Equine sperm refers to the male reproductive cells produced by stallions, essential for the process of fertilization and successful breeding in horses. The study of equine sperm encompasses various aspects, including morphology, motility, viability, and genetic integrity. These parameters are critical for assessing stallion fertility and improving breeding outcomes. Research in this field often focuses on understanding the factors that influence sperm quality, such as age, nutrition, and environmental conditions. Additionally, advancements in assisted reproductive technologies, such as artificial insemination and cryopreservation, rely heavily on the detailed study of sperm characteristics. This page compiles peer-reviewed research studies and scholarly articles that explore the biology, evaluation, and technological applications related to equine sperm.
Amann RP, Cristanelli MJ, Squires EL.Motility and fertility of frozen-thawed semen differs greatly amongst stallions. Differences in seminal plasma might be one cause of this variation. For 8 ejaculates from each of 17 stallions, seminal plasma was saved at -20 degrees C and spermatozoa were cryopreserved. Based on post-thaw sperm motility, seminal plasma samples from 7 stallions (2 good, 3 variable, 2 poor sperm motility) were selected for measurement of electrolytes, protein content and analysis by sodium dodecylsulphate gel electrophoresis (10% gel, Coomassie blue stain). Variation in seminal plasma was significant (P less tha...
Heiskanen ML, Pirhonen A, Koskinen E, Mäenpää PH.The role of various environmental conditions on sperm motility and ATP content was investigated by incubating raw and washed spermatozoa collected with an open-ended artificial vagina from 10 stallions in various biological and artificial media under different atmospheric conditions. Spermatozoa did not survive for more than 12 h when kept unextended in the original seminal fluid in any circumstances. The most favourable media tested for long-term sperm survival were Kenney's medium or Kenney's medium supplemented with 10 mM-theophylline and 10 mM-Hepes, pH 7.2. Centrifugation and slow cooling...
Arns MJ, Webb GW, Kreider JL, Potter GD, Evans JW.Bovine serum albumin (BSA) diluents containing lactose, raffinose or sucrose were not different (P greater than 0.05) in their ability to maintain stallion sperm viability, as determined by percentage motile spermatozoa (PMS) and their rate of forward movement (RFM), when stored at 37 or 5 degrees C for 24 h. These diluents did promote a higher (P greater than 0.05) PMS and RFM, when compared with BSA diluents containing arabinose or galactose. The BSA-arabinose and BSA-galactose diluents did not differ (P less than 0.05) in their ability to support sperm viability and were detrimental to sper...
Strzemienski PJ, Sertich PL, Varner DD, Kenney RM.Stallion semen was diluted in a Hepes-supplemented buffer (CM) (10(6) spermatozoa/ml) and placed in the upper well of a Sykes-Moore chemotaxis chamber. Chambers were incubated in a humidified atmosphere (5% CO2 in air) at 37 degrees C for 1 and 2 h and spermatozoa were allowed to swim through filters with a mean pore size of 3,5 or 8 micron. Spermatozoa entered filters of all three pore sizes. Distance travelled was greater for each increase in pore size (P less than 0.01) but did not differ (P greater than 0.05) between 1 and 2h of incubation. Extended semen from stallions of different fertil...
Klem ME, Kreider JL, Pruitt JB, Potter GD.Inclusion of either 1 or 3% (w/v) bovine serum albumin (BSA) in 8.6, 10, or 12% sucrose enhanced the maintenance of equine sperm motility in vitro at 38 degrees C for 8 h. There was a trend toward higher percent motile spermatozoa (PMS) at 16 and 24 h of incubation in semen samples containing BSA than in those that did not. The highest concentration of sucrose (12%) was slightly less effective in supporting PMS than either of the lower concentrations. However, sucrose concentrations had no apparent effect on rate of forward movement (RFM) of spermatozoa. Pregnancy and foaling rates were simila...
Garner DL, Pinkel D, Johnson LA, Pace MM.Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Van Huffel XM, Varner DD, Hinrichs K, Garcia MC, Strzemienski PJ, Kenney RM.A photomicrographic method for evaluation of stallion spermatozoal motility was developed, and spermatozoal image and velocity characteristics were defined. The photomicrographic method was compared with visual estimation of motility in the same semen sample over time. Using photomicrography, velocities and percentages of individual spermatozoal image characteristics were determined. Although there was a high correlation between results of the 2 methods, results of the photomicrographic method were more repeatable than were those of the visual method.
Zgórniak-Nowosielska I, Kosiniak K, Slagowska A.Eleven mycoplasma strains were isolated from the semen of 24 stallions. Eight of these strains were identified as Mycoplasma equigenitalium. Three strains which hydrolized arginine could not be identified. The growth inhibition test with immune sera against M. arginini and M. equirhinis was negative. Antibiotic sensitivity test showed that all strains were sensitive to four antibiotic of tetracycline group (oxytetracyclin, minocycline, transcycline and vibramycin). Lincomycin and gentamycin appeared to be the most active against all the strains. Comparative analysis of routine semen examinatio...
Province CA, Amann RP, Pickett BW, Squires EL.Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of can...
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...
Livolant F.The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chroma...
Froman DP, Amann RP.The effects of four vaginal lubricants on progressive spermatozoal motility were evaluated. Neat semen was exposed to 0, 5, or 10% (w/v) of H-R, sterile K-Y, nonsterile K-Y or Maxilube lubricating jellies for 10 min at 37 degrees C and then extended to 10x10(6) spermatozoa/ml. Spermatozoal motility was evaluated after 0, 1, 2, 4 and 6 or 8 h of incubation at 37 degrees C. For bovine spermatozoa, sterile K-Y jelly at 10% suppressed motility (P<0.05), but nonsterile K-Y, H-R and Maxilube jellies had no effect. Maxilube was toxic (P<0.01) to canine spermatozoa and is not recommended for use...
Arriola J, Foote RH.Because microfloral content of stallion semen tends to be high, and strains may be resistant to commonly used antibiotics, amikacin was tested with stallion semen and compared with bull semen. Nine ejaculates to stallion semen were incubated at 37 C in egg yolk-tris extender for 0, 2, 4, 6, 8 and 10 h in the presence of amikacin concentrations of 0, 50, 100, 250, 500, 1,000 and 10,000 microgram/ml, with penicillin and penicillin-streptomycin as controls. Averaged over all incubations, spermatozoal motility was 44, 48, 49, 46, 45, 45 and 19%, for increasing concentrations of amikacin, compared ...
Bader H.A total of 23 mares were inseminated once within 0-6 h after clinical detection of ovulation, 14 with fresh and 9 with deep-frozen semen containing 0.1 x 10(9) to 4.7 x 10(9) motile spermatozoa. Within these two groups, the mares were slaughtered 2, 4 or 6 h after insemination and their genital tracts removed. The utero-tubal junction, isthmus and ampulla ipsilateral to the ovary in which ovulation occurred were flushed separately for sperm recovery. In 1 or 2 mares of each group, the uterine horn and corpus uteri, the cervix and vagina were also flushed. Tissue samples were collected from the...
Rideout MI, Burns SJ, Simpson RB.Sterile filtrates were prepared from equine isolates of Klebsiella pneumoniae (genitalium), Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus zooepidemicus, Streptococcus equisimilis, Actinobacillus equuli, and Corynebacterium equi and mixed individually with extended stallion semen. When diluted in the extended semen the filtrates represented bacterial populations of 0.5 x 10(6), 1 x 10(6), 2 x 10(6) and 4 x 10(6) cells/ml. pH values were recorded for each filtrate. Specimens were monitored for percentage motile spermatozoa at 30-min intervals until they reached 10% o...
Guay P, Rondeau M, Boucher S.The effect of different glycerol concentrations (0 to 5.3 per cent) on motility, viability and aspartate aminotransferase (AST) release of stallion spermatozoa was studied before and after deep-freezing. Addition of glycerol to a TRIS-fructose-egg yolk diluent used to extend stallion semen had no effect on motility and viability of spermatozoa and it did not increase AST release. Inclusion of glycerol in the extender only partially preserved the motility and viability of stallion semen during deep-freezing. A fertility trial revealed that concentrating stallion semen by centrifugation, followe...
Dixon KE, Kreider JL.Fifty ejaculates, ten from each of 5 mature stallions, were utilized to study the effects of calcium and fatty acids on equine spermatozoa which were isolated in 3% Bovine Serum Albumin (BSA). The ejaculates were evaluated for percent motile spermatozoa, rate of forward movement, debris, primary abnormalities and secondary abnormalities. The isolation procedure consisted of layering 2 ml of diluted semen (100 x 10(6) spermatozoa/ml) over 6 ml of 3% BSA in 13 x 125 mm columns in a water bath (37 degrees C). After 30 min., the top semen layer and upper half of the BSA layer were withdrawn from a...
Chacon-Arellano JT, Woolley DM.Smooth muscle cells are present in the tunica albuginea testis of the horse, pig and sheep. typical fusiform muscle cells constitute a distinct layer up to 0.3 micrometer thick in the horse; there are fewer muscle cells, mainly of the branched form, in the pig; whereas in the sheep the muscle component is least well developed, with some cells intermediate in form between smooth muscle cells and fibroblasts (myofibroblasts). Attention is drawn to the continuity of this capsular muscle with the smooth muscle associated with the vasculature of the spermatic cord in the horse. This association sug...
Johnson L, Amann RP, Pickett BW.Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothrei...
Shideler RK, Squires EL, Pickett BW, Anderson EW.Twenty-three geldings were used to determine changes in seminal characteristics following castration and the effect of frequency of ejaculation on these seminal characteristics. In Exp. 1, semen was collected from 8 geldings every other day after castration until the number of spermatozoa per ejaculate was below 1% of the precastration value. An average of 3 ejaculates was required to reduce the number of spermatozoa below this level. In Exp. 2, 15 stallions were castrated and each stallion was assigned to 1 of 3 groups for seminal collection at 7, 14 or 21 days post-castration. The ejaculates...
Bielański W, Kaczmarski F.Semen samples were collected from 3 fertile stallions by means of an 'open' artificial vagina and examined under scanning and transmission electron microscopy. The stallion spermatozoon has many features in common with that of other mammals but differs specifically in that it has an asymmetric head, an abaxial position of the tail and an acrosome of small volume. The presence of microtubules in the neck is also a characteristic of stallion spermatozoa.
Amann RP, Thompson DL, Squires EL, Pickett BW.Extragonadal reserves totalled 89 X 10(9) spermatozoa for 5--16-year-old sexually rested stallions and 60 X 10(9) for 2--4-year-olds. Regardless of age, the cauda epididymidis contained 62% of the total reserves and the vas deferens, including the ampulla, contained 7% of the total reserves of spermatozoa. The caput plus corpus epididymidis from 5--16-year-old stallions (N = 41) contained 14.9 X 10(9) spermatozoa per side as compared (P less than 0.01) to 8.5 X 10(9) for 2--4-year olds (N = 30). Frequency of ejaculation did not influence the number of spermatozoa found in caput plus corpus epi...
Hemeida NA, Sack WO, McEntee K.Dutculi efferentes were studied by microdissection and histologic methods in 2 boar, 6 goat, 5 ram, 25 bull, and 19 stallion epididymides. On the basis of the present findings and the data of others and relating them to the total number of specimens examined in each species, an estimate (narrow range) was made of the minimal-maximal numbers of efferent ductules as follows: dog 13 to 15; cat 14 to 17; boar 14 to 16; goat 18 to 19; ram 17 to 20; bull 13 to 16; and stallion 14 to 17. The histologic appearance of the efferent ductules of the bull, including measurements of their diameter, lumen, a...
Loux SC, Macías-Garcia B, González-Fernández L, Canesin HD, Varner DD, Hinrichs K.Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeab...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Murphy C, English AM, Holden SA, Fair S.An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
Braun J, Hochi S, Oguri N, Sato K, Torres-Boggino F.Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF.Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato F.Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment...
Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró J, Yeste M.In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed pri...
Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, Paccamonti D, Graff KJ, Godke RA.In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilizat...
McCue PM, Matthews PM, Prell MJ, Bellone RR, Allen H.Genetic testing is required for the registration of foals of most equine breeds. Objective: To describe two clinical cases of marked delayed embryonic development or delayed fertilisation in pregnancies generated by embryo transfer. Methods: Case report. Methods: Donor mares were inseminated with semen from one stallion during one oestrous cycle and semen from a different stallion on the subsequent oestrous cycle. Embryo(s) were collected 8 days after ovulation during the second oestrous cycle and transferred into synchronised recipient mares. Genetic testing was performed to determine paren...
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...
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...
Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we e...
Ortega-Ferrusola C, González-Fernández L, Muriel A, Macías-García B, Rodríguez-Martínez H, Tapia JA, Alonso JM, Peña FJ.In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in sta...
White IG.When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsatur...
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...
Dietz JP, Sertich PL, Boston RC, Benson CE.Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial conce...
Hinrichs K.Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Roels K, Smits K, Ververs C, Govaere J, D'Herde K, Van Soom A.In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with det...
Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A, de Kruif A.Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following ce...
Dell'aquila ME, Fusco S, Lacalandra GM, Maritato F.The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with froz...
Sampaio BFB, Nogueira BG, Souza MIL, Silva EVDCE, Zúccari CESN.The aim of this experiment was to evaluate the effects of adding ascorbic acid 2-glucoside (AA2G), a water-soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat-soluble antioxidant α-tocopherol (α-Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid pero...
Graham JK.For many years, scientists have sought to develop laboratory assays that accurately predict the fertilizing capacity of a semen sample. This goal, however, has proven elusive and will most likely be very difficult to achieve, due to the complex nature of the problem. Part of the problem results from the many attributes that a spermatozoon must possess to fertilize an egg, and how laboratory assays can evaluate all of these attributes simultaneously. The percentage of motile sperm in a sample is most commonly used to evaluate semen quality. This assay, however, is not highly correlated with the...
Rossini JB, Rodriguez J, Bresnahan DR, Stokes JE, Carnevale EM.The clinical use of intracytoplasmic sperm injection (ICSI) in horses usually involves the transfer of embryos into recipient mares, resulting in substantial cost increases. This is essential when subfertile mares are oocyte donors; but some donors are fertile, with ICSI compensating for limited or poor-quality spermatozoa. Fertile oocyte donors could carry pregnancies, eliminating the need for a recipient. We assessed the potential of using oocyte donors as recipients for their own ICSI-produced embryos during the same cycle. Donors in oestrus and with large dominant follicles were administer...
Vieira LA, Matás C, Torrecillas A, Saez F, Gadea J.Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sp...
Rossetto L, Farcey MF, Bilbao MG, Bartolomé JA, Gallelli MF, Miragaya MH.The stress associated with training may reduce reproductive efficiency in Criollo stallions. The objective of this study was to compare semen quality and hormone concentrations in Criollo stallions under training or under regular field conditions. Criollo breed stallions (n = 18) were evaluated during the spring. The exercise group (n = 9) performed 1 hour of exercise per day and participated in competitions during the experimental period. The control group (n = 9) neither performed exercise nor participated in competitions. Serum and semen samples were obtained every 15 days (two separat...
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
Martins HS, Souza MR, Penna CF, da Silva GC, Côrtes SF, Stahlberg R, Lagares MA.Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml-1 , C4) C2 + Lf 500 μg ml-1 and C5) C2 + Lf 1000 μg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motili...
Ellington JE, Ball BA, Blue BJ, Wilker CE.Reliable capacitation of equine spermatozoa has been a major obstacle in the development of equine in vitro fertilization. Experiments were done to compare in vitro capacitation of equine spermatozoa by use of heparin/caffeine, calcium ionophore, uterine tube epithelial cell (UTEC)-conditioned medium, and direct culturing of spermatozoa with UTEC (coculturing). Capacitation-like changes, as determined by chlortetracycline membrane staining patterns, developed with UTEC-conditioned medium and coculturing, equivalent to that with calcium ionophore. Both of these treatments induced more (P < 0.05...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
