Carboxymethyl cellulose and glycerol act synergistically as cryoprotectant during cryopreservation of ram semen

Abstract

Wider implementation of AI in sheep in the field condition has not been possible till date due to very poor conception rate after cervical insemination with cryopreserved semen. Poor cervical penetrability in ewe and diminished sperm functions in cryopreserved semen are considered responsible for it. In the present study, effect of carboxymethyl cellulose (CMC) on post-thaw qualities of ram semen was investigated. Ejaculates from eight adult Malpura rams were pooled and diluted (800 × 106 sperm mL−1) with TES-Tris-fructose-egg yolk extender having either 5 or 6% glycerol and supplemented with 0, 0.25, 0.5, 0.75 and 1.0% (w/v) CMC and packaged into 0.25 mL French mini straws. The straws were progressively cooled to 5 °C inside a cold cabinet (5 °C) and then equilibrated for 22 h inside a refrigerator (2–5 °C). Straws were frozen at −25 °C min−1 up to −125 °C using a programmable cell freezer (Planer Biomed R-204, UK) and finally plunged into liquid nitrogen. The post-thaw progressive motility was higher (P < 0.05) in 0.75% CMC-treated group compared to control. Overall, both pre-freeze and post-thaw sperm kinetics was comparable between CMC-treated and control groups. The post-thaw sperm viability, acrosomal integrity and sperm with high mitochondrial membrane potential (hMMP) were relatively higher while sperm with high membrane cholesterol was significantly (P < 0.05) higher in presence of 0.25% CMC compared to the control. Both sperm having hMMP and non-capacitated sperm were significantly (P < 0.05) higher in presence of 5% glycerol than 6% glycerol. Similarly, functional membrane integrity (FMI) was higher in presence of 5% glycerol than 6% glycerol when CMC was added at 0.5% to extender. In conclusion, both 0.25% CMC and 5% glycerol resulted in improvement in several post-thaw sperm functions in cryopreserved ram semen. Thus CMC demonstrated cryoprotective effect on ram sperm in a synergistic manner with glycerol.