Abstract
Whole seminal plasma from ram semen collected before and after vasectomy was separated into 2 fractions, supernatant and pellet of vesicles, and their protein profiles characterized by one-dimensional (1D) gel electrophoresis. The effects of autologous whole seminal plasma and these fractions on motility characteristics (assessed subjectively and by computer-assisted sperm analysis), membrane status (assessed by chlortetracycline staining patterns), and in vitro fertility (assessed by fertilization success and timing of fertilization events) of washed frozen-thawed ram spermatozoa were studied. Regardless of vasectomy, whole seminal plasma and supernatant displayed similar protein patterns. These fractions, when included in the postthaw buffer, improved the motility characteristics (59.6% +/- 6.21% and 39.6% +/- 6.21% vs 31.7% +/- 6.46% and 15.5% +/- 6.46% total motility) and membrane integrity (36.6% +/- 8.52% and 31.2% +/- 8.19% vs 30.3% +/- 11.49% and 21.6% +/- 10.28% B staining pattern [characteristic of capacitated acrosome-intact cells] for whole seminal plasma and supernatant vs control at 3 and 6 hours of postthaw incubation, respectively) of frozen-thawed spermatozoa and improved their ability to fertilize in vitro-matured oocytes compared with control buffer without seminal plasma fractions (25.3%, 47.4%, and 37.4% vs 12.3%, 20.2%, and 20.5% oocytes fertilized for spermatozoa incubated with supernatant vs control at 2, 6, and 18 hours after insemination, respectively). Vesicles were absent from semen collected after vasectomy. Pellets of vesicles collected before vasectomy had no effect on spermatozoa at their normal protein concentration but marginally improved both motility characteristics and in vitro fertility, possibly due to contamination from supernatant proteins, when their concentration in the postthaw medium was increased by threefold. It was concluded that the vesicle-free supernatant fraction of seminal plasma, but not the seminal plasma membrane vesicles, improved the function and fertility of frozen-thawed ram spermatozoa when added to the postthaw medium.
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