24 EFFECT OF CULTURE OF SEMEN IN A LOW PRESSURE CONDITION AT ROOM TEMPERATURE ON VIABILITY AND CAPACITATION STATUS OF BOAR SPERM

Abstract

Sperm are affected by physical conditions, such as centrifugation and temperature. The objective of this study was to examine the effect of a low atmospheric pressure on viability and capacitation status of boar sperm during semen preservation at room temperature. Sperm-rich fraction from Berkshire boars was diluted at cells mL^–1 with modified Modena containing 20% seminal fluid after washing with centrifugation (300 × g for 35 min at room temperature) in a Percoll gradient (45%/90%). The sperm suspension was stored at a pressure of 0.5 or 1.0 atmospheres in the dark at room temperature (25°C). Following storage for 4 h or 4 days, the semen samples were analysed for viability, intracellular calcium level, and acrosome status of the sperm. Viability and intracellular calcium level of sperm were assessed by flow cytometry following staining with SYBR-Green/PI and Furo-3/PL, respectively. Sperm status associated with capacitation and acrosome reaction was analysed by CTC-assay under fluorescence microscope. Statistical analyses of data from 4 or 5 replicated trials were carried out by ANOVA and with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Viability of sperm was not different (P = 0.50) between 2 pressures (0.5 and 1.0 atm) 4 h and 4 days after the start of storage (94.6% v. 95.6% and 92.7% v. 94.3%, respectively). Although the percentage of live sperm with high intracellular calcium levels drastically increased (P < 0.01) 4 days after the start of storage (20.2% v. 23.4%) compared with 4 h of storage (5.5% v. 4.9%), there were no differences between sperm stored in 0.5 and 1.0 atm at 4 h (P = 0.80) and 4 days of storage (P = 0.40). After 4 h of storage, there were no differences in the percentage of intact (93.3% v. 94.7%), capacitated (5.5% v. 4.3%), and acrosome-reacted sperm (1.5% v. 1.5%) between sperm stored in 0.5 and 1.0 atm. After 4 days of storage, however, the percentage of intact sperm decreased when the sperm suspension was cultured in 0.5 atm (71.8%) compared with 1.0 atm (88.5%), and the incidence of capacitated sperm increased (14.3% v. 7.8%, respectively), whereas there was no difference in the acrosome-reacted cells. These results demonstrate that the status of sperm associated capacitation is stimulated in a low atmospheric pressure without any effects of the viability of sperm, during storage for 4 days.