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.
Delgado-Bermúdez A, Noto F, Bonilla-Correal S, Garcia-Bonavila E, Catalán J, Papas M, Bonet S, Miró J, Yeste M.Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen-thawed samples. In the presence of AC, different parameters were altered ( < 0.05), b...
Papas M, Catalán J, Fernandez-Fuertes B, Arroyo L, Bassols A, Miró J, Yeste M.While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. T...
Len J, Beehan D, Eilts B, Ebrahimie E, Lyle S.The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 106 sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator fo...
Canisso IF, Panzani D, Miró J, Ellerbrock RE.Donkeys are nonseasonal, polyestrous, territorial, and nonharem breeders. Although there are many similarities between horses and donkeys, there are also reproductive features that differ, from the longer cervix in the jenny to spermatogenic efficiency in the jack. Mules display reproductive cyclic activity but are rarely fertile. Frozen donkey semen has high pregnancy rates in mares, but lower rates in jennies. This article reviews key aspects of donkey and mule reproductive physiology, reproductive medicine, and assisted reproductive techniques that are useful for practitioners offering assi...
Ghosh S, Serafini R, Love CC, Teague SR, Hernández-Avilés C, LaCaze KA, Varner DD.Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P < 0.05)...
Morris LH, Maclellan LJ.The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable...
Ortiz I, Dorado J, Morrell JM, Diaz-Jimenez MA, Pereira B, Consuegra C, Hidalgo M.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
Snoeck PPDN, Pessoa THO, Pereira MGS, Bastos ICL, de Melo MIV.The objective of this study was to compare the BotuCrio extender with the Merk - egg yolk and the INRA 82 modified by the inclusion of acetamide, methyl cellulose and trehalose in substitution of glycerol for freezing equine semen. The semen was diluted after centrifugation to obtain 100 x 10 of sperm/ml in: BotuCrio (control); Merk - egg yolk or INRA 82 modified (Experiment 1). The extended semen was packaged in 0.5 ml straws, cooled and frozen in a freezing machine. The control extender was superior in preserving the motility, VCL, VSL, VAP, LIN, STR and the BCF when compared to the Merk - e...
Álvarez C, González N, Luño V, Gil L.The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sper...
Álvarez C, González N, Luño V, Martínez F, Gil L.The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated...
Neuhauser S, Bollwein H, Siuda M, Handler J.The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processe...
Gonzalez-Castro RA, Amoroso-Sanches F, Stokes JE, Graham JK, Carnevale EM.Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal p...
Greiser T, Sieme H, Martinsson G, Distl O.Objectives of the present study were to estimate genetic parameters for frozen-thawed semen traits of 271 fertile German Warmblood stallions and genetic correlations with raw semen quality traits. Semen samples were collected from stallions utilized for semen collection and artificial insemination (AI) on the Lower Saxon National Stud Celle and the North Rhine-Westphalian National Stud Warendorf. Semen quality variables were analyzed in 63,972 raw (gel-free volume, concentration, progressive motility, number of sperm) and 3681 frozen-thawed samples (motility, DNA fragmentation index (DFI), non...
Whitesell K, Stefanovski D, McDonnell S, Turner R.The stallion Breeding Soundness Examination (BSE), as proposed by the Society for Theriogenology, recommends that a stallion produce a minimum of one billion progressively motile, morphologically normal sperm (PMMNS) in the second of two ejaculates collected 1 h apart to be classified as a Satisfactory Prospective Breeder. With this in mind, the first objective of this study was to determine if the classification outcome of the traditional BSE differs depending on the methods used to evaluate sperm motility, morphology and concentration. We hypothesized that application of Computer Assisted ...
Del Prete C, Stout T, Montagnaro S, Pagnini U, Uccello M, Florio P, Ciani F, Tafuri S, Palumbo V, Pasolini MP, Cocchia N, Henning H.During cold storage stallion spermatozoa experience undergo oxidative stress, which can impair sperm function and fertilizing capacity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the main endogenous enzymatic antioxidants in stallion seminal plasma, and counteract reactive oxygen species. Semen dilution reduces the endogenous antioxidant concentrations. The aim of this study was to investigate whether addition of 15 IU/mL each of SOD, CAT, and GPX to diluted stallion semen would ameliorate a reactive oxygen-mediated decrease in semen quality during 72 h o...
Poirier M, Smith OE, Therrien J, Rigoglio NN, Miglino MA, Silva LA, Meirelles FV, Smith LC.Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetical...
Kumar P, Kumar R, Mehta JS, Chaudhary AK, Ravi SK, Chandra Mehta S, Ansari MM, Legha RA, Tripathi BN, Talluri TR.The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L),...
Papas M, Arroyo L, Bassols A, Catalán J, Bonilla-Correal S, Gacem S, Yeste M, Miró J.This study compared the activities of four antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GSR) in the seminal plasma of stallions and jackasses. Eighteen stallion ejaculates and 24 jack ejaculates were collected through an artificial vagina. Seminal plasma was obtained by several centrifugations at 3000×g and 4 °C for 10 min, and activities of SOD, CAT, GPX and GSR were subsequently determined. We also evaluated whether the collecting season had any influence on the activities of these four enzymes in both stallions ...
Tricyclic antidepressives, such as imipramine, indirectly induce ejaculation by increasing the noradrenaline concentration, which triggers an α-adrenergic response, whereas α-adrenergic agonists, such as xylazine and detomidine, directly trigger ejaculation by activating the α-1 adrenergic receptors. Furthermore, serum oxytocin concentrations in stallions increase drastically before ejaculation, but decline immediately thereafter, implicating the role of this hormone in emission. The objectives of the present study were to: 1) compare the efficiency of various protocols for inducing ex copu...
May CE, Guthrie AJ, Schulman ML.We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.
Moraes CR, Runcan EE, Blawut B, Coutinho da Silva MA.The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen anal...
Wilson M, Williams J, Montrose VT, Williams J.Most stallions within breeding programmes are expected to breed and compete concurrently. The exercising of stallions with regards to training regimes during the breeding season is a controversial subject. Daily exercise at low intensities is important for the mental and reproductive well-being of the stallion, however higher intensities of exercise, as seen in competing stallions, may have detrimental effects on seminal quality. To calculate if competition does affect semen quality, this study investigated the effect that equestrian discipline and timing of competition had on a range of stall...
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 ...
Walter J, Balzer HJ, Seeh C, Fey K, Bleul U, Osterrieder N.Equid herpesvirus 1 (EHV-1) is a highly prevalent pathogen in horse populations worldwide. Oronasal infection represents the classic route of disease transmission. Venereal shedding of EHV-1 is not regarded relevant in terms of virus spreading, which is in contrast to the close relatives of EHV-1, bovine and suid alphaherpesvirus, for which artificial insemination is a well-documented and accepted means of virus spread. Objective: Documentation of venereal EHV-1 shedding in 3 naturally infected stallions. Methods: Three stallions were infected during an acute outbreak by an EHV-1 strain with t...
Peña FJ, Ortega Ferrusola C, Martín Muñoz P.Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simul...
Silva MA, Silva KM, Jesus AL, Barros LO, Corteggio A, Altamura G, Borzacchiello G, Freitas AC.Papillomavirus (PV) are double-stranded DNA viruses that can cause both benignant and malignant tumours in mammals. Twelve genotypes of bovine papillomavirus (BPV1-12) have been identified so far. The presence of BPV1 and 2 has been found in the body fluids of cattle and horses. The aim of this study is to investigate the presence of BPV DNA and the expression of viral genes in the blood and sperm cells of healthy horses using PCR and RT-PCR. BPV-1 or 2 was detected in 14 of 70 blood samples (20%) and in 11 of 31 semen samples (35%). In five of fourteen blood samples, the E5 expression tested ...
Jeannerat E, Marti E, Berney C, Janett F, Bollwein H, Sieme H, Burger D, Wedekind C.The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm nu...
Kikuchi M, Takao Y, Tokuda N, Ohnami Y, Orino K, Watanabe K.Total 78 semen samples were obtained from 27 Thoroughbred stallions (aged 6 to 27 years), and were subjected to quantification of lactoferrin (Lf) in seminal plasma and examination of the seminal properties. The seminal plasma Lf concentration varied from 21 to 689 microg/ml, with a mean value of 244 +/- 151 microg/ml (S.D.). The seminal plasma Lf concentration and total seminal plasma Lf positively correlated with the sperm concentration (r=0.5938, P<0.001) and with the total sperm number (r=0.6959, P<0.001), respectively. There was no correlation between seminal plasma Lf and sperm mot...
Gravance CG, Champion Z, Liu IK, Casey PJ.The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculat...
Swegen A, Smith ND, Gibb Z, Curry BJ, Aitken RJ.Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibi...
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...
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 ...
Campos JR, Breheny P, Araujo RR, Troedsson MH, Squires EL, Timoney PJ, Balasuriya UB.Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days...
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)....
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...
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 ...
Burger D, Dolivo G, Wedekind C.Sperm competition theory predicts semen characteristics to be affected by the social environment. We used the polygamous horse (Equus caballus) to experimentally study within-subject plasticity in response to different social environments. Stallions were sequentially exposed, over a period of 8 weeks each, to other stallions and then singly to mares, or vice versa (in adjacent boxes separated by grills). Ejaculates were collected to determine semen characteristics. Highest sperm numbers were found in stallions that were first exposed to other stallions and then to mares, while lowest sperm num...
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...
Suliman Y, Becker F, Tuchscherer A, Wimmers K.Horses are seasonal breeders with a natural breeding season beginning in spring and extending through midsummer. In this study, quantitative and qualitative parameters of chilled stallion semen were compared between fertile and subfertile stallions and between the breeding and the non-breeding season. Semen quality parameters compared included ejaculate volume, sperm concentration, total sperm number, sperm morphology, and computer-assisted semen analysis (CASA)-derived sperm movement characteristics obtained from two groups of warmblood stallions ( ; four fertile stallions and four subfert...
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...
Madsen M, Christensen P.Semen samples were collected from 21 Danish Warmblood stallions by the Colorado artificial vagina (Colorado AV, 14 samples) or by the Missouri artificial vagina (Missouri AV, 7 samples). The semen was examined bacteriologically by direct plating (DP) on blood agar plates, and by plating of semen swabs stored in Stuart's transport media (TM) at 4 degrees C for 1-4 days. No significant differences were observed between results obtained by DP and cultures of identical TM samples. Of the 21 samples examined, only 1 TM (4.8%) and 2 DP samples (9.5%) were sterile, while the rest yielded a predominan...
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...
