Efficiency of melatonin on cryopreserved semen quality, hsp 70 and hsp 90 gene expression of swamp buffalo in different thawing methods

Abstract

The present study was undertaken with the objectives of observing the effects of melatonin on cryopreserved semen quality, hsp 70 and hsp 90 expression of swamp buffalo after thawing with different methods. Semen was collected from four swamp buffalo bulls (4years to 8 years of age) with the aid of artificial vagina. Semen was equally divided into two aliquots: one was extended and cryopreserved with tris-egg yolk-citrate-glycerol extender supplemented with 1mM of melatonin (MLT) and the other was extended with only tris-egg yolk-citrate-glycerol and cryopreserved at –196°C. Two thawing methods were applied: Method I:30°C for 60 seconds and Method II: 37°C for 30 seconds. Semen quality parameters for motility was measured by computer assisted semen analyser (CASA) and the sperm apoptosis was assessed by flow cytometry with Annexin V PE/7-AAD. The expression for hsp 70 and hsp 90 was assessed with reverse transcription polymerase chain reaction (RT-PCR). The results showed that the percentage of total motility, progressive motility, and curve line velocity of sperm in the 1 mM MLT treated groups were significantly higher than the untreated groups when thawed at 30°C for 60 seconds and 37°C for 30 seconds. However, there were no any significant differences in the other parameters such as straight line velocity (VSL), average path velocity (VAP), curve line distance (DCL), straight line distance (DSL), average path distance (DAP), amplitude of latera head (ALH), beat cross frequency (BCF), linearity (LIN), straightness (STR), wobble (WOB). However, all the sperm parameters values, excluding the WOB, were higher when 1mM treated frozen straws were thawed at 37°C for 30 seconds. The percentage of live spermatozoa in the 1 mM MLT group was significantly higher (p < 0.01) and significantly lower (p < 0.01) early apoptotic sperms than the untreated groups in every thawing conditions. Within the treatment groups, the highest percentage of live sperm was found when thawed at 37°C for 30 seconds which is significantly higher (p

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