Browsing by Subject "semen"
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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 Pregnancy Rates in Mares Inseminated with 0.5 or 1 Million Sperm Using Hysteroscopic or Transrectally Guided Deep-Horn Insemination Techniques(2013-11-08) Hayden, Shelby ShalenePlacement of sperm deep in the equine uterine horn allows fewer sperm to be inseminated while maintaining acceptable fertility, and has been promoted for use in circumstances when fertility would be expected to be low if standard insemination were used (e.g. semen from a subfertile stallion, or frozen-thawed semen). Two main deep- horn insemination techniques, transrectally guided (TRG) and hysteroscopic (HYS) insemination, have been developed for this purpose; however, there is some controversy regarding their comparative efficacy. This study was conducted to compare pregnancy rates when mares were inseminated by TRG or HYS, utilizing sperm numbers approaching and under the threshold for maximal fertility, resulting in reduced fertility. Pregnancy rates were not different between HYS and TRG techniques when 1 x 106 or 0.5 x 106 sperm were inseminated. Combined pregnancy rates for the two techniques were also not different. Pregnancy rates using a subthreshold number of sperm were not significantly affected by a deep-horn insemination technique. Dilution of semen to less than 20 x 106 sperm/mL has been reported to decrease semen quality in multiple species, a phenomenon known as the semen ?dilution effect.? The sperm concentrations utilized in Experiment 1 were 5 and 2.5 x 106/mL (1 and 0.5 x 106 sperm doses, respectively). This experiment was performed to evaluate whether the lower pregnancy rates obtained with 0.5 x 106 sperm was due to lower quality plasma membrane integrity (PMI) and sperm motion characteristics (TMOT, PMOT, VCL, VAP, VSL, STR). Treatments evaluated included 2.5 x 106 sperm/mL with the addition of 0, 7.5, and 25% seminal plasma, 30 x 106 sperm/mL, and 3:1 extender: semen. The 2.5 x 106 sperm/mL treatments have lower initial PMI, TMOT, and PMOT, but they maintain their initial quality following 24 and 48 h of cool-storage. The sperm velocity and straightness parameters suggest that sperm swim faster but have a more circular pattern as seminal plasma increases to 25% at a given concentration. Based on the findings from this experiment, the semen ?dilution effect? may not significantly alter stallion sperm characteristics when a commercially-available semen extender is used for semen dilution.Item Recovery and evaluation of somatic cells from ovine and bovine semen for use in nuclear transfer(2009-05-15) Liu, JieSomatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.