44 Boar epididymal semen variability in the sperm cryotolerance after cooling in 18°C at various holding times

This study investigated boar epididymal semen variability in the quality of pre-freeze and post-thaw semen cooled at 18°C at various holding times (0, 3, 6, 9, 12, 24, 48 h). A total of 50 testes of heterogeneous boars were collected (five testes per day) from the local abattoir (Molare Abattoir, Olifantsfontein, South Africa) and transported to the laboratory at 5°C within 30 min after slaughter. The testes were further stored at 5°C for an additional 2 h before being processed. Following storage, the cauda epididymides were removed from the testes and cleaned, and superficial blood vessels were punctured so that most of the blood could be wiped off. The slicing method was used to retrieve the semen from the cauda epididymides. Retrieved semen samples were diluted with a Beltsville thawing solution (BTS) extender. Diluted semen samples retrieved from five cauda epididymides in each day of collection were pooled in 50-mL centrifuge tubes (Whitehead Scientific). Diluted semen was cooled at 18°C for 48 h and frozen at different holding times (0, 3, 6, 9, 12, 24, 48 h). Following each holding time, cooled semen was resuspended with Fraction A (20% egg yolk + BTS) of the freezing extender and stored at 5°C for 45 min. Semen was further be diluted with Fraction B (3% glycerol + 20% egg yolk + BTS) of the freezing extender and loaded into 0.25-mL straws. Semen straws were then frozen with the aid of liquid nitrogen vapor for 20 min and transferred into the liquid nitrogen tank until further analysis. Thawing was accomplished by immersing the semen straws in warm (37°C) water for 1 min. The sperm motility was then evaluated using a Sperm class Analyzer® system (Microptic S.L.). The data were analyzed with the use of ANOVA. Greater than 70% sperm total motility was recorded from the semen cooled up to 24 h at 18°C before freezing (P < 0.05). Whereas, only greater than 70% sperm progressive motility was recorded from the semen cooled up to 6 h (P < 0.05). The highest post-thawed total (85.9%), progressive (60.3%), and rapid (33.2%) motilities were recorded from the semen samples cooled for 3 h before freezing (P < 0.05). Boar epididymal sperm survived up to 48 h of semen cooling with total sperm motility > 50% before freezing (P < 0.05); whereas, <40% post-thawed sperm total motility was recorded from the semen samples frozen after 9–24 h of cooling at 18°C (P < 0.05). Semen frozen immediately after retrieval (0 h) recorded less cryoresistance with lower post-thawed sperm total (27.2%), progressive (12.2%), and rapid (20.5%) motility (P < 0.05). The results from the present study show that semen cannot be cryopreserved immediately after retrieval. A holding time of 3 h improved the cryoresistance of the sperm cooled at 18°C. Therefore, these data suggest that cryopreservation of sperm from boar epididymal semen held at 18°C for 3 h can be used for conserving semen.