Effect of cooling to different sub-zero temperatures on boar sperm cryosurvival

Objective: To compare different cooling temperatures before ice formation on pig sperm quality, before and after cryopreservation. Methods: Semen diluted in BF5 was cooled from 23 °C to 5 °C (1% glycerol, 200 × 106 cells/mL). Sperm were packaged in plastic straws, and maintained at +5 °C per 16 h. 1. Freezing point of diluted spermatozoa was determined by exposing straws to nitrogen vapors. 2. Straws (at +5 °C) were further cooled to −3 °C, −5 °C, and −7 °C, and rewarmed. 3. Straws (at +5 °C) were further cooled to −3 °C and −5 °C, then frozen and stored in liquid nitrogen, and one month later thawed. Progressive motility (PM), viability (Eosin/Nigrosine), plasma membrane functionality (HOST), and acrosome integrity (phase-contrast microscopy) were assessed. Results: 1. Freezing point was −8.2 ± 0.3 (mean ± SEM); one of the ejaculates froze at different temperature from that of the others (P < 0.05). 2. PM (%) was 75%, 71%, 63%, and 40% (P < 0.05); viability (%) was 90%, 89%, 89%, and 81% (P < 0.05); HOST (%) was 49%, 43%, 40%, and 25% (P < 0.05); Acrosome integrity (%) was 90%, 89%, 83%, and 81% for +5, −3, −5, and −7 °C respectively. 3. PM (%) was 35%, 37%, and 39%; viability (%) was 57%, 60%, and 63%; HOST (%) was 22%, 22%, and 22%; acrosome integrity (%) was 86%, 85%, and 86% for +5, −3, and −5 °C respectively. Conclusions: Cooling of pig sperm to −7 °C (no freezing) damaged sperm function and structure; in contrast, cooling to either −3 °C or −5 °C did not change pig sperm survival after freeze-thawing