ARS | Publication request: THE POST-THAW QUALITY OF RAM SPERM HELD FOR 0 TO 48 HOURS AT 5°C PRIOR TO CRYOPRESERVATION

Submitted to: Animal Reproduction Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 6, 2005
Publication Date: June 1, 2006
Citation: Purdy, P.H. 2006. The post-thaw quality of ram sperm held for 0 to 48 hours at 5°c prior to cryopreservation. Animal Reproduction Sciences. 93:114-123.

Interpretive Summary: The effects of holding diluted ram semen at 5ºC for up to 48 hours prior to cryopreservation were investigated. Semen from 6 rams was collected in the autumn and again from 6 different rams in the spring. The samples were diluted in a one-step Tris-egg yolk-glycerol media and cooled to 5°C over 2 hours and maintained at 5°C for the duration of the experiments. Aliquots were loaded into semen straws at 0, 24 or 48 hours after cooling, frozen in liquid nitrogen vapor, and plunged into liquid nitrogen for storage. After thawing, samples frozen at times 0, 24, or 48 h in the autumn exhibited similar percentages of sperm motility, membrane integrities, and mean number of sperm bound to hen’s egg perivitelline membranes. Likewise, ram sperm collected in the spring and frozen at times 0, 24 and 48 h after cooling had similar motilities and membrane integrities, but the 0 h freeze time had significantly less sperm bound to a hen’s egg perivitelline membrane compared to the 48 h freeze time. The 24 h freeze time was not significantly different from either the 0 or 48 h freeze time. Comparison of freeze times across seasons resulted in no differences in sperm motilities or plasma membrane integrity, but the spring season had significantly more live acrosome reacted sperm at each freeze time and more sperm bound at the 48 h freeze time compared with the autumn season. These results suggest that ram sperm can be held at 5ºC for up to 48 hours prior to freezing with no injurious effects on motility, membrane integrity, or fertility potential as indicated by membrane binding ability, regardless of season.

Technical Abstract: The effects of holding diluted ram semen at 5ºC for up to 48 hours prior to cryopreservation were investigated. Semen from 6 rams was collected by electro-ejaculation in the autumn and again from 6 different rams in the spring. Sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis (CASA), respectively. Samples were diluted at 23ºC to 400 x 106 cells per mL in a one-step, Tris-egg yolk-glycerol medium and cooled to 5°C over 2 hours. The samples were maintained at 5°C for the duration of the experiments. Aliquots were loaded into 0.5 mL French straws at 0, 24 or 48 hours after cooling, frozen in liquid nitrogen vapor for 12 to 13 minutes, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, samples frozen at times 0, 24, or 48 h in the autumn exhibited similar percentages of motility (29, 31, 36%, respectively), progressive motility (16, 15, 17%), plasma membrane integrity (28, 35, 29%), live acrosome-reacted cells (0.4, 0.6, 0.8%) and mean number of sperm bound to a hen’s egg perivitelline membranes (155, 177, 106 sperm; P > 0.05) . Likewise, ram sperm collected in the spring and frozen at times 0, 24 and 48 h after cooling had similar (P > 0.05) total motility (21, 25, 20%), progressive motility (14, 15, 11%), plasma membrane integrity (26, 33, 31%) and live acrosome reacted cells (3.7, 3.5, 3.2%). The 0 h freeze time had significantly less sperm bound to a hen’s egg perivitelline membrane compared to the 48 h freeze time (250 and 470 sperm) but the 24 h freeze time was not significantly different from either the 0 or 48 h freeze time (281 sperm; P < 0.05). Comparison of freeze times across seasons resulted in no differences in total motility, progressive motility or plasma membrane integrity (P > 0.05) but the spring season had significantly more live acrosome-reacted sperm at each freeze time and more sperm bound at the 48 h freeze time compared with the autumn season (P < 0.05). These results suggest that ram sperm can be held at 5ºC for up to 48 hours prior to freezing with no injurious effects on motility, membrane integrity, or fertility potential as indicated by membrane binding-ability, regardless of season.