95 EFFICACY OF FIVE DIFFERENT SEMEN EXTENDERS FOR THE CRYOPRESERVATION OF BULL SEMEN

Abstract

Replacement of animal-origin components in extenders used for bull semen freezing is of high importance for individuals involved in cattle breeding. The experiment was designed to compare efficacy of 5 different semen extenders in cryopreservation of bull semen: sodium citrate-based extender containing egg yolk (CT), commercially available Bioxcell^® (IMV Technologies, L'Aigle, France), and 3 custom-made homogenized plant lipidsbased, egg yolk-free extenders (Y-1, Y-2, and Lipo) . The objective was to determine whether homogenization procedures of lipids improve the quality of the extender. Lipid homogenates of custom-made extenders were prepared in Tris buffer using a high pressure homogenizer (Nira Saovi, Parma, Italy). Ten (Y-1) or 5 (Y-2) homogenization cycles were applied and then 8% glycerol was added. Lipid liposomes were produced by simultanous high pressure homogenization of lipids and glycerol supplementation (Lipo). Semen was collected from young bulls of 3 different breeds (Simmental, Polish Red, and Holstein; 1 ejaculate/bull). Each ejaculate with at least 70% motility was split into 5 parts and processed further by a standard freezing protocol: semen was diluted at 35°C with each of the 5 extenders to a concentration of 100 × 10⁶ spermatozoa per mL, cooled to 4°C over 5 h, aspirated into 0.25-mL plastic straws, frozen in liquid nitrogen vapor to –140°C, and then plunged into LN₂. Straws were thawed in a water bath at 37°C for 20 s. Sperm motility was estimated microscopically immediately after thawing and after 5 h of storage at 22°C. Immediately after thawing, flow cytometry and SYBR-14/PI staining were used for examination of sperm membrane integrity (live/dead assay). A total of 20 000 spermatozoa of each sample were counted. Student's t-test was used to estimate statistical differences between experimental groups. The mean sperm motility after thawing ranged from 45.6% (SD = 13.7) for CT (egg yolk extender) to 57.8% (SD = 7.1) for Lipo. A significant difference (P < 0.05) was observed betweenY-1 (50.0%, SD = 9.7) and Lipo and Bioxcell (56.1%, SD = 8.6). After 5 h of storage at 22°C, the mean motility for all tested bulls ranged from 25.0% (SD = 7.1) for CT to 42.2% (SD = 7.5) for Lipo. Significant differences were observed between Lipo (P < 0.01), Y-2 (P < 0.05) and CT, and between Y-1 and Lipo (P < 0.01). Mean percentage of 'live' spermatozoa with intact membrane after freezing/thawing was 51.85% (SD = 11.49) for Y-1, 45.72% (SD = 9.36) for Y-2, 47.57% (SD = 7.93) for Lipo, 45.47% (SD = 8.35) for Bioxcell, and 49.06 (SD = 11.59) for CT. No significant differences were observed except forY-1 and Bioxcell extenders (P < 0.05). It can be concluded that both methods of lipid/glycerol homogenization can be successfully applied in the preparation of bull semen extender. In addition, extensive lipid homogenization (10 cycles) produced more transparent extender that in turn improved visualization of sperm. Custom-made plant origin lipids homogenization may provide a valuable alternative for the preparation of extenders that more closely match the membrane composition of bull sperm cells and thus contribute to development of an efficient extender free of animal-origin components for bull semen freezing.