Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml(-1). Freezing of semen samples was conducted at 30 degrees C min(-1) from temperature drop from 4 degrees C to -15 degrees C and -15 degrees C to -60 degrees C followed by 50 degrees C min(-1) from -60 degrees C to -140 degrees C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml(-1)) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 degrees C min(-1)[Protocol (PRT) I], 40 degrees C min(-1) (PRT II) and 50 degrees C min(-1) (PRT III) in the critical temperature fall zone of -15 degrees C to -60 degrees C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml(-1) in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml(-1) as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml(-1) as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml(-1), resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 degrees C min(-1) in the critical temperature fall zone of -15 to -60 degrees C perform better than the freezing rate of 30 degrees C min(-1). It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml(-1) improved post-thaw semen quality of cattle and buffalo.