Assessment of <i>Pisum sativum</i> agglutinin in identifying acrosomal damage in stallion spermatozoa

Abstract

The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.