PS3-1. Comparative Effects of Triladyl® and Andromed® Extenders and Pre-Freezing Cooling Methods on the Quality of Cryopreserved Ram Semen.

Abstract

Ram spermatozoa are highly vulnerable to cold chock during cryopreservation which negatively impacts post-thaw sperm quality. Semen extenders are solutions that provide nourishment and protect sperm cells against injury during the cooling and freezing process. Our aim was to assess the effects of two commercially available extenders (Triladyl® and Andromed®) and two cooling methods before freezing (cooling in 15 mL conical tube versus cooling in straws) on the quality of cryopreserved ram semen. Semen samples collected from five mature Katahdin rams once/week for three weeks were pooled and diluted in either Andromed® or Triladyl® to a final concentration of 800 × 106 spermatozoa/mL. Samples were assigned to four groups in a 2 × 2 factorial design: extender (Andromed® or Triladyl®) × cooling method (package-then-cool or cool-then-package). In the cool-then-package method, semen was cooled in 15 mL conical tubes before being loaded into 0.25 mL French straws, whereas in the package-then-cool method, semen was first loaded into the straws and then cooled. All groups were kept at 4 °C for 3 hours before freezing. Samples were subsequently frozen in a programmable freezer (MiniDigitcool, IMV®), stored in liquid nitrogen, and thawed later for analysis. Data were analyzed using two-way ANOVA to determine the main extender and cooling effects and their interaction and one-way ANOVA followed by LSD post-hoc test to detect differences among the four treatment groups. Following thawing, sperm motility (using CASA), viability and abnormalities (using trypan-blue stain), membrane integrity (by hypos-osmotic swelling (HOS) test), acrosome status (by Hoechst 33342/FITC-PNA), mitochondrial activity (using Hoechst 33342/Rhodamin 123), and lipid peroxidation (using BODIPY 581/591 C11) were evaluated. Total motility, acrosome status, mitochondrial activity, and lipid peroxidation showed extender effects (P < 0.05). However, progressive motility, and membrane integrity showed cooling effects (P < 0.05). Moreover, among the four treatment groups, total and progressive motility, % of live sperm, % of sperm with intact acrosome, and % of sperm with active mitochondria were the highest (P < 0.05) in the Triladyl® cool-then-package group (31.1±2.1, 26.7±2.3, 66.3±1.9, 76.2±1.1, and 78.0±1.2, respectively). Meanwhile, total sperm abnormalities and lipid peroxidation were the lowest (P < 0.05) in the same group (18.0±0.7 and 12.3±1.0, respectively). In conclusion, cryopreservation of ram semen using the Triladyl® extender in combination with the cool-then-package method resulted in superior post-thaw sperm quality than other extender and cooling procedures.