In vitro fertilization in the sheep: effect of elevated calcium concentration at insemination

Oocytes (n = 273), collected from superovulated ewes, were inseminated with in vitro capacitated spermatozoa from four rams [Crozet et al., 1987]. In each experiment, parallel insemination was performed using aliquants from a single ejaculate in either our standard fertilization medium DM-H-SS (a modification of Brackett's defined medium, buffered with HEPES and containing 20% v/v sheep serum) or in the same medium supplemented with calcium lactate (DM-H-SS + Ca). The measured total calcium concentrations were Ca++ T = 2.74 mM in DM-H-SS medium and Ca++ T = 8.74 mM in DM-H-SS - Ca; the ratio of free to total calcium in DM-H-SS was Ca++ F/Ca++ T = 0.85. Fertilization was assessed at 17 hours postinsemination. Variations in the ejaculates were observed for each of the four rams tested. When DM-H-SS--CA was used, the percentages of fertilized (75% vs. 50%) and monospermic (58% vs. 41% eggs were significantly enhanced compared with use of DM-H-SS. No improvement was observed in control medium DM-H-SS + lac containing neutralized lactic acid. Supplementing the fertilization medium with calcium had no apparent effect on the incidence of polyspermy. These experiments show that the fertilization rate achievable in vitro by individual ejaculates from various rams can be increased by raising the calcium concentration in the fertilization medium to a value much higher than that present in tubal fluids from estrous ewes. Extended incubation in such a high calcium concentration is unnecessary for in vitro capacitation of ram spermatozoa.