COMPARATIVE VIABILITY OF BOVINE SPERM FROZEN ON A CRYOMICROSCOPE OR IN STRAWS

The accuracy and repeatability of freezing rates and effects of evapor ation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as asses sed by using combinations of fluorescent stains and flow cytometry, wa s used in evaluating protocols for freezing spermatozoa on the cryomic roscope. Semen was diluted in Test-yolk (20%) extender containing 7% g lycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitro gen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicr oscope. Thawed samples were diluted with Hepes buffered medium contain ing 0.1% bovine serum albumin (BSA) and stained with either carboxymet hylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with p ropidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (s tained with CMFDA or SYBR-14), which were classified as plasma membran e-intact and 2) spermatozoa with intense red fluorescence, (stained wi th PI), which were classified as plasma membrane-damaged. Samples froz en using the cryomicroscope contained 29 and 26% plasma membrane-intac t (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. C ryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane-intact sperm cells, while spermatozoa frozen in 0.25- ml straws resulted in 34 and 31% PMI sperm cells for CWFDA and SYBR-14 , respectively. No significant difference was observed (P>0.05) for PM I spermatozoa stained with either CMFDA or SYBR-14. In addition, the a bility to recover spermatozoa after freezing on the cryomicroscope est ablishes the Linkam BCS 196 as a useful tool for the study of sperm ce ll cryopreservation. (C) 1997 by Elsevier Science Inc.