Effects of Various Cryoprotectants on Bull Sperm Quality, DNA Integrity and Oxidative Stress Parameters

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15×10(6)/ml. The extended samples were cooled slowly to 4°C and equilibrated for 4h. They were then loaded into 0.25ml French straws and frozen using a digital freezing machine at 3 programmed rates: -3°C/min from +4°C to -10°C, -40°C/min from -10°C to -100°C, and -20°C/min from -100°C to -140°C. Thereafter, the straws were plunged into liquid nitrogen at -196°C. Frozen straws were thawed individually at 37°C for 30s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7±2.92%) and progressive (26.4±2.64%) motilities when compared to the other groups (P<0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P<0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P<0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G+2% EG+2% DMSO yielded the lowest activities when compared to the other groups (P<0.001). In conclusion, no advantages were found in using EG or DMSO to replace G in bull sperm cryopreservation. Freezing with cryoprotectant 6% G yielded the best post-thaw sperm characteristics for Eastern Anatolian Red bull spermatozoa.