Journal articles on the topic 'Stallion sperm'
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Miró, Jordi, and Marion Papas. "Improvement of cryopreservation protocol in both purebred horses including Spanish horses." Spanish Journal of Agricultural Research 16, no. 4 (January 8, 2019): e0406. http://dx.doi.org/10.5424/sjar/2018164-13677.
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There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio.
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Chaudhary, A. K., G. N. Purohit, J. S. Mehta, S. K. Ravi, and T. R. Talluri. "144 Serum testosterone profile in Marwari stallions and its relationship with testicular parameters, semen characteristics, reaction time, stallion age, bodyweight, and height." Reproduction, Fertility and Development 32, no. 2 (2020): 198. http://dx.doi.org/10.1071/rdv32n2ab144.
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The present study investigated the serum testosterone profile of Marwari stallions before and after exposure to a mare and the relationship of serum testosterone profile with scrotal circumference, semen characteristics, reaction time, stallion age, bodyweight, and height. Marwari stallions (n=9) of three age groups (2-4 years, n=3; 4-6 years, n=3; and 6+ years, n=3) were used in the study. Scrotal circumference, height, and bodyweight of each of the stallions were measured. Semen was collected from each stallion twice per week in the early-morning hours using an artificial vagina and a mare in oestrus. Six ejaculates were collected from each stallion for evaluation of various seminal parameters (semen volume, sperm concentration, and progressive sperm motility). Reaction time for each stallion was also recorded. At every alternate semen collection (first, third, and fifth collections), blood samples were taken 15min before exposure to the mare and just before the semen collection for serum testosterone hormone assay using a horse testosterone enzyme-linked immunosorbent assay kit. The data obtained were analysed statistically using SPSS ver. 20.0 (IBM Corp.). The results showed that mean testosterone concentration was significantly different (P ≤ 0.01) among the stallions and was significantly lower (P ≤ 0.01) in the stallions below 4 years of age. No significant difference in testosterone level was observed before and after exposure to a mare. A positive correlation was detected between testosterone and both scrotal circumference (P ≤ 0.05) and sperm concentration (P ≤ 0.05), whereas a negative correlation was observed with reaction time (P ≤ 0.01). It was concluded that exposure to a mare does not change the testosterone level in stallion blood and that there is a relationship of serum testosterone concentration with scrotal circumference, sperm concentration, reaction time, and age but not with height and bodyweight.
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Nikitkina, E. V., A. A. Krutikova, and A. A. Musidray. "GRM8 GENE POLYMORPHISM AND STALLION SPERM QUALITY." International Journal of Veterinary Medicine, no. 3 (October 17, 2022): 200–203. http://dx.doi.org/10.52419/issn2072-2419.2022.3.200.
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Genome-Wide Association Studies fertility will allow further selection of animals at the genomic level, and genomic selection will allow the selection of animals with good spermatogenesis at an early age. After our GWAS, several candidate genes associated with stallion sperm quality were identified. One of these genes was the GRM8 gene. In the course of Sanger sequencing studies, four SNPs were identified in the exon of the GRM8 gene and their association with the quality of stallion sperm was carried out. For the rs1138419111 genotype, no significant differences were found in the studied parameters. According to the identified single nucleotide substitution rs1147388106, the largest volume of ejaculate was in stallions with the GG genotype. According to SNP rs395286150, stallions with the heterozygous CT genotype had the best sperm quality. Analysis of data on the SNP rs394524550 revealed a significant effect of the genotype on progressive motility. Three of the four SNPs identified in the exon of the GRM8 gene are significantly associated with such indicators of stallion sperm quality as ejaculate volume, concentration, and progressive motility. Project of the Ministry of Education and Science No. 121052600354-7.
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Heise, A., D. Gerber, D. H. Volkmann, W. Kähn, and N. K. Brouwer. "11 FERTILITY OF FRESH AND FROZEN - THAWED EPIDIDYMAL STALLION SPERM WITH OR WITHOUT EXPOSURE TO SEMINAL PLASMA." Reproduction, Fertility and Development 19, no. 1 (2007): 124. http://dx.doi.org/10.1071/rdv19n1ab11.
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The aim of this study was to determine if the addition of equine seminal plasma to epididymal semen enhances its fertility before or after freezing. Thirty-two mares were randomly assigned to 5 stallions; 3 stallions were kept in Pretoria, each having 7 mares, and 2 stallions were kept at Cornell, one having 6 mares and the other 5. Mares were synchronized using 10 daily IM progesterone and estradiol injections; an Ovuplant® implant (26 mg of deslorelin; Peptech Animal Health, Sydney, NSW, Australia) was inserted under the mucosa of the vaginal vestibulum once a follicle reached a diameter of 35 mm; implants were removed after ovulation. Mares were inseminated 30 h after implant insertion. Each insemination dose consisted of 200 million progressively motile sperm and was deposited into the uterine body. Following insemination, mares were examined for ovulation at 6 hourly intervals. Fourteen days after ovulation, mares were examined for pregnancy by transrectal ultrasonography and treated with PGF2α to induce the next estrus. Seminal plasma was collected from the stallions used in the trial prior to castration, frozen, and stored. In Pretoria, stallions were castrated and one epididymal tail was flushed with seminal plasma and the other with skim milk extender; in the first cycle, half of the mares were inseminated with one of the two sperm samples. In Cornell, testes of each stallion were removed 3 weeks apart, and all mares were inseminated first with one and 3 weeks later with the other semen sample. Mares were inseminated during consecutive estrous cycles using the following sperm types: fresh epididymal sperm that had been exposed to seminal plasma (G1: 4 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); fresh epididymal sperm that had never been exposed to seminal plasma (G2: 3 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); frozen–thawed ejaculated sperm (G3); frozen–thawed epididymal sperm that had been exposed to seminal plasma prior to freezing (G4); and frozen–thawed epididymal sperm that had never been exposed to seminal plasma (G5). The results of inseminations with fresh epididymal semen (G1–2) of 5 stallions and the preliminary results of inseminations with frozen–thawed epididymal semen (G3–5) of 2 stallions are summarized in the Table 1. Cycles where ovulation did not occur within 12 h after insemination were excluded. The pregnancy rate of mares inseminated with fresh epididymal sperm of G1 was significantly higher (chi-square test; P < 0.05) than that of mares of G2. The pregnancy rate of mares inseminated with frozen–thawed ejaculated semen (G3) was similar to that of mares inseminated with frozen–thawed epididymal semen of G4 and G5 (P = 0.3). Based on these preliminary results, we conclude that the fertility of fresh epididymal sperm can be enhanced by exposure to equine seminal plasma. To determine if the same holds true for frozen–thawed epididymal sperm, more inseminations must be performed. Table 1.Results of inseminations with various semen types
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Pessoa, G. A., J. M. Trentin, A. P. Martini, D. R. Dotto, L. A. M. Centeno, M. L. Jardim, K. V. Aires, and M. I. B. Rubin. "180 SPERM SELECTION OF STALLION PONIES THROUGH GLASS WOOL." Reproduction, Fertility and Development 26, no. 1 (2014): 204. http://dx.doi.org/10.1071/rdv26n1ab180.
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Two techniques of sperm concentration (centrifugation or filtering) and sperm separation technique with glass wool were applied to the sperm samples collected from 3 pony stallions (6 ejaculates; 2 from each stallion). Ejaculates were extended to a final concentration of 50 × 106 spermatozoa mL–1 using a nonfat dry milk-based extender and evaluations occurred at 24, 48, and 72 h after immediate ejaculate dilution and cooling. Each stallion was considered as a block, and semen from each stallion was assigned to 4 treatments: Group A (control): extended semen alone; Group B: extended-centrifuged semen; Group C: extended-sperm filtered semen; Group D: extended-glass wool-separated semen. All groups were tested for pH, osmolarity, motility, morphology, membrane functionality (hyposmotic), and cell viability (MTT assay). The experimental design was performed using a split-plot model. Data analysis at the level of 5% was performed using ANOVA and Bonferroni as post-hoc test. Data are presented as mean ± standard error. Group D had the highest rate of viable cells (P < 0.05) after the separation procedure (Table 1). Group B had a higher percentage of cells with tail defects after processing compared with the controls and Groups A, C, and D (P < 0.05). More than 60% of the cells retained on the filter showed defects (P < 0.001). Progressive motility was greater in group D at 0, 24, and 48 h (P < 0.05). Seventy-two hours after cooling, motility in groups A and B was lower than in Group D (P < 0.01). Group D showed a higher number of cells with mitochondrial activity during the cooling period. In conclusion, the technique of sperm selection by gravity using a glass wool filter resulted in an increased number of viable sperms after cooling pony semen for 24, 48, and 72 h. Table 1.Effect of sperm concentration and separation techniques on mean ± standard error percent of intact sperm from 3 stallions ponies (2 ejaculates/pony) stained with eosin-nigrosin
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Severa, L., L. Máchal, I. Křivánek, M. Machatková, and O. Mamica. "Characteristic of selected rheological parameters of stallion ejaculate." Archives Animal Breeding 51, no. 1 (October 10, 2008): 16–22. http://dx.doi.org/10.5194/aab-51-16-2008.
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Abstract. Dynamic viscosity of native (30 minutes after ejaculation) and 24 hours stored (at 4 °C) stallion ejaculate was measured. The ejaculate from 10 breeding stallions was examined in three different experimental series. The average value of dynamic viscosity at shear rate 1.02 s−1 was found to be 416.8 ± 10.1 mPa.s. The correlation between ejaculate volume, sperm concentration and viscosity was tested. The experiments resulted in finding a dependence between increasing viscosity and decreasing sperm concentration (rp = −0.67; P<0.05). Performed experiments with changing shear rate demonstrated non-Newtonian characteristics of stallion ejaculate with a clear shear-thinning behaviour. Stallion ejaculate appeared to be slightly time-dependent liquid.
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Oldenhof, Harriëtte, Anna Heutelbeck, Anne-Kathrin Blässe, Heinrich Bollwein, Gunilla Martinsson, Willem F. Wolkers, and Harald Sieme. "Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival." Reproduction, Fertility and Development 27, no. 2 (2015): 285. http://dx.doi.org/10.1071/rd13177.
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The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15–30°C temperature range, whereas membrane stability was found to be decreased at 4 and 37°C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30°C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22°C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg–1 among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.
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RAVAL, KATHAN, NILENDU PAUL, PRADEEP NAG, ELANGO K, YASH PAL, LEGHA R A, T. R. TALLURI, and ARUMUGAM KUMARESAN. "Asthenozoospermic stallions tend to have high acrosome reacted spermatozoa as evidenced by dual fluorescent staining assay." Indian Journal of Animal Sciences 92, no. 8 (August 22, 2022): 946–49. http://dx.doi.org/10.56093/ijans.v92i8.120018.
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Acrosome intactness of spermatozoa is the critical factor for establishing sperm reservoir in oviduct and for fertilizing an oocyte. However, frozen thawed spermatozoa tend to show higher proportion of acrosome reacted spermatozoa thereby compromising the fertility. Conventional staining techniques identify only sperm acrosome integrity and not precisely the acrosome reaction status. In this context, the current study was conducted to assess the acrosome status of cryopreserved spermatozoa using Fluorescein isothiocyanate conjugated peanut agglutinin and propidium iodide (FITC-PNA+PI) in stallions with varying sperm motility. Stallions were classified into high- (≥45%) and low-motile group (≤30%) based on their post-thaw sperm motility. The proportion of live acrosome intact (LAI) spermatozoa was significantly higher in high-motile group as compared to low-motile group. A significant positive correlation was observed between LAI and post-thaw sperm motility. In conclusion, the present study showed that FITC-PNA+PI combination could be used for rapid and accurate assessment of acrosome reaction status of stallion spermatozoa, and the proportion of LAI population in cryopreserved stallion semen had a strong correlation with sperm motility.
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Monteiro, G. A., P. N. Guasti, F. P. Hartwig, J. A. Dellaqua Jr., M. A. Alvarenga, and F. O. Papa. "Cooling of ejaculated and epididymal stallion sperm." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 65, no. 3 (June 2013): 681–86. http://dx.doi.org/10.1590/s0102-09352013000300010.
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After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.
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TKACHEV, A. V., YU I. KOROVIN, and O. L. TKACHEVA. "CRYORESISTANCE OF STALLION SPERM IN DIFFERENT MONTHS OF THE YEAR." Izvestiâ Timirâzevskoj selʹskohozâjstvennoj akademii, no. 2 (2022): 79–87. http://dx.doi.org/10.26897/0021-342x-2022-2-79-87.
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It is known that the seasonality of reproductive function is much more pronounced in mares than in stallions. The mare goes through an anestrus period in autumn and winter, while spermatogenesis in stallions continues throughout the year. Therefore, studies of cryoresistance of stallion sperm in different months of the year are relevant. The aim of the study was to study the cryoresistance of stallion sperm in different months of the year when creating cryopreservation of genetic material with intensive breeding use of sires. The cryoresistance of the sperm of stallions with their intensive breeding use was the best from September to December during sexual rest. The highest incidence of sperm was in December, which is 0.44% more than in October, 5.12% more than in November, 7.62% more (P<0.05) than in September, 20.53% more (P<0.01) than in January, 27.3% more (P<0.01) compared to February, 52.7% more (P<0.001) than in March, 137.9% more (P<0.05) than in April, 34.12% more (P<0.001) than in May, 23.5% more (P<0.001) than in June and 59.7% more (P<0.001) than in July. The survivability of sperms after thawing in a thermostat at 37°C for less than 3 hours was observed in March, April and July. The lowest sperm motility after defrosting was observed in April, which is 36.4% less (P<0.001) than in July, 38.6% less (P<0.001) than in May, 45.12% less (P<0.001) than in June, 50.1% less (P<0.001) than in in January, 52.6% less (P<0.001) than in February, 43.75% less (P<0.001) than in March, 53.8% less (P<0.001) than in September, 55% less (P<0.001) than in October, 54.4% less (P<0.001) than in November and 54.1% less (P<0.001) than in December. Based on the data obtained on the mobility of deconserved sperm doses, we mat expect the highest fertility from the genetic material that is harvested from September to December.
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Griffin, Róisín A., Mark Baker, Robert John Aitken, Aleona Swegen, and Zamira Gibb. "What makes a fertile sperm? Unique molecular attributes of stallion fertility." Reproduction 158, no. 4 (October 2019): R125—R137. http://dx.doi.org/10.1530/rep-19-0060.
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Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10–14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assays aimed at the accurate assessment of stallion fertility. This paper reviews our existing knowledge concerning the molecular mechanisms that underpin the functional competence of stallion spermatozoa, highlighting the relative importance of oxidative stress, DNA damage, sperm proteomics and RNA profile. We also consider the way in which fundamental improvements in our understanding of stallion sperm biology are informing the identification and development of possible biomarkers of fertility and thus avenues for the development of specific assays for fertility prediction.
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Morrell, J. M., A. Lundgren, P. Humblot, and A. Johannisson. "13 REACTIVE OXYGEN SPECIES IN STORED STALLION SEMEN." Reproduction, Fertility and Development 25, no. 1 (2013): 153. http://dx.doi.org/10.1071/rdv25n1ab13.
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The quality of cooled semen doses for AI varies considerably between stallions. One of the factors affecting sperm quality may be the content of reactive oxygen species (ROS), which are known to affect fertility in some species. The objective of this study was to measure the ROS content of cooled stored stallion semen doses for AI as part of an evaluation of sperm quality and to correlate it with pregnancy rates. Ejaculates (3 per stallion) were collected from 14 stallions at a commercial stud farm and were extended in INRA 96 (IMV Technologies) as standard cooled semen doses for AI. After transporting the semen doses overnight to the laboratory at the Swedish University of Agricultural Sciences (Uppsala, Sweden) in an insulated container with a cold pack, aliquots were evaluated for sperm quality and ROS content by staining with hydroethidine and dihydrodichlorofluorescein diacetate and measuring fluorescence by flow cytometry (Guthrie and Welch 2006 J. Anim. Sci. 84, 2089–2100). Seven sperm sub-populations were quantified: superoxide positive or negative, living (S+L and S–L, respectively); superoxide positive, dead (S+D); and hydrogen peroxide positive or negative, living or dead (P+L, P–L, P+D, and P–D, respectively). Sperm motility was measured by computer-assisted sperm motility analysis (Sperm Vision, Minitube), membrane integrity by flow cytometry, and chromatin structure using the sperm chromatin structure assay (all assays described in Morrell et al. 2011 Theriogenology 76, 1424–1432). Pregnancy rates following AI with cooled semen doses from the same stallions were available later in the year. The effect of stallion was tested by ANOVA. Correlations were calculated between the proportions of sperm stained for S or P and other sperm quality parameters and also with pregnancy rates (SAS version 9.1, SAS Institute Inc., Cary, NC, USA). The effect of stallion was significant on all variables measured (P < 0.01). There were no significant correlations between percentages of S+L or P+L spermatozoa and progressive motility, membrane integrity, or DNA fragmentation index (%DFI). There was a trend for the proportion of P-L spermatozoa to be correlated with progressive motility (r = 0.51; P < 0.07) whereas the proportions of S+D and P–D were negatively correlated with progressive motility (r = –0.66 and –0.61; P < 0.02). The proportion of S+D and chromatin damage were negatively correlated (r = –0.54; P < 0.05). There was a trend for the proportions of S+D and P+D to be negatively related to the overall pregnancy rate (r = –0.52; P < 0.07, and r = –0.58; P < 0.05, respectively). The proportions of superoxide and hydrogen peroxide-containing live spermatozoa were not correlated to stallion fertility or other parameters of semen quality in stored semen samples. In contrast, a significant negative relationship was found between the proportion of dead spermatozoa producing superoxide radicals and progressive motility, and between dead spermatozoa producing superoxide radicals and chromatin damage. However, many factors contribute to sperm quality and fertility, with many interactions between them. More work is needed to unravel these different effects and determine which factors can be used to predict stallion fertility.
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KUMAR, PRASHANT, ASHOK KUMAR, J. S. MEHTA, G. N. PUROHIT, S. K. RAVI, YASH PAL, R. A. LEGHA, B. N. TRIPATHI, and TR TALLURI. "Impact of supplementation of semen extender with antioxidants on quality of cooled or cryopreserved Marwari stallion spermatozoa." Indian Journal of Animal Sciences 90, no. 10 (April 5, 2021): 1356–61. http://dx.doi.org/10.56093/ijans.v90i10.111303.
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The aim of the present study was to evaluate the effects of supplementation of semen extender with two antioxidants namely Ascorbic acid (AA @ 0.9 g/L), Glutathione (GSH @ 2.5 mM), and combination of both (AA @ 0.9 g/L + GSH @ 2.5 mM) either in alone or in combination on the quality of cooled or cryopreserved Marwari stallion spermatozoa. For this purpose, a total of 24 ejaculates were collected from four adult and fertile Marwari stallions (6 ejaculates from each stallion) using an artificial vagina. Each freshly ejaculated semen sample was investigated for the semen quality parameters, viz. colour, consistency, total volume, gel volume, gel free volume, pH, progressive sperm motility, sperm concentration, sperm viability, sperm plasma membrane integrity, acrosomal integrity and DNA integrity. In the freshly ejaculated semen, no significant variation was found among individual stallions for various semen quality parameters except in sperm concentration. Pre-freeze and post-thaw semen evaluation revealed that the values for the most of the semen quality parameters were significantly higher in the semen extender being treated with the combination (AA @ 0.9 g/L +GSH @ 2.5 mM) of antioxidants group rather than AA and GSH alone or control. Addition of AA (0.9 g/L) and GSH (2.5 mM) to the freezing extender improved equine pre-freeze and post-thaw semen quality with the superiority of control group which indicates the beneficial role of supplementation of antioxidants to the stallion semen during cryopreservation process.
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Gibb, Z., C. G. Grupen, L. H. A. Morris, G. Evans, and W. M. C. Maxwell. "263. Substitution of skim milk with bovine serum albumin in a stallion semen diluent." Reproduction, Fertility and Development 20, no. 9 (2008): 63. http://dx.doi.org/10.1071/srb08abs263.
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Skim milk has long been utilised as a source of protective proteins in stallion semen diluents. However, skim milk is also thought to contain components that are toxic to sperm and reduces the clarity of sperm suspensions, which impedes sperm assessments. This may also reduce the effectiveness of staining procedures used to process sperm for flow cytometric sex-sorting. The aim of this study was to ascertain the optimal concentration of bovine serum albumin (BSA) to replace skim milk in a traditional stallion semen diluent, Kenney's Modified Tyrode's (KMT) Medium1, for handling and processing stallion sperm before flow cytometric sex-sorting. Two ejaculates were collected from each of three pony stallions. Each ejaculate was divided into five aliquots and diluted in either KMT with skim milk or KMT supplemented with 0, 0.25, 0.5 or 1% BSA. Diluted samples were further divided into two aliquots and either stored at 15°C for 18 h before incubation and assessment, or incubated and assessed immediately upon arrival. Samples were incubated at 34°C and evaluated at 0, 45 and 90 min for objective motility and acrosome integrity. No interactions were observed between any treatments over time. There was a lower percentage of intact and a higher percentage of detached acrosomes for sperm incubated in KMT containing 0% BSA than all other treatments. A greater proportion of sperm incubated in KMT with skim milk had partial acrosome damage compared with other treatments. There was no difference in % total motility for sperm incubated in KMT with skim milk, and KMT containing 0.5 and 1% BSA (Table 1). These results indicate that BSA may be suitable as an alternative protein source in stallion semen diluents. Further studies are required to compare sex-sorting rates and sperm quality after sex-sorting, incubation and staining in skim milk compared with BSA-based media. (1) Padilla, A W and Foote, R H 1991, ‘Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa’, Journal of Animal Science, vol. 69, no. 8, pp. 3308–331
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Suliman, Yara, Frank Becker, Armin Tuchscherer, and Klaus Wimmers. "Seasonal variations in quantitative and qualitative sperm characteristics in fertile and subfertile stallions." Archives Animal Breeding 63, no. 1 (May 14, 2020): 145–54. http://dx.doi.org/10.5194/aab-63-145-2020.
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Abstract. 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 (n=8; four fertile stallions and four subfertile stallions), which differ in the seasonal pregnancy rate 80 %–90 % (fertile) vs. 40 %–60 % (subfertile). A total of 64 ejaculates were collected from the stallions (n=8; four in the breeding season and four in the non-breeding season of each stallion). No significant differences in the semen quality parameters between the fertile and the subfertile stallions in the non-breeding season were observed. However, in the breeding season the proportion of morphologically normal sperm, total motility, progressive motility, average path velocity (VAP), and curvilinear velocity (VCL) were significantly higher in the fertile group (P<0.05) when compared with the subfertile group. In addition, a significant seasonal variation in the proportion of morphological normal sperm was found in the fertile group between the breeding and the non-breeding season (P<0.05). Moreover, significant seasonal variations (P<0.05) in CASA parameters of mean VAP, straight line velocity (VSL), and beat-cross frequency (BCF) were observed in the fertile and the subfertile stallions, which tended to be lower in the non-breeding season. In conclusion, differences between the fertile and the subfertile stallions were observed only in the breeding season, and a few of CASA-derived parameters seemed to be significantly lower during the non-breeding season in both the fertile and the subfertile stallions.
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Gonzalez-Castro, R. A., J. K. Graham, and E. M. Carnevale. "176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies." Reproduction, Fertility and Development 30, no. 1 (2018): 228. http://dx.doi.org/10.1071/rdv30n1ab176.
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Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on >85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P < 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P < 0.001; live: r = 0.71; P < 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.
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Spizziri, B. E., J. K. Graham, E. L. Squires, and M. R. Hudson. "216 IN VITRO CAPACITATION OF FROZEN/THAWED STALLION SPERM." Reproduction, Fertility and Development 20, no. 1 (2008): 187. http://dx.doi.org/10.1071/rdv20n1ab216.
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The inability of stallion sperm to capacitate in an in vitro setting has limited many equine assisted reproductive technologies such as gamete intrafallopian transfer (GIFT) and IVF. These experiments were conducted to develop methods for artificially capacitating frozen/thawed stallion sperm, using dilauroylphosphatidylcholine (PC-12), calcium ionophore A23187 (IONO), or methyl-β-cyclodextrins (MBC) so that sperm could be used in equine assisted reproductive technologies. Assisted reproductive technologies need to be established to maximize the number of offspring produced with cryopreserved spermfrom stallions with low cryosurvival rates or that have a very limited number of frozen sperm. Low concentrations of lysophosphatidylcholine (LPC) induce the acrosome reaction in capacitated sperm and, therefore, were used to detect fully capacitated sperm treated with PC-12, IONO, or MBC. Ejaculates from 8 stallions were diluted in a modified Tyrode's medium and centrifuged (1000g for 11 min). The sperm pellets were suspended to 400 million sperm mL–1 in EZ-Freezin LE, packaged into 0.5-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in a 37�C water bath for 30 s and washed through 35% Percoll (400g for 4.5 min) to remove egg yolk particles and seminal plasma. The sperm pellets were suspended in Medium 199 (0.6% BSA and 5 mm calcium chloride) to 50 million sperm mL–1, and stained with propidium iodide and FITC-peanut agglutinin (PNA) (1 mg mL–1 each; viability and acrosome reaction detection, respectively). Sperm were treated with PC-12 (13, 17, 20, 30, 40 µm), IONO (0.5, 1, 1.5, 2 µm), or MBC (0.4, 0.6, 0.8, 1 µm) and incubated in a 37�C water bath (20, 15, 20 min) to artificially capacitate sperm. Sperm were then challenged with LPC (2 or 5 µg), for 10 additional min, and analyzed by flow cytometry to determine the percentages of acrosome-reacted sperm. Data were analyzed by ANOVA and treatments were separated by Student-Newman-Keul's test. The PC-12 (20, 30, 40 µm), IONO (1.5, 2 µm), and MBC (0.4, 0.6, 0.8, 1 µm) artificially capacitated sperm equally well (59 to 70%, 52 to 59%, and 48 to 55%, respectively) and were higher than control sperm (13%, SEM � 9; P < 0.05). Sperm viability was similar at all IONO or MBC concentrations; however, PC-12-treated sperm (17 to 40 µm) had significantly lower sperm viability when compared with control (24 to 28% and 35%, respectively, SEM � 3; P < 0.05). In conclusion, frozen/thawed stallion sperm can be artificially capacitated by treating with PC-12, IONO, or MBC, and this may lead to practical applications for in vitro equine assisted reproductive technologies.
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Colleoni, S., R. Duchi, G. Lazzari, and C. Galli. "377 EMBRYO PRODUCTION BY OVUM PICKUP-INTRACYTOPLASMIC SPERM INJECTION-IVC IN AN EQUINE OVUM PICKUP PROGRAM USING SEMEN FROM FERTILE AND INFERTILE STALLIONS." Reproduction, Fertility and Development 22, no. 1 (2010): 345. http://dx.doi.org/10.1071/rdv22n1ab377.
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The introduction in equine reproduction of ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI), IVC, and embryo transfer, has allowed for the production of offspring from donors and stallions that could not reproduce by conventional techniques. For this reason, we used in our OPU-ICSI-IVC program both fertile stallions and stallions with field records of low or no fertility. Overall, 805 and 584 OPU oocytes were fertilized with sperm from fertile and infertile stallions, respectively. Cleavage rate was statistically lower in the latter group (65.94 v. 59.24%, chi square test; P < 0.05) but embryo development was similar (11.67 v. 8.20% blastocysts/injected oocytes, chi-square test). In order to further investigate the stallion effect on embryo development, we selected 3 stallions with low (A) or no (B, C) fertility in the field and we compared the results of the OPU program with embryo development obtained using oocytes recovered from abattoir ovaries and matured, fertilized, and cultured in vitro as the OPU oocytes. Part of the abattoir oocytes was fertilized with a stallion with known high fertility both in vivo and in vitro (abattoir fertile). Overall, the results (shown in the table) suggest a reduction in the efficiency of stallions A, B, and C compared with to the fertile stallion used as control (10.79, 7.69, and 5.0% v. 17.35%, respectively). For stallions A and B, the efficiency was further reduced in the OPU setting, indicating that the female component can play a role in the overall efficiency of the procedure. In particular, 4 mares out of 8 had a history of no pregnancy and all mares had some rate of inbreeding with the respective stallion used for the ICSI. Instead, the oocytes from the abattoir ovaries were collected in large pools from several mares, representing an average oocyte quality, and the mares were of different breed than the stallions. All data were analyzed by chi-square test and significance was set at P < 0.05. In conclusion, we demonstrated that, for those stallions in which fertility in the field is low or absent, OPU-ICSI-IVP is a suitable choice to obtain embryos, although the efficiency is variable depending not only on the stallion but also on the origin of the oocytes. Table 1.Stallion effect on embryo development of ovum pickup (OPU) and abattoir oocytes This work was supported by Fondazione Cariplo and Regione Lombardia.
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Coutinho da Silva, M. A., G. E. Seidel, E. L. Squires, Y. H. Choi, and E. M. Carnevale. "263BINDING OF STALLION SPERM TO EQUINE AND BOVINE ZONAE PELLUCIDAE: EFFECT OF MILK, MILK PROTEINS AND GLUCOSE." Reproduction, Fertility and Development 16, no. 2 (2004): 252. http://dx.doi.org/10.1071/rdv16n1ab263.
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The ability of sperm to bind to zonae pellucidae (ZP) has been correlated with fertilizing capacity of sperm in several species. Limited numbers of equine oocytes are available to perform such assays. Therefore, use of heterologous ZP to perform gamete binding tests with stallion sperm would be useful. We have found that addition of 10% of skim milk-based extender with glucose [EZ-Mixin®, Animal Reproduction Systems, Chino, USA;; (EZ)] to TALP significantly increased the number of stallion sperm bound to bovine ZP. Objectives of the present experiments were to determine: (1) if stallion sperm bind in similar numbers to equine and bovine ZP, and (2) the effects of skim milk, milk proteins and glucose on sperm binding to ZP. Denuded bovine (immature) and equine (mature) oocytes were stored at 5°C in salt solution (1.5M MgCl2, 40mM HEPES, 0.1% PVP). In Experiment I, 4 ejaculates from 2 stallions were centrifuged at 300g for 6min, and sperm pellets were resuspended in 1mL of TALP or EZ. Sperm were stained with Hoechst 33342, centrifuged, and resuspended to 2×106 sperm mL−1. Oocytes were placed into droplets of 45μL of TALP (7 to 10 oocytes/trt/ejac). Extended sperm (5μL) were added to oocytes, resulting in 2times105 sperm mL−1, and the mixutre was incubated for 2h at 38.5°C. Oocytes then were pipetted in TALP to remove loosely attached sperm and observed with fluorescence microscopy;; mean numbers of sperm bound to bovine and equine ZP for TALP were 29±1.9 and 36±2.6 (P>0.1) and for EZ, 149±5 and 152±6.3 (P>0.1), respectively. More sperm bound to ZP with EZ than to ZP with TALP (P<0.001). Experiment II used 4 ejaculates from 4 stallions. After initial centrifugation, sperm were resuspended in 1mL of each of six extenders: TALP, EZ, TALP containing 89.5mM glucose (TG), TALP containing 163.5mM glucose (THG), TALP containing 2.4mgmL−1 of skim-milk powder (TSM), and INRA 96® (IMV Technologies, L’Aigle, France) that contains 27mgmL−1 of native phosphocaseinate. Hoechst 33342-stained sperm and bovine oocytes were processed as described for Experiment I. Treatments containing milk proteins resulted in more sperm binding (P<0.01) than those without milk proteins (Table 1). In conclusion, use of bovine oocytes led to similar results for equine and bovine oocytes;; therefore, bovine oocytes can be used for binding assays with stallion sperm. High concentrations of glucose increased numbers of sperm bound to ZP;; however, presence of milk or milk proteins was more effective in enhancing binding of sperm to ZP. INRA96 contains relatively low glucose (67mM) and one milk protein. Therefore, we hypothesize that native phosphocaseinate may cause increased sperm binding to ZP. Table 1 Mean sperm bound per ZP±SEM (n=38–40/group)
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Parrish, J. J., C. Mueller, E. Ludwig, and J. L. Susko-Parrish. "226 DAY LENGTH DOES NOT AFFECT LIVE SPERM NUCLEAR SHAPE IN THE STALLION." Reproduction, Fertility and Development 20, no. 1 (2008): 192. http://dx.doi.org/10.1071/rdv20n1ab226.
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Fourier harmonic analysis (FHA) of sperm nuclei is a precise and objective method to evaluate shape of the sperm head, with the calculated harmonic amplitudes highly related to male fertility. The FHA approach has been developed for use in the bull and the boar but has not yet been applied to the stallion. Direct utilization of the previous fluorescent approaches to identify and image live sperm nuclei in the bull cannot be used in the stallion due to the increased thickness of the post-nuclear region and thin anterior region of the sperm head. An alternative approach was developed in which live and motile sperm were isolated after filtration of an ejaculate through a Sephadex G-15 column. The resulting live sperm were sonicated briefly to separate tails and heads. The heads were isolated on a 45–90 discontinous Percoll gradient, fixed with paraformaldehyde (0.2%), centrifuged onto glass slides, and dried. The slides were then stained with eosin (1%), cleared with water, and dried again; Permount was added, followed by a coverslip. Slides were imaged with phase contrast microscopy; digital images were acquired and evaluated with custom software to identify perimeter coordinates of sperm nuclei. The perimeter coordinates were next converted to Fourier harmonic amplitudes 0–5 (HA0–HA5) using trigonomic regression at 1 degree equally spaced angles. Fertility of bulls were previously reported to be most related to changes in HA0 and HA2 but no information is available on stallions. As fertility data on stallions is limited, to evaluate FHA in the equine, the day length (period of light) was increased in January from the ambient 9–10 h to 16. Semen samples from 5 light horse stallions were collected at weekly intervals for 8 weeks following the increase in light. It was hypothesized that fertility would increase for each stallion over the course of the experiment, as testosterone increases and spermatogenesis improves with increasing day length, as previously shown. Each week semen samples were evaluated for FHA live sperm nuclei, as described above. All parameter means and variations were recorded on 100 randomly selected sperm nuclei per semen sample evaluated. There was no difference relative to week 0 in the least squares mean or SD of HA0, HA1, HA2, HA3, HA4, or HA5 over the 8 weeks (P > 0.05). However stallions were consistently different for all HAs (P < 0.05). The overall mean HA0–HA5 � SEM were 1.973 � 0.38, 0.087 � 0.002, 0.721 � 0.021, 0.051 � 0.004, 0.208 � 0.014, and 0.031 � 0.002, respectively. Even though libido increased during the experiment, confirming the effect of light on the stallions, no affect on sperm nuclear shape or its variation was detected using FHA. Based on work in other species involving numbers of sperm inseminated, if large numbers of sperm from these stallions were inseminated in mares, we would predict no change in fertility due to season. Further research is needed to confirm this prediction.
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Gaitskell-Phillips, Gemma, Francisco E. Martín-Cano, José M. Ortiz-Rodríguez, Antonio Silva-Rodríguez, Maria C. Gil, Cristina Ortega-Ferrusola, and Fernando J. Peña. "Differences in the proteome of stallion spermatozoa explain stallion-to-stallion variability in sperm quality post-thaw†." Biology of Reproduction 104, no. 5 (January 12, 2021): 1097–113. http://dx.doi.org/10.1093/biolre/ioab003.
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Abstract The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those with a fold change > 3, a P = 8.2e-04, and a q = 0.074 (equivalent to False discovery rate (FDR)) were selected, and the following proteins were identified in fresh samples as discriminant variables of good motility post-thaw: F6YTG8, K9K273, A0A3Q2I7V9, F7CE45, F6YU15, and F6SKR3. Other discriminant variables were also identified as predictors of good mitochondrial membrane potential and viability post-thaw. We concluded that proteomic approaches are a powerful tool to improve current sperm biotechnologies.
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Papa, F. O., C. Ramires Neto, Y. F. R. Sancler-Silva, H. L. Resende, G. A. Monteiro, C. P. Freitas-Dell'aqua, and M. A. Alvarenga. "66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN." Reproduction, Fertility and Development 27, no. 1 (2015): 126. http://dx.doi.org/10.1071/rdv27n1ab66.
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The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-CryoTM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 106 of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 106 total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P > 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.
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Härtlová, Helena, Radko Rajmon, Iva Krontorádová, Jiří Mamica, Lukáš Zita, Petra Klabanová, and Antonín Černocký. "Semen quality, lipid peroxidation, and seminal plasma antioxidant status in horses with different intensities of physical exercise." Acta Veterinaria Brno 82, no. 1 (2013): 31–35. http://dx.doi.org/10.2754/avb201382010031.
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The aim of this study was to compare markers of semen quality, sperm membrane damage, and the seminal plasma antioxidant activity in warmblood stallions with and without sport workload stress. Four stallions were used for breeding only (control) and four both for breeding and competition in jumping. Semen samples were collected at 14-day intervals (from June to August) from each stallion (5 ejaculates per stallion). Immediately after sperm collection, a conventional examination of the ejaculate was processed. Catalytic activities of enzymes aspartate aminotransferase, alanin aminotransferase, glutathione peroxidase, superoxide dismutase and indicator of lipoperoxidation - F2α isoprostanes were measured in samples of seminal plasma. Contrary to basic semen quality indicators, the values of seminal plasma pH, aspartate aminotransferase and alanin aminotransferase were significantly (P < 0.05) impaired in the physically stressed stallions. Also, the level of F2α isoprostanes and the activity of superoxide dismutase were significantly (P < 0.05) increased by stress. The antioxidant activities of superoxide dismutase and glutathion peroxidase increased during the monitored period and reflected changes in F2α isoprostane concentration. We can conclude that even the conventional basic sperm indicators stay within the reference ranges of the biochemical indicators of seminal plasma such as pH or AST/ALT activity may be negatively influenced by sport workload stress. Increased concentrations of F2α isoprostanes indicate that lipoperoxidation can be a mechanism of cell membrane destabilization, which is counteracted by an increase of antioxidant enzyme activities. This is the first report of oxidative stress symptoms in normospermic equine semen in relation to stallion sport workload.
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Hidalgo, M., I. Rodriguez, J. Dorado, J. Sanz, and C. Soler. "Effect of sample size and staining methods on stallion sperm morphometry by the Sperm Class Analyzer ." Veterinární Medicína 50, No. 1 (March 27, 2012): 24–32. http://dx.doi.org/10.17221/5593-vetmed.
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Computer-assisted sperm morphometry analysis has improved the assessment of sperm morphology, but the results depend on the use of adequate evaluation and staining procedures of spermatozoa from individual species. In this study, the morphological module of the Sperm Class Analyzer®was used for the morphometric analysis of stallion sperm heads and midpieces. Semen samples were obtained from six fertile stallions in order to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris’ Haematoxylin) on the accuracy of image processing and sperm morphometry, and the effect of the sample size on sperm morphometric measurements. Harris’ Haematoxylin was the staining technique of choice on the accuracy of the image processing with an optimum contrast of sperm cells with the surrounding background that allows an efficient boundary detection and segmentation which results in the highest proportion of sperm heads and midpieces assessed (80.47%). The results indicate that the staining methods affected significantly the sperm dimensions with increased values from Diff-Quik than Hemacolor and Harris’ Haematoxylin respectively (Diff-Quik > Hemacolor > Harris’ Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. In conclusion, to obtain objective and accurate sperm morphometric measurements by the Sperm Class Analyzer® system in the stallion, it’s recommended the analysis of 100 spermatozoa from slides which have been previously stained with Harris’ Haematoxylin.
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Medica, Ashlee J., Robert J. Aitken, Garth L. Nicolson, Alecia R. Sheridan, Aleona Swegen, Geoffry N. De Iuliis, and Zamira Gibb. "Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro." Reproduction and Fertility 2, no. 3 (August 11, 2021): 199–209. http://dx.doi.org/10.1530/raf-21-0028.
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Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro. Lay summary Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules – called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.
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Bertelsmann, H., S. Keppler, M. Höltershinken, H. Bollwein, D. Behne, D. Alber, G. Bukalis, A. Kyriakopoulos, and H. Sieme. "Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality." Reproduction, Fertility and Development 22, no. 5 (2010): 886. http://dx.doi.org/10.1071/rd10032.
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The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion–1) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = –0.42, respectively; P ≤ 0.05), and the selenium concentration in spermatozoa (nmol g–1) was correlated with PRC (r = 0.40, P < 0.03). The results of the present study show that the determination of an adequate selenium status for the male equine reproduction requires the analysis of selenium in spermatozoa. Furthermore, selenium is associated with improved sperm quality and fertility in the stallion.
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Papa, F., M. Carmo, P. Papa, J. Dell'Aqua, and M. Alvarenga. "157 EFFECT OF DENSITY GRADIENT SELECTION ON EMBRYO RECOVERY RATES OF FRESH AND COOLED STALLION SEMEN." Reproduction, Fertility and Development 25, no. 1 (2013): 227. http://dx.doi.org/10.1071/rdv25n1ab157.
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The aim of this study was to improve the spermatic kinetic parameters from stallions with poor quality of fresh and chilled semen by the use of density gradient Equipure™ (Nidacon, Mölndal, Sweden). Semen from 5 Quarter Horse stallions aged 8 and 16 years with history of low embryo recovery rates were used. The kinetics sperm evaluation was performed by computerized semen analysis (CASA) and plasma membrane integrity with fluorescent probes. The average motility parameters for fresh semen before selection were total motility (MT) 60%, progressive motility (PM) 30%, and plasma membrane integrity (IMP) 60% and for cooled semen (24 h at 5°C) were: MT 50%, MP 18%, and 50% IMP. For the group of fresh and cooled semen with no density gradient selection (NS), mares were inseminated with 1 billion viable sperm diluted in skim milk extender in the uterine body, 24 h after ovulation induction with 1 mg of deslorelin. For EquipureTM selection group (SE) semen was concentrated through Spermfilter membrane™ and resuspended in 5 mL of BotuSemenTM (BotupharmaTM, Brazil). In a 15 mL conic tube 5 mL of EquipureTM was added and another part containing 5 mL of the resuspended sperm was slowly added over the EquipureTM column. The 15 mL conic tube was centrifuged at 400g for 20 min. After centrifugation, the sperm pellet was carefully aspirated and to the pellet was resuspended in 4 mL of BotucrioTM. The sperm recovery rate with EquipureTM was 40%. Deep uterine AI was performed 24 h after ovulation induction with 1 mg of deslorelin. Data were analyzed by ANOVA followed by Tukey’s test (P < 0.05). The analysis of semen after EquipureTM selection resulted in average: 75, 35, 65, and 40%, respectively, for MT, MP, and IMP index and sperm recovery. The embryo recovery rate from the 5 stallions showed the following results: Stallion 1 (fresh semen): 12 mares (NS)/4 embryos (33%) and for group SE 22 mares/16 embryos, (72%; P ≤ 0.05). Stallion 2 (fresh semen): 9 mares (NS)/4 embryos, (44%) and for group SE, 12 mares/8 embryos (66%; P > 0.1). Stallion 3 (fresh semen): 4 mares (NS)/1 embryo (25%) and 7 mares for SE/4 embryos (57%; P > 0.1). Stallion 4 (chilled semen): 4 mares (NS)/0 embryo 0% and for SE group 8 mares/6 embryos 75%; P ≤ 0.001). Stallion 5 (chilled semen): 10 mares (NS)/2 embryos (20%) and for SE 6 mares/3 embryos (50%; P > 0.1). For the overall results, 39 inseminations were performed on the no selected group and 11 embryos recovered (28%) for the selected group 55 inseminations and 37 embryos recovered (67%; P < 0.01). The results clearly showed that selection by sperm density gradient EquipureTM was a very effective technique that allowed an improvement on semen quality and fertility. The authors acknowledge support from FAPESP and Botupharma.
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Naumenkova, V. A., M. M. Atroshchenko, and A. V. Kalinova. "Stabilization of stallion sperm parameters in the post-vaccination period due to a complex of vitamins." BIO Web of Conferences 37 (2021): 00128. http://dx.doi.org/10.1051/bioconf/20213700128.
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The aim of our research was to diminish the damaging effect of vaccination on the quality of stallion sperm and its cryostability. The task was to study the effect of the complex vitamin of tetrahydrovit on sperm parameters under conditions of vaccination against leptospirosis. Stallions that were vaccinated against leptospirosis were divided into 3 groups: the first –control, without vitamin supplements, the second – with the addition of 2 ml of tetrahydrovit vitamin daily, the third group – intramuscular injection of tetrahydrovit 1 time in 7 days. The reaction of the body to the introduction of the vaccine in the group without vitamins proceeded with a significant decrease in the quality of sperm in the first two weeks. Later, the motility and the number of whole membranes of fresh sperm gradually improved, but the survival rate of chilled and frozen sperm remained low until the end of the observations. Among stallions in the experimental groups, which were given tetrahydrovit vitamin (orally or intramuscularly), the decrease in sperm parameters was insignificant, within the usual fluctuations, which allowed one to avoid culling spermodoses. Tetrahydrovit vitamin promotes a stabilizing effect on the quality indicators of sperm of stallions-producers after vaccination.
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Shitikova, Anna M., Mikhail M. Atroshchenko, Lidia V. Krokhotina, Mariya G. Engalycheva, and Mariya N. Dmitrieva. "Effect of Testosterone, Dihydrotestosterone, Estradiol and Cortisol on the Quality of Fresh and Cryopreserved Stallion Sperm." Journal of Experimental Biology and Agricultural Sciences 10, no. 3 (June 26, 2022): 619–27. http://dx.doi.org/10.18006/2022.10(3).619.627.
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The effect of steroid hormones on the quality of fresh and cryopreserve sperm has not been fully understood. This study aimed to evaluate the effect of testosterone, dihydrotestosterone, estradiol, and cortisol on the quality of fresh and cryopreserved stallion sperm. The study was conducted on 40 Equus caballus stallions, including Arab (n=20), Oryol trotting (n=4), Standardbred (n=4), and Soviet Heavy Draft (n=12) breeds. The average age of the experimental animals was 9.9 ± 0.7 years. We determined standard quality indicators in fresh and cryopreserved sperm and the concentration of steroid hormones in the blood plasma of stallions. Results of the study suggested a negative correlation between the level of testosterone with total (r=-0.41; p<0.01) and progressive (r=-0.44; p<0.01) sperm motility in cryopreserved sperm as well as in fresh sperm (r=-0.38; p<0.05 and r=-0.39; p<0.05 correspondingly). While the level of estradiol showed a positive correlation with survival rate in cryopreserved (r=0.35; p<0.05) and in fresh (r=0.33; p<0.05) sperm. Further, the level of cortisol in the blood plasma of stallions did not show any statistically significant correlations with the qualitative characteristics of sperm. A positive relationship was found between the concentration of dihydrotestosterone with the volume of ejaculate (r=0.37; p<0.05) and the total number of sperm in the ejaculate (r=0.43; p<0.01). Results of the study can be concluded that steroid hormones have different effects on the quality indicators of fresh and cryopreserved sperm of stallions and their concentration in the blood should be considered when selecting stallions for cryopreservation of sperm.
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Aurich, Jörg, Juliane Kuhl, Alexander Tichy, and Christine Aurich. "Efficiency of Semen Cryopreservation in Stallions." Animals 10, no. 6 (June 13, 2020): 1033. http://dx.doi.org/10.3390/ani10061033.
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Differences in the cryotolerance of spermatozoa exist among stallions, but it remains to be determined to what extent such differences are affected by breed. In this study, post-thaw semen quality in stallions presented for semen cryopreservation was analysed retrospectively (1012 ejaculates from 134 stallions of 5 breeds). The percentage of frozen–thawed ejaculates acceptable for artificial insemination (AI) and the number of insemination doses per cryopreserved ejaculate was calculated. Logistic regression analysis revealed sperm motility in raw semen as the most important explanatory variable for the percentage of cryopreserved ejaculates with a post-thaw quality acceptable for AI. Of the other variables included into the model, stallion age was the most important parameter with more acceptable ejaculates in younger than in older stallions. Logistic regression also showed more acceptable frozen–thawed ejaculates in Arab stallions versus Warmbloods, Quarter Horses and Icelandic horses. The analysis thus demonstrates differences in the percentage of acceptable cryopreserved ejaculates among horse breeds. Season was a less relevant explanatory variable for percentage of acceptable cryopreserved ejaculates. Logistic regression revealed total sperm count as the most important variable determining the number of cryopreserved semen doses obtained per acceptable ejaculate. In conclusion, logistic regression analysis revealed stallion age and breed as explanatory variables for the percentage of cryopreserved ejaculates acceptable for AI.
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Warnke, Christina, A. Tuchscherer, Hannelore Alm, W. Kanitz, S. Blottner, and H. Torner. "Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation." Acta Veterinaria Hungarica 51, no. 3 (July 1, 2003): 395–408. http://dx.doi.org/10.1556/avet.51.2003.3.13.
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The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 ± 12.7% vs. 66.8 ± 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 ± 5.8% in May and 15.6 ± 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 ± 43.9 vs. 164.4 ± 66.4 µm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) ≯ stallion (P < 0.001) ≯ season (P < 0.05).
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Bubenickova, Filipa, Jiří Šichtař, Linda Nováčková, Jitka Sirohi, and Ondřej Šimoník. "The structure of subpopulations of stallion spermatozoa after thawing differs between good and poor freezers." Czech Journal of Animal Science 65, No. 11 (November 27, 2020): 403–10. http://dx.doi.org/10.17221/127/2020-cjas.
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The aim of this study was to evaluate differences in the presence of sperm subpopulations in frozen-thawed semen in stallions with different freezability. The motility of individual spermatozoa of 24 stallions from 15 breeds was evaluated using computer-assisted sperm analysis (CASA) immediately after thawing (T0) and after 30 min of incubation (T30). In accordance with our previous studies, samples were initially divided based on their total motility into categories of good (GF) and poor (PF) freezers. K-means cluster analysis of kinematic parameters of spermatozoa was used to divide motile sperm (n = 57 630) into three subpopulations. Analysis of variance was used to evaluate differences in the subpopulations between GF and PF stallions at the times of incubation T0 and T30. Statistically significant differences were found in most kinematic parameters between PF and GF stallions as well as between the times of incubation T0 and T30 (P < 0.05). Spermatozoa of good freezers are represented more frequently in the fast and medium fast subpopulations and are faster and more linear than those of poor freezers (P < 0.05). Sperm from PF stallions were more strongly affected by longer incubation. The percentage of sperm in the fast and medium fast subpopulations was lower in samples from PF stallions, but assessment of the motility parameters in particular sperm subpopulations revealed that these sperm had good velocity. Poor freezer samples had lower sperm quality due to a reduced total proportion of motile sperm, and these samples were more sensitive to prolonged time after thawing. Thus, an efficient sperm selection method or a special insemination technique should be used for obtaining doses from stallions with poor freezability. Our study showed that the CASA system and cluster analysis are promising tools for better understanding the significant differences in the individual stallion freezability, and further research should be focused on their application in the field.
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Pirosanto, Y., M. Valera, A. Molina, J. Dorado, and S. Demyda-Peyrás. "23 Sperm quality of Pure Spanish stallions is affected by inbreeding coefficient and age." Reproduction, Fertility and Development 32, no. 2 (2020): 137. http://dx.doi.org/10.1071/rdv32n2ab23.
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Inbreeding depression, a genetic condition produced by the mating of close-related individuals, has been associated with a reduction of fertility in several species. However, a loss in sperm quality was also associated with age. In horses, the few existing reports have described a tendency of both parameters to produce a negative effect on sperm quality. However, those reports were performed using a subjective evaluation of sperm motility. In the present study, a total of 692 ejaculates from 86 Pure Spanish stallions (PRE), aged between 3 and 22 years, were evaluated using a computer-assisted methodology to determine the effect of inbreeding in four semen parameters: free-gel volume (V), sperm concentration (C, by haemocytometer), and total (TM) and progressive (PM) sperm motility (by Spermvision sperm class analyser; Minitube). The inbreeding coefficient (F) was estimated using 300 000 PRE pedigree records approximately (minimum pedigree depth, eight equivalent complete generations; range, between 1 and 30.1%). Stallion, age, ejaculate, and season of semen collection were the variables included in the statistical model (general linear model), with ejaculate and season being the variables with a major effect (by variance components analysis). Our results showed that sperm concentration (r=−0.18; P<0.0001) and volume (to a lesser extent) were reduced with advancing age, both showing a major decline after 15 years of age. To the contrary, sperm motility was not affected by age of the stallion. We also found a negative correlation between the inbreeding coefficient and ejaculate volume (r=−0.14; P<0.001), with a marked decrease seen when F was between 7 and 20%. Also, a negative correlation was observed in PM (r=−0.08; P<0.05), although to a lower extent. Conversely, C and TM were not affected by inbreeding depression (P>0.05). In conclusion, our results demonstrated that high levels of inbreeding can compromise severely the sperm quality of the PRE stallion, which, subsequently, may have a negative influence on fertility. Ongoing studies using genomic data will help to detect genetic variants associated with stallion semen quality and how it is influenced by inbreeding in specific genomic regions.
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Morrell, J. M., A. Johannisson, L. Juntilla, K. Rytty, L. Bäckgren, A. M. Dalin, and H. Rodriguez-Martinez. "Stallion Sperm Viability, as Measured by the Nucleocounter SP-100, Is Affected by Extender and Enhanced by Single Layer Centrifugation." Veterinary Medicine International 2010 (2010): 1–7. http://dx.doi.org/10.4061/2010/659862.
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On-stud assessment of stallion sperm quality can be problematic. A new instrument, the Nucleocounter SP-100, was validated for measuring stallion sperm concentration and viability. It was subsequently used to evaluate sperm viability in Kenney's extender and INRA96. There was a strong correlation between sperm concentrations measured by the Nucleocounter SP-100 and by the Bürker counting chamber (; ). Similarly, there was a good correlation between sperm viability results from the Nucleocounter SP-100 and flow cytometric results (; ). Sperm viability at 24 hours was significantly better for samples extended in INRA96 than in Kenney's extender (). Furthermore, sperm kinematics were better for stored samples in INRA96 than in Kenney's extender. Single Layer Centrifugation selected spermatozoa that maintained their viability better during storage for 24 hours than the uncentrifuged samples. In conclusion, the type of semen extender used and Single Layer Centrifugation were found to influence both the kinematics and viability of stallion spermatozoa. The Nucelocounter-SP100 was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability.
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Leemans, Bart, Tom A. E. Stout, Ann Van Soom, and Bart M. Gadella. "pH-dependent effects of procaine on equine gamete activation†." Biology of Reproduction 101, no. 5 (August 2, 2019): 1056–74. http://dx.doi.org/10.1093/biolre/ioz131.
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Abstract Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5–10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway.
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Felmer, R., C. Arroyo-Salvo, F. Fuentes, P. Cabrera, F. Treulen, M. Silva, and M. E. Arias. "156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation." Reproduction, Fertility and Development 31, no. 1 (2019): 203. http://dx.doi.org/10.1071/rdv31n1ab156.
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Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm. Funding support was received from FONDECYT 1160467 CONICYT, Chile.
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Papas, Marion, Jaime Catalán, Beatriz Fernandez-Fuertes, Laura Arroyo, Anna Bassols, Jordi Miró, and Marc Yeste. "Specific Activity of Superoxide Dismutase in Stallion Seminal Plasma Is Related to Sperm Cryotolerance." Antioxidants 8, no. 11 (November 9, 2019): 539. http://dx.doi.org/10.3390/antiox8110539.
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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. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability.
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Severa, Libor, Ivo Křivánek, and Ladislav Máchal. "On the specific conductivity of stallion ejaculate." Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 55, no. 4 (2007): 63–68. http://dx.doi.org/10.11118/actaun200755040063.
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Ejaculate specific conductivity σ (S.m – 1) was measured for 10 breeding stallions. The set of measurings was performed three times for native ejaculate and three times for ejaculate stored for 24 hours at 4 °C. The average value of specific conductivity of native ejaculate was found to be 1.22 ± 0.11 S.m – 1, the same quantity for stored ejaculate was calculated to be 1.11 ± 0.11 S.m – 1. The values of coefficients of variation have been calculated for several quantities, and qualitative (ejaculate volume, sperm concentration, and sperm motility) and quantitative parameters have been compared by way of phenotype coefficients of correlation. Two dependencies, namely specific conductivity vs. sperm concentration (rp = – 0.79; P < 0.99) and specific conductivity vs. motility (rp = – 0.78; P < 0.99), have been evaluated as relevant and enabling supplementary description of stallion ejaculate quality by means of other (physical) quantity.
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Ткачова, О. Л., Л. Т. Добродєєва, Л. В. Россоха, В. І. Россоха, and О. В. Ткачов. "Цитогенетична та біотехнологічна оцінка жеребців тракененської та ганноверської порід." Вісник Полтавської державної аграрної академії, no. 2 (June 28, 2013): 64–66. http://dx.doi.org/10.31210/visnyk2013.02.16.
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Проведено порівняльну цитогенетичну і біотехнологічну оцінку обстежених жеребців тракененської та ганноверської порід за загальною хромосомною нестабільністю й кількісними та якісними показниками сперми після відтавання. За загальної хромосомної нестабільності обстежених жеребців ганноверської породи 4,9 % біотехнологічна придатність сперми становила 76,19 %, у жеребців тракененської породи біотехнологічна придатність сперми становила 72,73 % за хромосомної нестабільності 5,91 %. На біотехнологічну придатність сперми обстежених жеребців впливає також наявність парних і кольцевих аберацій.
The comparative citogenetic and biotechnological evaluation of the inspected Traken and Hannover stallions by their general chromosomal instability and by the quantitative and qualitative parameters of sperm after thawing have been conducted. The general chromosomal instability of the investigated stallions of Hannover breed was 4,9%, the biotechnological fitness of sperm was 76,19% and the biotechnological fitness of sperm in the stallions of Traken breed was 72,73 and theirchromosomal instability was 5,91%. The presence of pair and circular aberrations also influences the biotechnological fitness of sperm of the inspected stallion.
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Coutinho da Silva, M. A., C. R. F. Pinto, J. M. Young, and K. Cole. "8 THE USE OF ANNEXIN V MAGNETIC-ACTIVATED CELL SORTING TO SEPARATE APOPTOTIC SPERM FROM THE EJACULATE OF STALLIONS." Reproduction, Fertility and Development 23, no. 1 (2011): 110. http://dx.doi.org/10.1071/rdv23n1ab8.
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Magnetic-activated cell sorting (MACS) has been used successfully in humans to remove apoptotic sperm from the ejaculate. Annexin V-conjugated microbeads recognise sperm with externalized phosphatidylserine, which is considered one of the features of apoptosis, and the labelled sperm is separated by MACS. The goals of the study were to determine if MACS can be used to separate apoptotic sperm from the ejaculate of stallions; and to determine if removal of apoptotic sperm improves the quality of stallion sperm. Our hypothesis was that MACS would improve semen quality by removing apoptotic sperm, resulting in samples with higher motility and viability. Two ejaculates from three different stallions of good fertility were used. Sperm were diluted with Tyrode’s albumin lactate pyruvate (TALP) and incubated with annexin V-conjugated microbeads for 15 min at 37°C. Control samples were incubated in the absence of annexin V microbeads. The suspension was then loaded into the separation column containing iron globes, which were fitted in a magnet (MiniMACS; Miltenyi Biotec Inc., Auburn, CA, USA). The effluent sample containing annexin-negative sperm was collected and then, the column was removed from the magnetic field and rinsed with TALP to collect the annexin-positive cells. Sperm viability, motility, morphology and caspase activation were determined in all three samples: control, annexin-negative, and annexin-positive. Data were evaluated by ANOVA and individual comparisons were performed by Tukey’s hsd test. Significance was set at P < 0.05 and data is presented as means ± SEM (Table 1). The main effect of stallion was significant only for sperm motility parameters. Sperm recovery rate following MACS was 46 ± 3%. In conclusion, the use of MACS was effective in removing apoptotic sperm from the ejaculate. The annexin-positive population displayed a higher proportion of sperm with activated caspases and lower membrane integrity and motility. However, removal of apoptotic sperm from the ejaculate did not improve sperm parameters in the annexin-negative group compared to control group. In addition, sperm morphology was not affected by MACS. Further studies are necessary to determine if MACS could be used successfully to improve sperm quality from subfertile stallions and frozen semen. Table 1.Sperm parameters following annexin V MACS (mean ± SEM) The authors are thankful to Mark Williams at Miltenyi Biotec Inc. for providing supplies; and Dr Ashok Agarwal at The Center for Reproductive Medicine, Cleveland Clinic, for scientific input.
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Choi, Y. H., D. D. Varner, C. C. Love, D. L. Hartman, and K. Hinrichs. "Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse." REPRODUCTION 142, no. 4 (October 2011): 529–38. http://dx.doi.org/10.1530/rep-11-0145.
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Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P<0.05). In Experiment 3, blastocyst development rate was 6–11% after injection of sperm lyophilized from fresh or frozen–thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze–thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.
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Amoroso-Sanches, F., R. Gonzalez-Castro, J. Stokes, and E. Carnevale. "180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes." Reproduction, Fertility and Development 31, no. 1 (2019): 214. http://dx.doi.org/10.1071/rdv31n1ab180.
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Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (> 70%), DNA integrity (<8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P<0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P<0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.
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USUGA, ALEXANDRA, BENJAMIN ROJANO, and GIOVANNI RESTREPO. "Lyophilized seminal plasma can improve stallion semen freezability." Indian Journal of Animal Sciences 90, no. 2 (March 6, 2020): 171–75. http://dx.doi.org/10.56093/ijans.v90i2.98769.
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The aim of this study was to evaluate the effect of lyophilized seminal plasma (LSP) on stallion semen freezability. Seminal plasma from 30 stallions was lyophilized to obtain a pool of LSP. Fifteen ejaculates from five stallions were supplemented before freezing with 0 mg/mL (Control), 1.44 mg/mL (LSP1), 5.04 mg/mL (LSP2) or 8.68 mg/ mL (LSP3) of LSP. Total antioxidant capacity (TAC) of LSP was assessed using Oxygen Radical Absorbance Capacity (ORAC) assay. Post-thaw motility and kinetics, sperm viability, normal morphology and membrane integrity were evaluated. Completely randomized mixed models were fitted for data analyses. The results was analyzed based on freezability of semen samples. TAC for LSP pool was 13679.4±911.6 μmol Trolox 100/g (ORAC units). Semen supplementation with LSP1 and LSP2 showed a positive effect on post-thaw total motility and membrane integrity. Supplementation with LSP3 showed a decrease in post-thaw total and progressive motility, straight line velocity and sperm viability. For poor freezability semen samples, supplementation with LSP1 and LSP2, showed higher post-thaw total motility and membrane integrity than good freezability semen samples. In conclusion, supplementation with LSP can improve the post-thaw seminal quality of stallion semen with poor freezability.
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Papa, F. O., C. M. Melo, E. G. Fioratti, J. A. Dell’Aqua, F. S. Zahn, and M. A. Alvarenga. "Freezing of stallion epididymal sperm." Animal Reproduction Science 107, no. 3-4 (September 2008): 293–301. http://dx.doi.org/10.1016/j.anireprosci.2008.05.003.
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Brito, Leonardo F. C. "Evaluation of Stallion Sperm Morphology." Clinical Techniques in Equine Practice 6, no. 4 (December 2007): 249–64. http://dx.doi.org/10.1053/j.ctep.2007.09.004.
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Olaciregui, M., L. Gil, A. Montón, V. Luño, R. A. Jerez, and J. I. Martí. "Cryopreservation of epididymal stallion sperm." Cryobiology 68, no. 1 (February 2014): 91–95. http://dx.doi.org/10.1016/j.cryobiol.2013.12.009.
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Alimardonov, A. Sh, and M. M. Safarov. "The effectiveness of artificial insemination in horse breeding." Agrarian science, no. 1 (March 7, 2020): 31–33. http://dx.doi.org/10.32634/0869-8155-2020-334-1-31-33.
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As a result of achievements in genetics and in technology of artificial insemination` cattle breeders received a powerful instrument of improving animals productive qualities, the possibilities of selecting stallions with a high genetic potential of productivity were expanded` and the rate of genetic improvement of entire populations was accelerated. Sperm from stallions is taken on an artificial vagina. Evaluation of the quality of sperm is carried out for 3 consecutive days. Insemination of mares is carried out at artificial insemination stations. 10 days after ovulation the mares are checked again with a probing stallion. If the mare for 35–40 days has not shown signs of sexual hunting again then it is followed by rectal test for foal.
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Nikitkina, Elena, Artem Musidray, Anna Krutikova, Polina Anipchenko, Kirill Plemyashov, and Gennadiy Shiryaev. "Efficiency of Tris-Based Extender Steridyl for Semen Cryopreservation in Stallions." Animals 10, no. 10 (October 4, 2020): 1801. http://dx.doi.org/10.3390/ani10101801.
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The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl® (Minitube) for freezing sperm of stallions. Steridyl is a concentrated extender medium for freezing ruminant semen. It already contains sterilized egg yolk. Semen was collected from nine stallions, aged from 7 to 12 years old. The total and progressive motility of sperm frozen in Steridyl was significantly higher than in semen frozen in LCCY. The number of spermatozoa with normal morphology in samples frozen in LCCY was 60.4 ± 1.72%, and with Steridyl, 72.4 ± 2.10% (p < 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing–thawing. No differences among the extenders were seen with the DNA integrity of spermatozoa. Six out of ten (60%) mares were pregnant after artificial insemination (AI) by LCCY frozen semen, and 9/12 (75%) by Steridyl frozen semen. No differences among extenders were seen in pregnancy rate. In conclusion, Steridyl was proven to be a good diluent for freezing stallion semen, even though it was developed for ruminants.
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Mellisho, E., R. Olmos, E. Ancco, H. Celiz, and C. Quispe. "15 THERMORESISTANCE SPERM TEST FOR EPIDIDYMAL STALLION SPERM FROZEN WITH TWO CRYOPROTECTANTS." Reproduction, Fertility and Development 26, no. 1 (2014): 121. http://dx.doi.org/10.1071/rdv26n1ab15.
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The Peruvian Paso Horse has become a flagship product of Peru, for which it is very important to work on promoting and preserving their genetic material through semen. The objective of this study was to evaluate the thermoresistance of epididymal sperm of stallion frozen with 2 cryoprotectants (dimethylformamide and glycerol). The testes with the epididymides (n = 16) of 8 adult stallions in good health condition were obtained in the slaughter house and transport at isothermal conditions (30°C). Spermatozoa from the epididymis were recovered and incubated with lactated ringer's solution (Hartman solution) for 30 min, centrifuged at 2000 RPM for 15 min, and resuspended with an extender based on sucrose (7%) and egg yolk (3%) with different levels of cryoprotectants: dimethylformamide-DMF (T4%), glycerol-GLI (4%), and a combination of dimethylformamide-DMF (2%) + glycerol-GLI (2%) with an equilibrium time of 20 min at 5°C. The final dilution was 160 million motile spermatozoa placed on 0.5-mL straws. These straws were kept over liquid nitrogen fumes for 15 min over a tripod stand. After cryopreservation, the straws were stored in liquid nitrogen tanks (–196°C). The thawing process of the straws was initiated at 75°C for 7 s, immediately followed by an evaluation of sperm motility at 4 times 0, 30, 60, and 90 min in which sperm was incubated on water bath at 37°C (thermoresistance test). The data was evaluated (ANOVA) with the SAS 9.12 program (SAS Institute Inc., Cary, NC, USA) and, for the media, Tukey's test was carried out. The results showed an effect of the treatment on the motility of the frozen-thawed semen; dimethylformamide, the cryoprotectant, had the best performance. Table 1.Motility of frozen-thawed stallion epididymal sperm (thermoresistance sperm test)1
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Atroshchenko, Mikhail M., Ekaterina Arkhangelskaya, Dmitry A. Isaev, Sergey B. Stavitsky, Alexander M. Zaitsev, Valery V. Kalaschnikov, Sergey Leonov, and Andreyan N. Osipov. "Reproductive Characteristics of Thawed Stallion Sperm." Animals 9, no. 12 (December 9, 2019): 1099. http://dx.doi.org/10.3390/ani9121099.
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The main goal of our study was to determine a set of thawed stallion sperm characteristics that have predictive value for the pregnancy rate (PR) of mares after artificial insemination (AI). DNA fragmentation and survival of sperm during hypothermic storage were studied in addition to routinely determined semen characteristics such as concentration, percentage of motile spermatozoa, and morphology. To estimate DNA fragmentation, a modified hallo assay was applied. Sperm survival was determined within hours as the ability of spermatozoa to maintain progressive motility (PM) during the storage of ejaculate diluted with lactose-chelate-citrate-yolk (LCCY) medium at +4 °C. Strong positive correlation between PR and thawed sperm motility (r = 0.90, p < 0.05) as well as between PR and sperm survival (r = 084, p < 0.05) was revealed. There was also a strong negative correlation between PR and DNA damages in spermatozoa (r = −0.94, p < 0.05). We found no dependence of PR on normal morphology spermatozoa percentage in thawed semen. We concluded that the sperm activity, survival, and DNA fragmentation should be considered as the sufficient reproductive characteristics of semen to evaluate the quality of frozen/thawed sperm and prediction of PR.