FROZEN-THAWED BOVINE SPERMATOZOA DILUTED BY SLOW OR RAPID DILUTION METHOD: MEASUREMENTS ON OCCURRENCE OF OSMOTIC SHOCK AND SPERM VIABILITY zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR J.R. Correa and P.M. Zavos Department of Animal Science, University of Kentucky Lexington, KY, 40546, USA Received for publication: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP March Z, 1995 Accepted: May 3, 1995 ABSTRACT This study was undertaken to investigate the occurrence of osmotic shock, sperm viability and membrane functional status of frozen-thawed bovine spermatozoa during a short-term incubation period (2 h) in vitro after dilution by 2 methods. Frozen semen from 10 bulls (OS-ml plastic straws, 7% glycerol) was thawed and diluted by slow or rapid dilution method with Ham’s F-10 medium containing 0 or 7% glycerol and assessed for sperm motion parameters, percentage of spermatozoa with coiled tails and reactivity to the hypoosmotic swelling (HOS; percentage of spermatozoa swelling) test at 60 min intervals during a 2 h incubation period (37’C). Post-thaw sperm viability, as reflected by percentage and grade of motility (0 to 4) did not differ between the 2 dilution methods (P~0.05) at the beginning of incubation (Time 0). However, differences were apparent (P<O.OS) as the incubation time increased. Slow dilution with medium containing 0% glycerol caused less increase (PcO.05) in percentage of spermatozoa with coiled tails; Moreover, these spermatozoa showed greater reactivity to the HOS test. When contrasting slow vs rapid dilution methods, the occurrence of osmotic shock was less frequent, and response to the HOS test was greater for spermatozoa diluted slowly, regardless of the glycerol content of the incubation medium. Rapid deglycerolization of frozen-thawed bovine spermatozoa in a single step, induces damage which is not detected on the basis of spermatozoa1 motility but is clearly evident after several hours of incubation by using the HOS test to detect damage. Key words: spermatozoa, glycerol, dilution, osmotic shock, viability INTRODUCTION Current success in the cryopreservation of mammalian sperm is attributable, in large part, to the fortuitous observation made by Polge in 1949 that glycerol carries some unique Acknowledgements The authors wish to express their appreciation to the American Breeder Services for their support and for providing the semen for this research effort. Theriogenology 44:963-971, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0093-691 X/95/$1 0.00 SSDI 0093-691X(95)00283-9