162 FREEZING OF SEMEN FROM ENDANGERED ASTURIANA DE LA MONTAÑA BULLS IN ZWITTERONIC LIPIDS-BASED EXTENDERS

Abstract

This experiment was designed to test the efficacy of 2 different preparation protocols of zwitteronic soyabean-origin lipids for the production of lipidsglycerol liposomes for use in bull semen cryopreservation. Lipids liposomes were prepared at 10% concentration in Tris buffer by 1. highpressure homogenization (Panda 2K, Parma, Italy) and then 8% glycerol were added, extender-1 (E-1); Lipids were homogenized together with glycerol, extender-2 (E-2). Bioxcell extender (E-3) was used as control. Semen was collected 3 times from 3 endangered Asturiana de la Montaña bulls by the means of an artificial vagina. Ejaculates with at least 70% motility were processed further by a standard freezing protocol used in our AI station. Semen was diluted at 37°C with each of the 4 extenders to a concentration of 92 × 10⁶ spermatozoa per mL, cooled to 4°C over 4 h, aspirated into 0.25-mL plastic straws (IMV Technologies, Aigle, France), frozen in a bio-freezer (IMV Technologies) in 3 steps from 4 to –140°C, and then plunged into liquid N₂. Straws were thawed in a water bath at 37°C for 30 s. Sperm motility was analyzed microscopically immediately after collection, after dilution, and after 4, 24, 48, and 72 h of storage at 4°C. Post-thaw semen progressive motility was assessed microscopically, and sperm movement characteristics were analyzed by computer-assisted semen analysis (CASA) (SCA^®, Microptic, Barcelona, Spain). Data were compared between extenders and bulls by 2-way ANOVA; percentages were transformed by arcsine transformation before ANOVA. Total and progressive sperm motility at 0 h after dilution ranged from 90 to 70% and was not different between extenders and bulls. There was no difference between bulls in total and progressive motility after 24 h of cold storage; however, both were significantly greater (P < 0.05) for Control (62.4 ± 14.7 and 41.4 ± 14.9) and E-1 (70.1 ± 12 and 33.8 ± 7.0) extenders than for the E-2 extender (22.5 ± 17 and 1.2 ± 1.3). Average post-thaw sperm motility was not different between bulls for either extender, but motility for Bioxcell (Control, 48.1 ± 14.6%) and E-1 extenders (43.2 ± 13.0%) were significantly greater (P < 0.05) than for E-2 extender (18.7 ± 8.8%). There were no differences between bulls for all kinematic semen parameters: curvilinear (VCL), straight line (VSL), average path (VAP) velocities, linearity (LIN) and straightness (STR), evaluated by CASA before and after freezing; however, all were lower (P < 0.05) for the E-2 extender and not different between Control and E-1 extenders. Sperm movements derived from heads (VCL) and linearity of sperm(LIN), both closely related to field fertility, were in the range of 90.9 ± 2.1 and 63.0 ± 5.5 for E-3 (Control) extender; 99.1 ± 3.4 and 49.4 ± 3.5 for E-1; and 21.8 ± 2.2 and 29.9 ± 4.0 for E-2. In summary, zwitteronic soyabean lipid liposomes are an effective egg yolk substitute for the cryopreservation of Asturiana de la Montaña bull semen; however, the homogenization protocol of the lipids-glycerol mixture must be improved.