73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING

Abstract

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15°C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 × 10⁹ cells mL^–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5°C over 2–3 h. At 5°C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37°C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37°C under 5% CO₂ in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR^®14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.