FLOW CYTOMETRIC EVALUATION OF CRYOPRESERVED BOVINE SPERMATOZOA PROCESSED USING A NEW ANTIBIOTIC COMBINATION

Bovine spermatozoa cryopreserved in media containing two combinations of antibiotics were analyzed using a FACS analyzer and an EPICS V flow cytometer. Duplicate samples from 10 bulls with information on nonreturn rates were examined. Semen was processed using either penicillin-streptomycin-polymyxin-lincospectin (Regimen I) or gentamicin-tylosin-lincospectin (Regimen II). Spermatozoa were thawed at 37°C and incubated for 0, 1.5, and 3.0 h in 10 mM HEPES buffer at 39°C. Aliquots were then stained using a combination of 5(and-6)-carboxy-4′,5′ dimethyl fluorescein diacetate and hydroethidine. Flow cytometric analysis of the stained samples identified five fluorescent populations for the FACS analyzer and three for the EPICS V. There were no significant differences in the number of spermatozoa within similar fluorescent populations between antibiotic Regimens I and II for all incubation times with one exception. One spermatozoal population (FACS Analyzer), after 1.5 h of incubation, contained more cells with decreased viability (P<0.05). All single and combined populations identified as being related to nonreturn rates were similar between antibiotic groups with one exception. The treatment of spermatozoa with the new antibiotic regimen for the control of microorganisms did not alter spermatozoal viability or fertilizing ability. © 1990.