Effect of exogenous melatonin implant on post-thaw semen quality of buffalo bulls.

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.

Sodium dodecyl sulphate, N-octyl ß-D glucopyranoside and 4-methoxy phenyl ß-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls.

Sodium Dodecyl Sulphate (SDS), N-Octyl ß-D Glucopyranoside (NOG), 4-Methoxy Phenyl ß-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -40 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); ii) Moderate P25 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -50 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); and iii) Rapid P26 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -60 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

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 °C min-1 from temperature drop from 4 °C to -15 °C and -15 °C to - 60 °C followed by 50 °C min-1 from - 60 °C to -140 °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 °C min-1[Protocol (PRT) I], 40 °C min-1 (PRT II) and 50 °C min-1 (PRT III) in the critical temperature fall zone of -15 °C to -60 °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 °C min-1 in the critical temperature fall zone of -15 to -60 °C perform better than the freezing rate of 30 °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.