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.
Varner DD, Ward CR, Storey BT, Kenney RM.Equine spermatozoa were incubated in a chemically defined medium for 8 hours. The medium preserved spermatozoal viability, as assessed by total spermatozoal motility, progressive spermatozoal motility, and spermatozoal exclusion of eosin stain. Effects of time and divalent cation ionophore, A23187, on the occurrence and character of the spermatozoal acrosome reaction were determined. Two light microscopic assays, a triple-stain technique and a chlortetracycline fluorescence assay, were calibrated with transmission electron microscopy for detection of the acrosome reaction. Incubation time and ...
Gross MK, Toscano DG, Toscano WA.Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by...
Bélaïche D, Loir M, Kruggle W, Sautière P.Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.
Francl AT, Amann RP, Squires EL, Pickett BW.The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with inseminatio...
Kosiniak K, Bittmar A.Physiological processes connected with sexual maturation of stallions were observed on 10 half-breed Anglo-Arab stallions beginning from 8 months of age, until 4.5 years of age. It was found that there is full somatic and sexual development in the stallion reached around the age of 3.5 years, and the sperm morphology stabilized in the range of the physiological norm around 3.0 years of age. On the other hand biochemical components of the semen plasma such as glycerylphosphorylcholine (GPC), ergothioneine (EGT), total protein (PRT), up to age 4.5 years, reach significantly lower value than in m...
Boyle MS, Cran DG, Allen WR, Hunter RH.The morphology of the uterotubal junction (UTJ) and caudal isthmus during the peri-ovulatory period, and the distribution of spermatozoa within the region, were studied in 10 Pony mares. The proximal tip of the uterine horn and caudal 1-2 cm of the isthmus were removed during oestrus or shortly after ovulation from animals mated or artificially inseminated within the previous 24 h. The tissues were incised longitudinally and fixed for scanning electron microscopy. Analysis of micrographs showed deep longitudinal and oedematous folds in the preovulatory samples. After ovulation, much of the fol...
Okólski A, Babusik P, Tischner M, Lietz W.Comparisons were made between 2 methods of oocyte recovery from the ovarian follicles of slaughtered mares: 500 oocytes (3 per mare) were obtained by aspiration of follicular fluid from ovaries of 162 mares, and 120 oocytes (8 per mare) by isolation and rupture of follicles from ovaries of 14 mares. In the oocytes recovered after rupture of follicles, 89.2% were morphologically unchanged, in comparison to 29.3% obtained by aspiration of follicular fluid. Stallion spermatozoa capacitated in vitro were tested on zona-free hamster oocytes. The stallion spermatozoa were washed in TCM-199 and prein...
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...
Wessel MT, Ball BA.Osmotic stress is an important component of the damage to spermatozoa during cryopreservation. Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating...
Morris LH.The generally recommended minimum number of spermatozoa required for conventional artificial insemination in the mare is in excess of 200 x 10(6) progressively motile spermatozoa. Recent developments in different insemination techniques such as deep uterine, hysteroscopic and oviductal insemination, which have been designed to use significantly fewer spermatozoa, are reviewed in this paper. A number of studies have demonstrated that ultrasound guided deep uterine insemination of 5 x 10(6) fresh spermatozoa can produce satisfactory pregnancy rates. The use of hysteroscopic insemination enables ...
Clément F, Vincent P, Mahla R, Meriaux JC, Palmer E.The aim of the present study was to determine which artificial insemination results in fertilization when mares are inseminated several times before ovulation. Mares in oestrus were inseminated over 62 cycles with fresh semen at 48 h intervals from when a follicle > or =30 mm in diameter was detected until ovulation. The number of inseminations was limited to three. Three fertile stallions were used and a different stallion was used for each artificial insemination. The order of the three stallions was changed for each cycle. Embryos were collected between day 10 and day 12 after ovulation and...
Meyers SA, Rosenberger A, Orpneck K.Three protein bands with hyaluronidase activity and molecular masses of 87, 48 and 43 kDa were isolated from purified equine sperm plasma membranes. Indirect immunofluorescence was used to assess sperm labelling patterns using a polyclonal antibody to sperm hyaluronidase. In ejaculated spermatozoa, surface-associated hyaluronidase was localized to the posterior head region of 98 +/- 2% of spermatozoa (n=10). Epididymides were isolated from mature stallions (n=5) and divided into caput, corpus and cauda epididymides in separate Petri dishes. The epididymidal tubules were dissected and washed us...
Schembri MA, Major DA, Suttie JJ, Maxwell WM, Evans G.To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. Methods: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. Results: Following centrifugation, spermatozoa ...
Coutinho da Silva MA, Seidel GE, Squires EL, Graham JK, Carnevale EM.Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 comp...
Guignot F, Ottogalli M, Yvon JM, Magistrini M.The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5% CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 microM) and cultured individ...
Silva GF, Cunha R, Carvalho F, Ribeiro M, Rocha A, Amorim I, Guimarães T.A 30-year-old Lusitano stallion presented with an enlarged right epididymis. The ultrasound scan revealed a cyst-like formation and the histopathological examination was compatible with epididymal cyst located at the body/tail transition, epididymal spermatocele and sperm granuloma and epididymitis. However, these conditions did not seem to affect the animal's reproductive performance, nor did the semen parameters analyzed over the 8 years after the diagnosis show significant changes. Nevertheless, since the ejaculate contains mostly sperm cells from the tail of the epididymis, where fertile s...
Carnevale EM.Methods for the collection and transfer of equine oocytes have been developed, and uses of these techniques have resulted in new clinical and research possibilities. Because oocyte transfer avoids reproductive problems associated with the oviduct, uterus, and cervix, pregnancies can be produced from many mares that cannot carry a pregnancy or produce embryos. Oocytes for clinical transfers are usually collected from preovulatory follicles and cultured for a short interval or transferred directly into a recipient's oviduct. For oocyte transfer, the recipient is inseminated within the uterus. A ...
Fernández-Hernández P, García-Marín LJ, Bragado MJ, Domingo A, González-Fernández L, Macías-García B.In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three s...
Jasko DJ, Smith K, Little TV, Lein D, Foote RH.A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analy...
Schumacher J, Varner DD, Schmitz DG, Blanchard TL.A urethral defect, presumed to communicate with the corpus spongiosum penis, caused hematuria in seven geldings and hemospermia in three stallions. Hematuria in geldings occurred at the end of urination. Hematuria was not observed in stallions with hemospermia. A linear urethral defect was identified, by endoscopic examination, on the convex surface the urethra at the level of the ischial arch of each horse. Cause of the defect was not determined. Two stallions were successfully treated for hemospermia, one by temporary subischial urethrostomy combined with sexual rest for 10 weeks, and the ot...
Pérez-Marín CC, Requena FD, Arando A, Requena L, Requena F, Agüera EI.The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment I...
Schmid RL, Kato H, Herickhoff LA, Schenk JL, McCue PM, Chung YG, Squires EL.In Expt 1, compact cumulus oocyte complexes (COCs) were matured in: (i) control medium (Hepes-buffered TCM-199 with 10% oestrous cow serum (OCS) + oestradiol, LH and FSH); (ii) Hepes-buffered TCM-199 with 20% follicular fluid; or (iii) control medium containing 250 ng progesterone ml(-1). Mature oocytes were collected by transvaginal aspiration as a positive control for the in vitro maturation (IVM) treatments. Oocytes were fertilized by ICSI and cultured in Menezo's B2 + 5% fetal calf serum (FCS). There were no significant differences among IVM treatments. In Expt 2, oocytes with expanded COC...
Elkhawagah AR, Nervo T, Poletto M, Martino NA, Gallo D, Bertero A, Vincenti L.The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 10 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferre...
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...
Araujo JF, Righini AS, Fleury JJ, Caldas MC, Costa-Neto JB, Marques N.An attempt has been made to define semen seasonality in a horse in the Southern Hemisphere. Repeated measurements of three variables in the semen were made for 36 months (Jan/90-Dec/92) in a 21-year old "Mangalarga" stallion living under natural photoperiod and temperature conditions in a farm situated in São José do Rio Pardo, São Paulo, Brazil (latitude 21 degrees) 36'S; longitude 46 degrees 53' W). The horse fed on natural pasture and a nutritionally balanced feed twice a day (11:00 and 17:00 h). Water and mineral supplement were available ad libitum. Semen was collected almost daily by ...
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...
Voge J, Varner DD, Blanchard TL, Meschini M, Turner C, Teague SR, Brinsko SP, Love CC.Urine-contaminated stallion semen is a clinical problem due to a variety of causes. The effect of the level of urine contamination on the longevity of sperm quality has not been evaluated. The aim of this study was to determine the effects of urine concentration level (0%, 10%, 20%, 30%, and 40%) and cushioned centrifugation and resuspension of the sperm pellet in fresh extender, on measures of sperm quality, immediately after semen collection (T0), after 1 hour of storage at room temperature (T1), and after 24 hours of cooled storage (T24). In general, most sperm quality measures declined w...
Katila T.In this review, effects of the composition of the inseminate on uterine response and pregnancy rates in mares are discussed. The inseminate can differ for volume, sperm concentration, total sperm numbers, presence, absence, or proportion of seminal plasma, and extender composition. Semen can be used as fresh, cooled, or frozen. The site of semen deposition also plays a role; semen is deposited either into the uterine body (standard artificial insemination (AI)) or into the tip of the uterine horn ipsilateral to the preovulatory follicle (deep AI) using the hysterocopical or transrectally guide...
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...
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...
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...
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...
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...
Merkies K, Buhr MM.Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calc...
Bresnahan DR, Lupole RE, Stilz CR, Carnevale EM.Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment...
Azuma T, Choi YH, Hochi S, Oguri N.The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or ...
Retamal CA, Dias AJ, Brasil FC, Lanzana FR, López ML.The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P<0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two cat...
