Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium

Abstract

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen-thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l-1 and 5 mmol caffeine l-1. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml-1, more penetrated oocytes were observed 14 h after insemination with 7-12 x 106 cells ml-1 than with 4-5 x 106 cells ml-1 and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO3 concentration but maintaining the sperm concentration at 8 x 106 cells ml-1 resulted in significantly more oocytes being penetrated in media containing 45.83-50.25 than 37.0-41.42 mmol NaHCO3 l-1; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l-1, the inclusion of PVA at 0-5 mg ml-1 had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour. Smaller changes in the distribution of CTC patterns occurred during the later stages, suggesting that the rapidly responding cells were non-fertilizing, owing to damage by freeze-thawing, and that the fertilizing spermatozoa were drawn from the remaining pool of cells which underwent capacitation more slowly. This is the first report indicating that capacitation of frozen-thawed ejaculated boar spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, protein-free medium.