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. Manuscript profile

The effects of five levels of osmolality and pH as well as three temperatures on Awassi ram sperm motility parameters assessed by computer-aided sperm analyzer(CASA)in tyrode albumin lactate pyruvate (TALP) and egg-yolk-tris (YET) media were studied. Semen obtained from five adult Awassi rams were pooled and diluted in the above two media at 100, 200, 300, 400 and 500 mOsm/kg at temperatures of 4˚, 20˚ and 37˚ degree celsius and also at pH levels of 5, 6, 7, 8 and 9 at the same temperatures. The osmolality, pH and temperature had a significant effect (P<0.05) on the percent motility (MOT %) and the percent of sperm showing progressive motility (PMOT %). With the exception of lateral head displacement, all the other CASA motility parameters were also significantly affected (P<0.05). Semen incubated in YET was able to tolerate osmolarities within 200 to 400 mOsm/kg range for MOT %. Alkaline condition at pH 9 had higher negative effect than acidic condition at pH 5. Compared to the 20 ˚C and 37 ˚C, the 4 ˚C had negatively affected MOT % and PMOT % and this was more obvious in TALP medium. In conclusion, our results suggest: 1) the significant effects of osmolality, pH and temperature on Awassi sperm incubated in both TALP and YET media and 2) the need for careful selection of temperature by which the semen may be manipulated during artificial insemination and in vitro fertilization experiments in these two media. Manuscript profile