Comparison of Flow Cytometric Analysis and Eosin-Nigrosin Staining Methods for Determining some Morphological Characteristics of Bull Epididymal Spermatozoa

Abstract

The aim of this study is to investigate necrosis and apoptosis in epididymal bull spermatozoa before freezing and after thawing using the flow cytometric method and to compare this with eosin-nigrosin dyeing, which is the conventional method used in assessing of spermatozoa. The testicles from fourteen bulls at local slaughterhouse were used for this study. The proportions of live spermatozoa, total apoptotic, necrotic and early necrotic spermatozoa levels were observed via flow cytometry. Annexin V/ PI fluoruscence dyeing was used to investigate the proprotions of apoptotic, necrotic, early necrotic and live spermatozoa for flow cytometry. The proportion of dead spermatozoa and protoplasmic droplets were determined using the eosin-nigrosin conventional dyeing method in fresh and frozen-thawed spermatozoa. The average dead spermatozoa count with flow cytometry was less than with the eosin-nigrosin method (P < 0.05). Some morphological characteristics such as protoplasmic droplets could be determined with the eosin-nigrosin method; however, sperm subpopulations entering the death process (apoptotic, necrotic and early necrotic) could be defined clearly only with the flow cytometric method. As a result, combination of eosin-nigrosin dyeing method and flow cytometric analysis of sperm morphological evaluation could give better results of bull epididymal semen in comparison to eosin-nigrosin dyeing method alone.