Removing Seminal Plasma Improves Sex-Sorting of Bovine Sperm.

Abstract

REMOVING SEMINAL PLASMA IMPROVES SEX-SORTING OF BOVINE SPERMExperiments 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 sexsorting bovine sperm. In Experiment I, ejaculates were collected by artificial vagina, two each from 10 bulls with an average of one hour between collections. Semen was centrifuged to separate sperm from seminal plasma, and then sperm were re-diluted to 160x10 6 sperm per ml with staining TALP (Tyrode's albumin, lactate, pyruvate) and 0, 5, 10 or 20% seminal plasma from the same ejaculate or reciprocally from first/second ejaculates. Following incubation with Hoechst 33342, sperm were sorted and analyzed with a flow cytometer/cell sorter. The % live-oriented sperm was higher for treatments with 0% seminal plasma (64.4%) than 5 (59.6%), 10 (59.0%) and 20% (57.8%) seminal plasma (p<0.01). The % live-oriented sperm was higher for second (63.0%) than first ejaculates (56.2%). Sort rate was higher for samples with 0% seminal plasma (p<0.05).The percentages of membrane-impaired sperm were lower for 0% (16.5%) than 5 (21.9%), 10 (23.6%) or 20% (23.4%) seminal plasma (p<0.003), and for second iii ejaculates (18.2%) compared to first ejaculates (25.9%). Whether seminal plasma was from first versus second ejaculates had no effect (p>0.1). 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. Sperm were stained with Hoechst 33342 as in Experiment I, but with 10% seminal plasma in staining TALP.Only seminal plasma from one bull resulted in differences for any sorting parameters; 31% of sperm were membrane-impaired with seminal plasma from this bull compared to 16-19% for seminal plasma from other bulls (p<0.05). Sort rate was decreased (3.02x10 3 sperm per sec) compared to a range of 3.64-3.97x10 3 sperm per sec with the other seminal plasmas (p<0.05). This may have been due to contamination of the original semen sample, since this effect was only seen with one of six bulls.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 conta...

Seminal Plasma and its Effect on Ruminant Spermatozoa During Processing

“…Maxwell et al (1996) found the presence of seminal plasma in simple sorting diluents had no effect during Hoechst staining, but reported supplementation of the collection medium to be beneficial. However, more recent studies (on high speed sorters and in more complex media) reported decreases in the number of viable and correctly orientated spermatozoa when seminal plasma (5-20% v ⁄ v) was present in the staining medium (Burroughs 2011), which caused a reduction in the speed at which spermatozoa could be sex-sorted. In addition, this detrimental effect of seminal plasma prior to sorting was evident after sorting (where membranedamaged sperm are removed) and freezing, indicating a persistent destabilization of the sperm membrane.…”