioned ...
Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, Hinrichs K, Álvarez-Barrientos A....Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-...
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
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...
Ijaz A, Ducharme R.Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respective...
Rota A, Furzi C, Panzani D, Camillo F.This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and u...
Love CC.Semen collection techniques in the stallion have evolved considerably over the last 70 to 80 years and are used today primarily for artificial insemination. Semen can be collected from stallions that are otherwise unable to breed, allowing continued use of valuable animals. There are many options for collection of semen from stallions that present with ejaculatory dysfunction (see the article by McDonnell elsewhere in this issue.) Although there are many advantages to the use of artificial breeding, the collector must understand each step of the collection procedure as well as stallion prefere...
Samper JC, Hellander JC, Crabo BG.Studies were designed to investigate whether sperm motility determined with a Hamilton-Thorn HTM-2000 motility analyzer (HTM), or the percentage of spermatozoa that passed through glass wool (GW), Sephadex (S), or glass wool/Sephadex (GWS) filters could be used to predict the fertilizing potential of fresh or frozen semen. In the fresh semen study, 10 randomly selected ejaculates from 4 stallions exclusively used for A.I. breeding were assayed during the season. The 521 mares used were inseminated with 500 x 10(6) motile spermatozoa after gynaecological examination every 2 days. In the frozen ...
Hernández-Avilés C, Serafini R, Love CC, Teague SR, LaCaze KA, Lawhon SD, Wu J, Blanchard TL, Varner DD.Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05)....
Allen WR.Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallio...
de Andrade AF, Zaffalon FG, Celeghini EC, Nascimento J, Bressan FF, Martins SM, de Arruda RP.The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma ...
Vidament M.Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ...
Alghamdi AS, Funnell BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC, Foster DN.We performed a series of comparative studies of bull and stallion seminal plasma (SP) and its role on sperm-neutrophil binding as well as the interaction between semen extender and seminal DNase. Because of contrasting roles of SP on sperm-neutrophil binding between horses and cattle, it was suspected there were some species-specific differences on sperm interaction with SP proteins due to the variations in the natural location of semen deposition (uterus compared to vagina). Bull frozen-thawed sperm removed from egg yolk extender showed similar results to fresh sperm, but this also caused ext...
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...
Metcalf ES.Despite the numerous benefits of having the capability to transport semen internationally, there are serious potential ramifications if that semen is contaminated with a communicable disease. Bacteria: Many commensal bacteria colonize the exterior of the stallion penis and are not regarded as pathogenic. They may be cultured from an ejaculate. Alterations of the normal bacterial flora on the exterior genitalia may cause the growth of opportunistic bacteria such as Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcus zooepidemicus, which, if inseminated, may cause infertility in suscepti...
Papa FO, Felício GB, Melo-Oña CM, Alvarenga MA, De Vita B, Trinque C, Puoli-Filho JN, Dell'Aqua JA.The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment...
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...
Troedsson MH, Liu IK, Crabo BG.Following the deposition of semen in the mares 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 mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that sperm...
