Zona pellucida penetration assay for capacitation of bovine sperm

Abstract

In vitro capacitation and in vitro zona penetration systems are described for bovine spermatozoa. The criterion for capacitation was penetration by sperm of either 1) the zona pellucida of dead bovine oocytes, or 2) live, intact oocytes in which subsequent development was observed. Sperm were washed free of seminal plasma by centrifugation and incubated in a modified Tyrode's medium (TALP) in a 5% CO₂-air atmosphere at 38°C. Dead bovine oocytes were obtained from > 1 mm follicles from ovaries stored at −20°C. Spermatozoa at a final concentration of 10⁷ ml were coincubated with the oocytes for 3 hr after capacitation treatments. Initial experiments established that: 1) dead bovine oocytes can be penetrated by bovine sperm capacitated in the rabbit reproductive tract; 2) oocytes should be used for the penetration test within 24 hr of thawing; 3) oocytes could be stored for 24 hr at 4°C after penetration before evaluation; and 4) penetration of zonae pellucidae of dead bovine oocytes by sperm is species specific. The percentages of dead oocytes penetrated after incubating frozen-thawed sperm for 0, 6, 12, 24, and 30 hr prior to incubation with oocytes were 17, 24, 34, 10, and 0% respectively; the corresponding percentages for fresh semen were 1, 2, 5, 42, and 25%. Addition of 1% bovine seminal plasma to suspensions of washed, capacitated sperm decreased oocyte penetration percentages from 48% for the control (no seminal plasma) to 23%. Spermatozoa capacitated in vitro for 24 hr fertilized 15 of 22 ovulated oocytes (68%) from superovulated cows. Three embryos developed to the morula stage, but no pregnancy resulted after transfer of these embryos to bovine recipients. The present results provide the basis for a reliable (interassay C.V. = 13.4%) in vitro assay system for capacitation based on sperm penetration of the zona pellucida of dead bovine oocytes.