Artifactual disruption of sperm midpiece in extended stallion semen

Abstract

Microscopic examination of stallion sperm morphology provides a useful criterion for assessing semen quality and reproductive capabilities of a stallion. In previous investigations, personnel in our laboratory noted an apparent artifactual defect of the sperm midpiece when stallion semen was mixed with various commercial extenders prior to formalin fixation for sperm morphologic analysis. We coined this defect “stippled midpiece.” Objective was to determine the etiology of this previously unreported defect by investigating the induced morphologic change using light and electron microscopic techniques. Sixteen diluents/extenders in 2 sperm exposure times were compared prior to fixation for their effect on the incidence of stippled midpieces. Raw semen was used as a control. The abnormality was most prevalent (p < 0.05) when semen was mixed with diluents containing casein, or milk and egg yolk. In contrast, diluents containing albumin or no protein were similar (p > 0.05) and exhibited the lowest (p < 0.05) incidence of stippled midpieces among diluent groups. Diluents containing protein exhibited a higher (p < 0.05) incidence of stippled midpieces compared to raw semen. Intrinsic sperm quality was not correlated with the incidence of stippled midpieces when semen was formalin-preserved in raw form. However, sperm quality was correlated with the incidence of stippled midpieces when semen was mixed with certain diluents prior to formalin fixation. This relationship was most pronounced when diluent contained milk or casein. Transmission electron microscopic findings corroborated that of light microscopy for the location and structural features of the stippled midpieces.