70 REFREEZING POST-THAWED GOAT SEMEN

Abstract

The ability to successfully refreeze caprine sperm could provide a means of salvaging semen that was mistakenly thawed. The objective of this study was to compare treatment post-thaw semen parameters of twice-frozen caprine semen. Frozen semen from six mature Boer bucks (range in age from 2 to 6 years) was utilised for this experiment. Semen from each buck was extended in an egg yolk-based extender and packaged in 0.5-mL plastic straws before freezing and stored in liquid nitrogen. Three units of frozen semen from each buck was randomly allotted to each of four treatments as follows: (A) thaw and evaluate (control), (B) thaw, then plunge into liquid nitrogen, thaw, and evaluate, (C) thaw, incubate for 3 min at 37°C, slow cool and freeze, thaw, and evaluate, and (D) thaw, incubate for 5 min at 37°C, slow cool and freeze, thaw, and evaluate. Post-thaw parameters included total motility (TM), progressive motility (PM), membrane integrity (MI), and sperm abnormalities (AB). To obtain MI and AB, samples were stained with an eosin-nigrosin stain. A computerized programmable freezer was used to refreeze semen samples in treatment (Trt) C and Trt D. During the slow cooling portion of the protocol, samples were allowed to equilibrate at 38°C, then cooled to 4°C at a rate of 0.30°C min^–1, and then held for 5 min. Samples were then cooled to –8°C at a rate of 15°C min^–1, seeded, and cooled to –10°C at 15°C min^–1, samples were then ramped to –80°C at 30°C min^–1 before plunging into liquid nitrogen. Results indicate that post-thaw TM was significantly greater for Trt A (60%) when compared with Trt B, C, and D (0.05, 35, and 39%, respectively). Mean TM were not different between Trt C (35%) and Trt D (39%) but were greater than that for Trt B (0.05%). The PM for post-thaw semen in Trt A was also significantly greater (P < 0.05) when compared with that for Trt B and C (0.05 and 25%); however, no difference was found for mean PM for Trt A (47%) and Trt D (30%), nor were differences found between Trt C (25%) or Trt D (30%). Membrane integrity was higher in Trt A (27%) when compared to Trt B (2.2%). No differences in membrane integrity where found between Trt A, C, and D (27, 13, and 14%, respectively). Additionally, no differences were found between Trt B, C, and D for membrane integrity. Sperm morphology were not different were found with across all treatment groups. These results (i.e. Trt C and D) indicate that semen from mature Boer bucks can undergo a second freeze thaw cycle and still retain motility without dramatically affecting sperm morphology and membrane integrity. These findings indicate that directly plunging recently thawed semen back into liquid nitrogen should not be used for artificial insemination.