Removing seminal plasma improves sex-sorting of bovine sperm

Experiments for this thesis evaluated how characteristics of bovine ejaculates affect efficiency of sorting X- from Y-chromosome bearing sperm by flow cytometry/cell sorting, and further, document that removal of seminal plasma improves efficacy of sex-sorting bovine sperm. In Experiment I, ejaculates were collected by artificial vagina, two each from 10 bulls with an average of one hour between collections. Effects of seminal plasma originating from bulls different from those whose sperm were sorted was evaluated in Experiment II. Semen collection and initial analysis were performed as in Experiment I and for all successive experiments. Seminal plasma from ejaculates of 6 bulls (3 that sorted well; 3 that sorted poorly) used in Experiment I was used with sperm collected for Experiment II as well as seminal plasma from sperm donors.;Experiment III investigated effects of adding bovine serum albumin (BSA) during staining with Hoechst 33342 using semen from 10 bulls. BSA was added at 0, 0.3 or 0.9% to staining TALP with either 0 or 10% seminal plasma. Although BSA has been shown to be beneficial to sperm, there was a possibility that BSA would bind Hoechst 33342, thereby reducing sorting efficiency. There was no evidence of either effect; however, sperm with 0% seminal plasma had higher % live oriented cells (65%) than sperm incubated with 10% seminal plasma (61%; p<0.05). In addition, samples containing 0% seminal plasma had only 16% membrane-impaired sperm compared to 20% for samples containg 10% seminal plasma (p<0.05).;Experiment IV explored current industry dogma that ejaculates with initial sperm concentrations of <109 sperm per ml sort poorly. Seminal plasma volume was added or removed to create sperm concentrations of 0.7, 1.4 and 2.1x109 sperm per ml, replicated with 10 ejaculates. Samples were sorted at 0h or stored at 16°C and sorted at 4h. While sorting parameters and sperm quality deteriorated from 0 to 4h, there was no interaction between storage time and sperm concentration; means presented are averaged over 0 and 4h. The % live-oriented sperm was higher for samples stored at 2.1x109 sperm per ml (66.4%) than 1.4x109 sperm per ml (64.1%) or 0.7x109 sperm per ml (62.7%; p<0.01). The % membrane-impaired sperm was lower for samples containing 2.1x10 9 sperm per ml (15.1%) than 1.4x109 per ml (17.0%) or 0.7x109 per ml (18.0%; p<0.01). The X sort rate was lower for samples of 0.7x109 per ml (3.45x103 sperm per sec) than for samples containing 1.4x109 and 2.1x10 9 sperm per ml (3.85 and 3.94x103 sperm per sec; p<0.05).;For Experiment V, sperm from 10 bulls were diluted to 0.7, 1.4 and 2.1x10 9 sperm per ml using staining TALP containing 0 or 10% seminal plasma and stored for 1h. After bulk sorting, sperm were cryopreserved in 20% egg yolk TRIS extender with 6% glycerol. Post-thaw analysis was performed by flow cytometry for % membrane compromised sperm, and by computer assisted sperm analysis for motility. Samples containing 0% seminal plasma had greater % live-oriented cells (54.0%) than samples containing 10% seminal plasma (50.3%). Samples with 0% seminal plasma also had lower % membrane-impaired sperm than samples containing 10% seminal plasma (18.8% vs. 22.2%; p<0.01). Post-thaw motility was higher for sperm incubated with 0% seminal plasma (41.0%) than for sperm incubated with 10% seminal plasma (35.5%; p<0.05). No differences were observed for sperm stored at 0.7, 1.4 or 2.1x109 when all samples contained 0 or 10% seminal plasma during storage and staining. Therefore, when combined with Experiment IV, seminal plasma, not initial sperm concentration, impairs sort efficiency.;Experiment VI evaluated various combinations of sperm, seminal plasma, and Hoechst 33342 concentrations during staining. Two ejaculates were collected from 11 bulls on different days. After seminal plasma removal, sperm were re-suspended in TALP at 160 or 240x106 sperm per ml with 0 or 10% seminal plasma. Hoechst 33342 was added for final concentrations of 49, 65 or 81muM. Staining sperm with 0% seminal plasma resulted in higher % live-oriented cells (57.4% vs. 53.7%) and higher sort rates (3.60x10 3sperm per sec vs. 3.28x103sperm per sec) compared to sperm in 10% seminal plasma (both p<0.01). There was an interaction between sperm concentration and H33342 concentration for ability to separate X and Y populations and for sort rate. Using 65muM H33342 was sufficient to optimally stain 160x106 sperm per ml, while 240x10 6 sperm required 81muM H33342 to reach similar degrees of separation (peak to valley ratio) and sort rates. (Abstract shortened by UMI.)...