Browsing by Subject "stallion"
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Item Cushioned centrifugation of stallion semen: factors impacting equine sperm recovery rate and quality(2009-05-15) Waite, Jessica ArleneCentrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire? Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire? Component B, OptiPrep?, or Cushion Fluid?. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ? 90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.Item Dietary supplementation of omega-3 fatty acids and subsequent effects on fresh, cooled, and frozen seminal characteristics of stallions(2009-05-15) Grady, Sicilia TatianaThe use of cooled and frozen/thawed semen offers many advantages to breeders. However, many stallions produce spermatozoa that are unable to endure the stresses of cooling/storage and freezing/thawing. Improving the quality and viability of equine spermatozoa via appropriate dietary manipulation could make these stallions commercially viable for cooling or cryopreservation. To evaluate whether spermatozoa quality and viability can be improved by supplementation of omega-3 fatty acids, and if improvements can be made by altering the sources of these fats, nine miniature stallions were placed into 1 of 2 treatment groups and fed either a fish- or algae/flaxseed-based supplement which was added to the basal concentrate. Motion characteristics, membrane integrity and morphology of spermatozoa in fresh, cooled/stored (24 and 48 h), and frozen/thawed semen samples were analyzed. When comparing spermatozoa obtained from stallions in each treatment, no differences were found (P > 0.05) in motility, percentage of membrane intact spermatozoa, and percentage of morphologically normal spermatozoa of stallions. Overall, omega-3 supplementation did not appear to have a beneficial effect on offsetting the harmful effects of the cooling and freezing processes. However, when analyzing the data of one stallion that had < 40% progressive motility (PMOT) after 24 h of cooling and storage, a significant increase was observed in total motility, and progressive motility of fresh and 24 h cooled/stored spermatozoa was observed when supplemented with the fish-based supplement. Thus, omega-3 fatty acid supplementation may be most beneficial for stallions that produce lower quality ejaculates. However, further studies should be conducted, with a larger sample size, in order to substantiate these findings.Item Flow cytometric evaluation of acrosome function/dysfunction in the stallion(2009-06-02) Bosard, Tegan S.The objective of this study was to establish a rapid and efficient assay that would assess acrosomal status and function of the stallion acrosome. Ejaculates from fertile and subfertile stallions were extended to 25x106/mL and divided into aliquots (1mL) treated with no ionophore (control) or 10?M A23187 and incubated at 37?C for 0, 1, 2, and 3h. Following incubation, samples were fixed with 2% paraformaldehyde for 10 minutes at room temperature; then stored at 4?C in Dulbecco?s Phosphate-buffered saline (DPBS) for 0, 24, and 72 hours (i.e. post-fixation storage). After post-fixation storage samples were then permeabilized with 95% ethanol at -20?C for 10 minutes. Samples were resuspended in 20% fetal bovine serum in DPBS, labeled with fluorescein isothiocyanate for 10 minutes, and analyzed by flow cytometry. Post-fixation storage produced fewer (P<0.05) acrosome intact (AI) spermatozoa and a higher (P<0.05) fluorescence intensity than respective fresh samples. Regardless of incubation time or treatment, cool-stored samples averaged ~6% lower (P<0.001) AI spermatozoa than the corresponding fresh semen; however, cooled storage did not alter (P>0.2) the overall fluorescence properties as compared to fresh semen (730?8.08 vs. 734?8.01 fluorescence intensity units, respectively). For fertile stallions, the percentage of AI spermatozoa was higher (p<0.01) in control samples than A23187 samples at incubation times 1, 2, and 3h (Control-59, 56, and 51% vs. A23187- 46, 29, and 23%, respectively), but not at Time 0. For subfertile stallions, the percentage of AI spermatozoa was not affected by ionophore treatment (P>0.05) or incubation period (P>0.05). The results suggest that post-fixation storage in DPBS for up to three days is still representative of the acrosomal competence of the original sample. In addition, spermatozoa stored for 24 hours in an Equitainer? exhibited a small (~6%) but significant decrease in the percentage AI spermatozoa. Storage conditions may therefore, affect acrosomal integrity and contribute to reduced fertility when cooled-semen is used. Subfertile stallions exhibited little response [<11% acrosome reacted (AR)] after 3h of A23187 exposure, while the fertile stallions demonstrated a substantial response (? 36% AR) as soon as 1h after ionophore exposure. This assay diagnosed acrosomal dysfunction in stallions with unexplained subfertility.Item Hyperactivated Motility of Stallion Spermatozoa(2013-12-02) Loux, Shavahn CIn vitro fertilization does not occur readily in the horse. Recent evidence suggests that this is due to failure to initiate hyperactivated motility in vitro; however, little is known about the induction of hyperactivated motility in equine sperm. In mice, hyperactivated motility requires the CatSper channel, a pH-gated calcium channel, therefore we investigated this channel and its related intracellular changes, alkalinization and calcium influx, in equine sperm. Motility was assessed by computer-assisted sperm motility analysis, andchanges in intracellular pH and calcium were determined via the calcium and pH-specific fluorescent probes, BCECF-AM, Fluo3-AMand Fluo4-AM. Additionally, a demembranated sperm model was developed to investigate the direct effect of major regulators of sperm motility on axonemal function. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper, in equine sperm. Hyperactivation was induced by treatment with high-pH medium, procaine and 4-aminopyridine. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. Sperm treated with procaine in calcium-deficient media both maintained motility and underwent hyperactivation, suggesting that extracellular calcium was not required for hyperactivation. CATSPER1 protein was localized to the principal piece of equine sperm on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. Demembranated equine sperm required ATP for reactivated motility, but did not require cAMP. Motility of demembranated equine sperm was not inhibited by elimination of calcium (chelation to below 20 pM). Excess calcium inhibited motility at concentrations lower than those reported in other species. Calcium-inhibited sperm arrested with a straight tail rather than in a curve, as seen with calcium arrest in other species. Hyperactivated-like motility was not induced at any pH or calcium concentration. Equine sperm were not inhibited by cadmium at concentrations that profoundly inhibit motility in demembranated sperm in other species. These findings indicate species-specific differences in calcium regulation of sperm motility which may relate directly to the inefficiency of functional capacitation of equine sperm under standard in vitro conditions.Item Plasma Concentrations of Testosterone, Luteinizing Hormone, and Estrone Sulfate in Stallions Following Hemicastration(2013-11-25) Valdez, RaulHemicastration is a veterinary surgical procedure in stallions and may be needed for removal of a diseased testicle. The effects of hemicastration on the neuroendocrine system and the hormonal response of the remaining testicle are unclear. In this study, blood plasma concentrations of testosterone, luteinizing hormone, and estrone sulfate were assessed following hemicastration. Miniature stallions (n=8) were used in this study and blood was drawn 7 d prior to hemicastration, and 12 h, 48 h, 14 d, 30 d, and 90 d post hemicastration. Blood samples from all stallions were drawn every 15 min (0, 15, 30, 45, 60 min) for 1 h each sampling period. Plasma was analyzed by RIA for concentrations of testosterone, luteinizing hormone, and estrone sulfate. Compared to pre-surgerical concentrations, plasma luteinizing hormone at 12 h, 48 h, 14 d and 60 d were greater (P < 0.05). Compared to 12 h, plasma testosterone values at 48 h, 14 d, and 60 d were higher (P < 0.05). Compared to pre-hemicastration values, plasma concentrations of estrone sulfate were lower (P < 0.05) at all time periods, but tended to increase up to 60 d. After 30 d, stallions were housed together rather than individually creating a harem group. Luteinizing hormone and testosterone values increased dramatically compared to previous time periods following the housing modification. These results provide insight to better understand the hormonal profiles and compensatory response of the remaining testicle following hemicastration.Item Testicular function in normal and poor semen quality stallions(Texas A&M University, 2006-04-12) Bryan, Tina MichelleThe chromosomal location of endocrine genes was established, and relationships between expression of specific endocrine genes and measures of testis function in normal and poor semen quality stallions was assessed. Consensus primer sequences for glucocorticoid receptor (GR) and luteinizing hormone receptor (LHR) were used to screen the CHORI-241 equine bacterial artificial chromosome (BAC) library. The identity of PCR-positive BAC clones was confirmed by sequencing. Verified BACs were mapped to horse metaphase chromosome spreads by fluorescence in situ hybridization (FISH). The BACs containing the GR and LHR were localized by FISH to ECA 14q16-q21 and ECA15q22-q23, respectively. In addition to FISH mapping, the 5000rad horse x hamster radiation hybrid (RH) panel was screened in duplicate. Two-point linkage analysis placed GR 0 cR from LEX047, while LHR was 36.67 cR from TKY011 on ECA14 and ECA15, respectively. Total testicular parenchymal weight, mean daily sperm production (DSP) per gram parenchyma and mean apoptotic rate (406.05 ?? 24.33g vs. 180.01 ?? 34.41g, 15.29 ?? 0.87 vs. 10.24 ?? 1.10, 6.70 ?? 0.88 vs. 14.25 ?? 1.11, respectively) differed (P<0.05) between normal (n=8) and poor semen quality (n=5) stallions. Also, plasma estradiol and inhibin concentrations were higher (P<0.05) in normal stallions than in poor semen quality stallions. Testicular expression of estrogen receptor beta (ER beta), βB inhibin, prolactin receptor (PRLR), growth hormone receptor (GHR) and insulin-like growth factor I receptor (IGF-IR) mRNAs were all lower (P<0.05) in poor semen quality stallions than in normal stallions. The BACs and primers developed in this study will facilitate future investigations of GR and LHR gene structure in the horse as well as providing a resource for physiological investigation of these two genes that are primary regulators of stress responsiveness and fertility. These data add important endocrine genes to the horse cytogenetic map. Also, important hormonal and gene expression changes have been identified in poor semen quality stallions for further investigation.Item The role of testicular germ cell apoptosis during equine spermatogenesis(Texas A&M University, 2007-04-25) Heninger, Noah Leland, IIIApoptosis in testicular germ cells has been demonstrated in many species. Features of apoptosis reported in other species were used to confirm use of the TUNEL assay in stallion testes. Eight stallions with normal testicular size and semen quality were evaluated to determine the germ cell types and stages where apoptosis most commonly occurs. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV and V of the seminiferous epithelial cycle corresponding to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Round and elongated spermatids were labeled less frequently by the TUNEL assay. To examine the relationships between germ cell apoptotic rate and spermatogenic efficiency, seminal traits were assessed to classify stallions into normal or reduced quality semen groups. Apoptotic rates were higher for stages IV-VI and stage VIII seminiferous tubules in stallions with reduced semen quality. Daily sperm production (DSP) per gram and per testis were lower for stallions with reduced semen quality. Regression analyses revealed negative linear relationships for germ cell apoptotic rate with DSP/g, DSP/testis, daily sperm output, progressively motile sperm and morphologically normal sperm in ejaculates. Mean circulating concentrations of inhibin were lower for stallions ejaculating reduced quality semen. Apoptotic rate was negatively correlated with concentrations of inhibin and estradiol-17b and positively correlated with concentrations of LH and FSH. To study germ cell apoptosis and formation of the Sertoli cell barrier during the initiation of spermatogenesis, tubule development was classified based on lumen score. Formation of a seminiferous tubule lumen was consistent with events leading to development of a Sertoli cell barrier. A primary wave of apoptosis removed early differentiating germ cell types prior to the formation of a tubule lumen facilitating both the formation of a tubule lumen and a Sertoli cell barrier. A second wave of apoptosis occurred after the formation of a lumen but before seminiferous tubule cross-sections contained a full complement of germ cells. In conclusion, apoptosis is an essential mechanism during normal spermatogenesis. Apoptosis also accounts for low numbers of normal sperm seen in the ejaculates of some stallions.Item Thermoregulation of the testicle in response to exercise and subsequent effects on seminal characteristics in stallions(2012-07-16) Mawyer, Jeannette DianeStudies performed on stallions have characterized detrimental effects on semen quality resulting from thermal stress by testicular insulation, but few have investigated the effects of exercise-induced increases in core body temperature on stallion semen parameters. To our knowledge, this is the first study that correlates subcutaneous scrotal temperature and stallion spermatozoa quality using a subdermal scrotal thermal sensory device. Stallions were assigned to a non-exercised (non-ex; control; n=4) or exercised (ex; n=4) group. A motorized equine exerciser was used to work stallions 30 min/d for 4 d/wk during a 12-wk period from July through October. Temperatures (subcutaneous scrotal, subcutaneous neck, rectal, and ambient) were recorded before exercise, immediately after exercise, and 60 and 120 min post-exercise. Humidity data were obtained later to determine THI. No deleterious effects were observed from implantation of thermal sensory devices. An interaction of treatment and time (P < 0.0001) was evident for rectal and neck temperatures. The relationship between scrotal and rectal temperatures was highest (rs =0.761), and other correlations existed between scrotal, neck, and ambient temperatures, as well. Mean rectal temperature in the ex group increased 1.9?C (P < 0.0001), while there was a slight increase in scrotal temperature of 0.8?C (P > 0.05) from 0 min to 22 min. Although an increase in core body temperature was successfully induced by exercise protocol, scrotal temperatures were not significantly affected, and no treatment effects were found in any of the semen parameters measured (P > 0.05). Therefore, no significant changes in fresh or cooled semen parameters resulted from exercise or increases in core body temperature resulting from exercise protocol used in this study. Results of this study indicate that thermal sensory devices are a safe and effective way to measure subcutaneous scrotal and neck temperatures. Although an increase in core body temperature was successfully induced by exercise protocol, scrotal temperatures were not significantly affected, indicating efficient thermoregulation of the testes by the scrotum. Since the testes experienced no significant thermal insult during the exercise protocol, no significant changes in fresh or cooled semen parameters were evident as a result of exercise or elevated core temperature.