Formation of Hydrogen Peroxide by Chilled Goat Spermatozoa and the Effects of Dead Spermatozoa on Motility Characteristics

In this study, the formation of hydrogen peroxide (H2O2) by chilled goat spermatozoa was measured. Furthermore, the effects of dead spermatozoa on motility characteristics were studied. Fresh collected ejaculates from five Shami bucks were centrifuged and virtually all seminal plasma was removed. A part of the collected spermatozoa was killed by two ways: the first by repeated freezing in liquid nitrogen and thawing at 37 ˚C in water bath and the second by adding the spermatozoa to double distilled water.Two experiments were conducted after two hours of samples incubation intris-egg yolk (TEY) medium at 5 ˚C. In the first experiment, a fluorometric assay with 10-acetyl-3,7-dihydroxyphenoxazine agent as a probe for H2O2 detection was used to measure H2O2 formation. In the second experiment, the effects of adding 0 (control), 25, 50 and 75 % (V/V) of dead spermatozoa to live ones on sperm motility were assessed using computer-aided sperm analysis (CASA). Hydrogen peroxide was generated from live and dead chilled spermatozoa and the amounts of this agent increased with time. Moreover, clear significant differences (P<0.05) were observed between the generated levels from dead spermatozoa compared to live ones. The dead spermatozoa by repeated freezing-thawing treatment produced the higher H2O2 amount (P<0.05). The values of percent motile spermatozoa (MOT %), percent of progressively motile spermatozoa (PMOT %) and average path velocity (VAP) were significantly (P<0.05) reduced compared to controls when dead spermatozoa were added. The negative effect on the previous CASA parameters was increased when the percentages of dead spermatozoa were increased whatever the way of sperm death was. In conclusion, the high formation of H2O2 from dead chilled goat spermatozoa may be responsible for motility decreased. The removal of dead spermatozoa from incubation medium could help to improve the motility characteristics of chilled goat sperm.