A technique for preserving retained distal cytoplasmic droplets in situ for immunofluorescence evaluation of ejaculated porcine spermatozoa.

Cytoplasmic droplets (CD) associated with mammalian sperm have traditionally been investigated after isolation from ejaculated sperm cells, or in fixed tissue sections from the epididymides. Many of the current techniques for preparing spermatozoa for immunofluorescence assay (IFA) induce separation of distal cytoplasmic droplets, particularly when membrane permeabilization is required. This article describes a technique capable of maintaining distal cytoplasmic droplets in situ on ejaculated porcine spermatozoa throughout the process of permeabilization and immunostaining. Key steps in this technique include fixation with 0.2%, glutaraldehyde, permeabilization with 0.05% TX-100, and microtube incubations for IFA. Utilizing glutaraldehyde fixation, this technique yielded a mean retention rate of 88% for distal cytoplasmic droplets on ejaculated porcine spermatozoa.