Examination of the binding ability of bovine spermatozoa to the zona pellucida as an indicator of fertility

Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R-2 = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R-2 = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.