Characteristic and DNA Integrity of Spermatozoa Cryopreserved in the Presence of Glutathione and Hydrogen peroxide of Nilli Ravi Buffalo

Abstract

Cryopreservation affects (damages) sperm plasma membrane, acrosomal membrane and the DNA integrity and consequently reduces the sperm motility and fertilizing ability and also reduces the intracellular level of thiols specifically glutathione (GSH) which is an important antioxidant. The addition of GSH prior to cryopreservation for prevention of the sperms from these damages is gaining interest for researchers. Nili Ravi buffalo is an important breed for dairy industry and meat production that is why the semen production units produce semen commercially. The aim of this study is to investigate the effects of cryopreservation, preventive role of GSH addition and adverse role of H2O2 addition prior to cryopreservation on Nili Ravi buffalo bull sperm characteristics (Motility, plasma membrane integrity, acrosomal integrity, DNA integrity). The different combinations of these two additives were also used to determine the effect of GSH detoxification against H2O2 on sperm parameters. To conduct this study semen was collected from five bulls and diluted individually with EYTG extender and 10 groups were prepared from each semen sample. One group studied as fresh without any additives, the 2nd group cryopreserved without any additives and other groups were cryopreserved in the presence of additives (1 mM GSH, 5 mM GSH, 100 μM H2O2, 200 μM H2O2) and combination of these additives (1 mM GSH + 100 μM H2O2, 1 mM GSH + 200 μM H2O2, 5 mM GSH + 100 μM H2O2 and 5 mM GSH + 200 μM H2O2). After thawing sperm parameters were observed such as total motility, motility gradation i.e. 4/4, 3/4, 2/4 1/4 by wet preparation method, sperm membrane integrity by HOS test, acrosomal activity i.e. halo formation rate, halo diameter and acrosin index by gelatin digestion test and DNA integrity by single cell gel electrophoresis (comet assay). The parameters of DNA comet i.e. comet length, height, head diameter, DNA percentage in head and tail, tail length, tail diameter, tail moment, olive tail moment were measured through tritek comet score soft ware. Cryopreservation reduced significantly mean percentage sperm total motility from 80.4±0.84 to 71.28±2.65%. Additive 100 μM H2O2 maintained total sperm motility percentage at 71.51±2.18 whereas, both concentrations of GSH maintained total sperm motility at 56 and 51%. Combination GSH + H2O2 with different concentrations did not maintain sperm motility above 50% except additive 1 mM GSH + 200 μM H2O2 that supports sperm motility at 55%. In sperm motility gradation 4/4 represents the progressive motility that showed no significant difference in fresh semen, cryopreserved semen. All additives maintained motility at 50 – 69% except 5 mM GSH additive, showed significantly low 4/4 motility grade about 46%. This additive (5 mM GSH) showed significant increase (36.56%) in 3/4 motility grade which represents somewhat jerky and slow movement. Cryopreserved sperms also showed increased (26.84%) motility in grade 3/4 than fresh semen. No significant difference was observed in other two (2/4 and 1/4) categories. Cryopreservation maintained mean sperm membrane integrity at 75.35±1.74% which did not significantly decrease than fresh sperms (79.75±1.55%). The additive 5 mM GSH maintained sperm membrane integrity more than 50% i.e. 52.39±1.01. Sperm damaged membrane was observed 51-74% with all other additives. Sperm acrosomal activity parameters were significantly lowered by cryopreservation than fresh, in cryopreserved semen mean sperm halo formation rate 38.84±2.85, mean halo diameter 11.91±0.44 μm and acrosin index 4.6±0.34 were observed and in fresh these were 92.86±1.45%, 16.38±1.04 μm and 15.0±2.4. Additives 5 mM GSH, 5 mM GSH + 200 μM H2O2 increased sperm halo formation rate up to 5.85±1.58; 85±1.04 with mean halo diameter 12.78±0.58; 12.29±0.57 and acrosin index 10.1±0.1; 9.9±0.13 respectively. A highly significant increase 14.8±0.63 in halo diameter was observed with additive 1 mM GSH, whereas, halo formation rate and acrosin index decreased than that of 5 mM GSH additive 69.21±2.02 and 8.0± 0.23. All other additives significantly decreased halo formation rate and acrosin index, however, no significant increase was observed within all additives than cryopreserved sperms. Cryopreservation significantly reduced sperm DNA integrity to 55.99±1.55%, in fresh sperm it was 85.63±0.65%. A dose dependent decrease in DNA damage %age was observed with additives 5 mM GSH, 1 mM GSH that was 83.43%; 60.01% respectively. Whereas, H2O2 additives increased DNA damage up to 45%, however, combinations showed better effect on DNA integrity percentage that was highest (89.12%) with additive 5 mM GSH + 200 μM H2O2 and other combinations also maintain DNA integrity up to (60-70%). DNA comet tail parameters (tail DNA, tail length, tail moment, olive tail moment); represent degree of DNA damage i.e. increase in all four tail parameters shows greater DNA damage. The additive 1 mM GSH proved highly protective role to reduce significantly (P<0.0001) DNA damage compared to control. Additive 5 mM GSH + 100 μM H2O2 also reduced significantly (P<0.0001) DNA damage. Additives 200 μM H2O2 and 1 mM GSH + 200 μM H2O2 were highly significantly (P<0.0001) increased DNA damage compared to control in all four tail parameters. It is concluded that the sperm cryopreservation of Nili Ravi buffalo bull decreased total motility, acrosomal activity and DNA integrity but did not decrease sperm functional plasma membrane integrity and intracellular sperm glutathione levels. Addition of antioxidant (GSH) either 1 mM or 5 mM prior to sperm cryopreservation showed protective effect on sperm DNA integrity and acrosomal activity but did not protect sperm total motility and plasma membrane.