ASSESSMENT OF RAM SPERM MEMBRANE INTEGRITY FOLLOWING DIFFERENT THAWING PROCEDURES

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The seme n was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws(approx. 100 mill./straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the followi ng thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluoro chromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), t he hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane int egrity. Significant differences between thawing treatments were found for post-thaw motility (P<0.05) and membrane integrity (P<0.01), and v ariation among rams was statistically significant. Post-thaw sperm mot ility as well as the percentage of spermatozoa showing intact membrane s were significantly higher (P<0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1% , and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corre sponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec in stead of 70 degrees C for 5 sec without further reducing sperm motilit y or membrane integrity. This lower thawing temperature would facilita te the widespread use of frozen/thawed ram semen under farm conditions in Sweden. (C) 1997 by Elsevier Science Inc.