Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin

Abstract

Bovine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa which were either preincubated for 5-5.5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 micrograms/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1-5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.

Initiation, Persistence, and Cessation of the Series of Intracellular Ca2+ Responses during Fertilization of Bovine Eggs.

“…Sperm were capacitated in two ways: 1) treatment with 10 µM calcium ionophore A23187 for 3 min [13], or 2) treatment with 10 mM caffeine-sodium benzoate (Sigma) and 20 µg/ml heparin (170 USP units/ml; Sigma) for 60 min [14]. After washing the chemicals out, the corresponding sperm suspension 1 or 2 was prepared at the concentration of 5 or 25 10 6 sperm/ml, respectively.…”

Caffeine in Fertilization Medium Is Not Essential for Bovine IVF by Fully Capacitated Spermatozoa

“…Under the anaerobic conditions in the medium covered with mineral oil, the respiration of preincubated spermatozoa introduced into fertilization medium with 5 mM caffeine might be over-stimulated by 5 mM caffeine, and consequently the motility of the spermatozoa may be decreased. In previous studies on bovine in vitro fertilization showing high fertilization rates in fertilization medium with caffeine [1][2][3][4][5][6][13][14][15][16][17][18], the sperm concentrations at the site of fertilization were very high and these high sperm concentrations might have made up for the disadvantage of 5 mM caffeine at fertilization.…”

“…Takahashi and Hanada have reported that caffeine in the medium appears to enhance the capacitating action of I-A [4]. Synergistic effects of caffeine and heparin on IVF have been reported in cattle [5,6]. Furthermore, caffeine in the fertilization medium accelerates sperm penetration in vitro in pigs [7][8][9] and mice [10].…”

Evaluation of laser-assisted lentiviral transgenesis in bovine

“…Unlike in vivo, the cumulus cells are tightly attached to the in vitro matured oocyte (Park et al, 1989). Several studies confirm that removal of cumulus cells strongly decreases developmental capacity (Cox et al, 1993;Zhang et al, 1995 …”