Submitted to: Journal Of Reproduction, Fertility And Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 26, 1996
Publication Date: N/A
Interpretive Summary: Gender preselection technology is based on measuring the DNA in the X and Y-chromosome bearing sperm cell. Development of a practical protocol for field use is dependent on the optimization of the technology. One means of optimizing gender preselection technology is dependent upon increasing the survival of sperm after they pass through the flow cytometer cell sorter. Membrane integrity plays a large role in sperm survival. This study evaluated membrane integrity of sorted sperm. The results showed that the addition of seminal plasma to the sort collection media was helpful to maintaining membrane integrity in boar sperm. Addition of seminal plasma to test-yolk collection media was the most helpful. This information will be used to elucidate complementary factors associated with the maintenance of sperm plasma membrane during flow cytometric sperm sorting.
Technical Abstract: Boar, bull and ram spermatozoa were examined after staining with Hoechst 33342 and flow cytometric sorting in the presence of absence of seminal plasma. The spermatozoa were assessed for viability with flow cytometry using live/dead staining with SYBR-14/PI, membrane integrity using FITC- PSA/PI, and motility and acrosome integrity were estimated by microscopy. Test Yolk and Test Yolk + P were more effective at maintaining motility of boar spermatozoa, and the motility, viability and acrosome integrity of bull spermatozoa, than the other media. More boar spermatozoa had normal apical ridges (NAR), and more bull spermatozoa were live and motile, when the collection meddium contained seminal plasma than in its absence. There was a small but significant reduction in the percentage of live boar spermatozoa during the 4 hr holding after dilution and flow sorting, but holding time had no effect on bull sperm viability. More boar spermatozoa were live when both the extender and the collection medium contained 10 compared with 0, 5 or 20% seminal plasma; motility of spermatozoa was also better when the extender contained 10% seminal plasma, but was unaffected by the concentration of seminal plasma in the collection medium. The presence of 20% seminal plasma in the extender substantially reduced the proportion of live and motile boar spermatozoa after flow sorting. The results of this study indiciate that the in vitro viability and membrane integrity of spermatozoa would be improved if seminal plasma (10%) was routinely included in the BTS and Hepes-BSA staining extenders for boar and ram spermatozoa respectively, when used in preparation for flow cytometric sorting, and if 10% and 50% seminal plasma were included inthe Test-Yolk (2%) collection medium for boar or bull and ram spermatozoa respectively.