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Osmotic Tolerance Limits and Effects of Cryoprotectants on Motility of Bovine Spermatozoa¹

^a Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705 ^b Comparative Medicine Center, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211

This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen from beef bulls was collected by electroejaculation and extended 1:3 in TL-Hepes containing 100 µg/ml pyruvate and 6 mg/ml BSA. In solutions of 150–1200 mOsmolal (mOsm), bull spermatozoa behaved as linear osmometers (r² = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 µm³. Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270–360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92–103% of their isosmotic cell volume. Motility following a one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively, compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol as a CPA.

¹ This work was supported, in part, by the Cryobiology Research Institute and the U.S. Department of Agriculture (USDA). Mention of a trade name or proprietary product does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of others mentioned.

² Correspondence: J.K. Critser, Comparative Medicine Center, College of Veterinary Medicine, 1600 East Rollins Road, University of Missouri-Columbia, Columbia, MO 65211. FAX: 573 848 7521; critserj{at}missouri.edu

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