Abstract
Cryopreservation can adversely affect the quality of ram spermatozoa, reducing their suitability for artificial insemination. Therefore, optimizing cryopreservation protocols, including extender composition and cooling procedures, is important for improving post-thaw sperm quality. 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 four weeks were pooled and diluted in either Andromed® or Triladyl® to a final concentration of 800 × 106 spermatozoa/mL. Samples were allocated into four treatment groups in a 2 × 2 factorial design: Triladyl® package-then-cool (T1), Triladyl® cool-then-package (T2), Andromed® package-then-cool (A1), and Andromed® cool-then-package (A2). 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 h before freezing. Samples were subsequently frozen in a programmable freezer (MiniDigitcool, IMV®), stored in liquid nitrogen, and thawed later for analysis. 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, the percentages of motile sperm, live sperm, sperm with intact acrosomes, and sperm with active mitochondria were the highest (p < 0.05) in the Triladyl® cool-then-package (T2; 31.1 ± 2.1, 26.7 ± 2.3, 66.3 ± 1.9, 76.2 ± 1.1, and 78.0 ± 1.2, respectively) group. Meanwhile, the percentages of abnormal and lipid peroxidation-positive sperm 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.