An Investigative Immunoassay Targeting Two Osteopontin Epitopes in Boar Semen

Abstract

The identification of superior sire is largely dependent on the semen quality evaluations, currently through computer-aided sperm class analyser (CASA). Immunoassays present a viable method for analysis of proteins associated with fertility, such as osteopontin. We have targeted osteopontin at an upstream epitope at position 249 - 267 amino acids and a downstream epitope at position 59 - 74 amino acids of full osteopontin protein in raw, BTS, Kobidil, Tris and Citrate liquid preserved Large white and Kolbroek boar semen. The Genetool analysis software was used to quantify the amount of osteopontin detected. Three boars per breed were used and experiments were repeated five times. Our results revealed that the downstream 15 amino acid epitope was more sensitive to osteopontin antibody. Between total motility and osteopontin, Kolbroek boar semen preserved in Kobidil, Citrate, BTS and Tris extenders revealed positive correlations of 0.564, 0.471, -0.960 and 0.330, respectively. Large white boars semen showed correlations of -0.043, 0.655, 0.778, and 0.202 Kobidil, Citrate, BTS and Tris, respectively. For rapid motility and osteopontin, Kolbroek boar semen revealed positive correlations of 0.842, 0.601, 0.164 and 0.202, respectively while large white boars semen showed correlations of -0.909, -0.144, -0.210, and 0.089, respectively. Only BTS and Kobidil showed high negative correlations between osteopontin and viability in Kolbroek (-0.975) and Large white (-0.746) boar semen, respectively. No significant correlations with osteopontin were observed when Tris extender was used in both breeds. In conclusion, Kolbroek boar semen requires osteopontin in Kobodil extender while large white boar semen requires osteopontin in BTS extender.