Effects of Hydrogen Peroxide (H₂O₂) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°CIn Vitro

Abstract

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H2O2 was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris-egg yolk-citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5 degrees C) and cryopreserved in 0.5-ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen-thawed semen was separated by centrifugation (1500 g; 15 min) and were washed with sperm TALP. The sperm cells were re-suspended in incubation TALP at the rate of 10(8) sperm cells per millilitre and incubated with 0, 10, 25, and 50 microm H2O2 per ml at 37 degrees C. Sperm motility, viability and intact acrosome percentages were assessed at 15-min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H2O2. Among the different levels of H2O2, the 50-microm H2O2-incorporated group had significantly (p<0.05) higher malonaldehyde (MDA) level than the other groups. In the 50-microm H2O2-incorporated group, the MDA levels in fresh, equilibrated and frozen-thawed semen after incubation for 60 min were 961.6+/-12.7, 991.8+/-10.3 and 1234.9+/-9.6 nm per 10(9) spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H2O2 and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H2O2 was significantly (p<0.05) higher in frozen-thawed than fresh and equilibrated spermatozoa.

Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm

“…These results confirmed the earlier reports regarding the deleterious effects of H 2 O 2 on sperm functions [29,11]. The sperm from various species behave differently in high or low concentrations (10 lM to 5 mM) of H 2 O 2 [29,11]. In some species a very low concentration of H 2 O 2 (10 lM) is sufficient to reduce the sperm motility [5] whereas, in some others even the highest H 2 O 2 concentration of H 2 O 2 (5 mM) is proved to be ineffective [4].…”

“…It has been noted that H 2 O 2 significantly decreased sperm motility, viability and membrane integrity during 12 h incubation period. These results confirmed the earlier reports regarding the deleterious effects of H 2 O 2 on sperm functions [29,11]. The sperm from various species behave differently in high or low concentrations (10 lM to 5 mM) of H 2 O 2 [29,11].…”

“…In some species a very low concentration of H 2 O 2 (10 lM) is sufficient to reduce the sperm motility [5] whereas, in some others even the highest H 2 O 2 concentration of H 2 O 2 (5 mM) is proved to be ineffective [4]. It is described that the increased ROS generation reduces the antioxidant enzymes level in the media which in turn lower viability, membrane and DNA integrity of the sperm [17,11]. As the cell membrane is the principal target site for ROS due to the presence of high polyunsaturated fatty acids contents [8].…”

“…The low concentrations of ROS (Superoxide anion; O 2À and H 2 O 2 ) in semen initiate the sperm capacitation [6], whereas high concentrations of ROS reduce the acrosin activity [48], motility [40], acrosome reaction, fertilizing ability [16] and increase the DNA fragmentation [17]. The earlier studies in different species demonstrated that oxidative stress induced by H 2 O 2 markedly suppresses the sperm parameters due to excessive generation of ROS [1,10,11,21,29].…”

Effects of the Insemination of Hydrogen Peroxide-Treated Epididymal Mouse Spermatozoa on γH2AX Repair and Embryo Development

“…Although numerous reports have described DNA damage to sperm caused by exogenous ROS (H 2 O 2 ) or cryopreservation, only sperm motility variables and intracellular ROS and nitric oxide levels were examined [13], [14], [15]. The DNA damage to sperm needs to be repaired after fertilization to ensure correct genetic message transfer to progeny.…”