Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux.

Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 106 sperm mL-1) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL-1 catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at -25 °C min-1 up to -125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.

Supplementation of cauda epididymal plasma improves sperm characteristics following liquid preservation of ram semen at 3-5°C.

Mammalian spermatozoa remain immotile and metabolically inactive in the cauda epididymidis, thus maintaining fertility for several weeks. The aim of this study was to functionally characterise and evaluate the effect of cauda epididymal plasma (CEP) on liquid preservation of ram spermatozoa. Four experiments were conducted to investigate the effects of: (1) CEP and its fractions on sperm motility; (2) CEP (10%, 15%, 20% v/v) on liquid preservation of ram spermatozoa; (3) seminal plasma (SP; 20%, 30%, 50% v/v) on liquid-preserved spermatozoa; and (4) both CEP and post-storage SP treatment on sperm characteristics. Biochemical characterisation of ram CEP revealed high protein (30.9mgmL-1), catalase (68.9IUmL-1), alkaline phosphatase (17.5IUmL-1) activities and total antioxidant capacity (1112µM Trolox equivalent). Progressive motility of prewashed cauda spermatozoa was reduced (P<0.05) by CEP or its protein-rich fraction compared with protein-free plasma or phosphate-buffered saline. After 48h storage, total motility, rapid motility (average path velocity >75µms-1; 53.9%, 73.5% and 71.8% with 0, 15% and 20% CEP respectively) and straight line velocity (86.3, 102.1 and 102.4µms-1 with 0, 15% and 20% CEP respectively) were significantly (P<0.05) higher in the CEP-treated groups than the control. Viability and acrosomal integrity were similar between groups; however, functional membrane integrity was higher (P<0.05) in the 15% CEP-treated group. Treatment of liquid-preserved spermatozoa with either 20%, 30% or 50% SP improved (P<0.05) rapid motility and kinematics at each time point of storage compared with control. In conclusion, liquid preservation of ram spermatozoa in the presence of 15% or 20% CEP and post-storage treatment with SP significantly improve sperm characteristics.