SPERM MEMBRANE FUNCTIONAL INTEGRITY AND RESPONSE OF FROZEN-THAWED BOVINE SPERMATOZOA DURING THE HYPOOSMOTIC SWELLING TEST INCUBATION AT VARYING TEMPERATURES

The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at va rying temperatures during and after thawing by exposing the spermatozo a to standardized hypoosmotic conditions. The hypoosmotic swelling (HO S) test was employed to measure changes in sperm membrane functional s tatus and permeability. Frozen specimens (from 5 bulls) were thawed at 37 degrees C for 10 sec and transferred to a water bath at 37 (Aliquo t 1), 21 (Aliquot 2) or 5 degrees C (Aliquot 3) to complete thawing(1 to 2 min). The specimens were maintained and processed at these temper atures for additional 5 to 10 min. Specimens were slowly diluted 1:1 ( v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml o f a 100 mOsm/L HOS diluent. The following treatments were performed: 1 ) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 degrees C for 5 min; 2) Aliquot 2, specimens were incubated in HOS so lutions at 21 or 37 degrees C for 5 min; and 3) Aliquot 3, specimens w ere incubated in HOS solutions at 5 or 37 degrees C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min i ntervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specim ens processed at temperatures below 37 degrees C was higher when sampl es were incubated in HOS diluents at 37 degrees C. This finding indica tes that the potential for sperm swelling (measurement of sperm membra ne functional status) can be maintained when spermatozoa are processed at temperatures below 37 degrees C. The highest response to the HOS t est was observed in spermatozoa processed at 21 degrees C and incubate d in a HOS solution at 37 degrees C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 degrees C throughout. Thawing of spermatozoa at 37 degrees C, follo wed by processing at 21 degrees C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of froze n-thawed bovine spermatozoa. (C) 1997 by Elsevier Science Inc.