85 THE COMPARISON OF SPERM FREEZABILITY USING TWO EGG YOLK-FREE DILUENTS IN ZANDI RAM

Abstract

The aim of this study was to investigate the effect of 2 different commercial egg yolk-free extenders, Bioxcell and AndroMed, on post-thaw ram sperm motility and recovery rate. A total of 10 ejaculates were collected from 5 adult Iranian Zandi rams using artificial vagina during 3 weeks, once per week. Preexamination was conducted on semen, and semen motility >70% were selected. Semen samples were pooled and diluted with Bioxcell or AndroMed. Pooled semen was split into 2 treatments and extended to a final concentration of 200 × 10⁸ spermatozoa per mL with extenders. The extended semen was manually packed into 0.25-mL mini-straws and sealed with PVC powder. After 4 h of equilibration in a waterbath at 5°C, the straws were dried with paper tissue, placed horizontally on a rack, and transferred to an isothermal box to be frozen in vapor 5 cm above liquid nitrogen. After 10 min, the straws were plunged into liquid nitrogen. Freezing was conducted using IMV semi-automatic equipment. The percentage of motility and progressive motility of sperm were evaluated before freezing (at 37°C), after refrigeration (at 5°C) and thawing, and recovery rate was calculated. To evaluate indices of sperm motility including the percentage of motile sperm and the progressive forward motility, semen was diluted with 0.9% NaCl w/v (1:100). Ten μL of diluted semen was placed on the prewarmed (37°C) microscope slide (76.2 mm × 24.5 mm; Pearl, China) covered with a cover slip (24 × 24 mm; Menzel-Glaser, Germany) and examined 10 different fields under a phase contrast microscope (Nikon, Tokyo, Japan), at ×200. Dilution factor 1:5 was used. The microscope was equipped with warm plate set at 37°C. Data were analyzed using Proc GLM of SAS. The effect of extender on sperm qualitative characteristics after thawing was significant. Least squares mean percent motility and progressive motility of AndroMed (36.05% ± 0.75; 29.0% ± 0.84) was significantly higher (P < 0.01) than Bioxcell (8.02% ± 0.35; 4.0% ± 0.60); least squares mean recovery rate in AndroMed (34.23% ± 1.05) also was higher (P < 0.01) than Bioxcell (10.0% ± 0.92). Results suggested that AndroMed in comparison with Bioxcell had more ability to preserve sperm quality during the freezing and thawing process.