Assessment of ram sperm membrane integrity following different thawing procedures

Abstract

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 semen 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 following thawing procedures: 1) 70 °C, 5 sec; 2) 50 °C, 9 sec and 3) 35 °C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 °C than for those thawed at 35 °C (67.0 ± 1.1 and 63.0 ± 1.1%, and 50.5 ± 1.5 and 41.7 ± 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 °C and 50 °C thawing were compared. It was concluded that sperm can be thawed at 50 °C for 9 sec instead of 70 °C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.