IMPACT OF MELATONIN AND CRYOPROTECTANTS ON BUCK FROZEN SEMEN DURING HOT AND COLD MONTHS

Abstract

This research was designed to assess the improvement of the freezability of buck semen using two different types of cryoprotectants supplemented with melatonin as antioxidant in cold and hot months. Pooled samples from four sexually mature Egyptian Baladi Bucks were used in this experiment. Semen was extended (1:8) with Tris-fructose-citric containing egg yolk using glycerol and dimethyl sulfoxide (DMSO) supplemented with two doses of melatonin (10-6M and10-3M) in addition to control group. Computer assisted semen analysis (CASA) was used to evaluate semen after cryopreservation. While, enzymatic activity was measured using spectrophotometer technique. Real-time PCR was used for expression profile of selected genes. The results revealed that the progressive motility percentage was higher (P<0.05) in samples supplemented with low dose of melatonin (10-6 M) compared to high dose (10-3M) in glycerol (74.4±2.4 vs. 64.4±2.5) and DMSO based extender (35.5 ±2.4 vs. 32.9 ±2.5) in cold months. The same trend was found in samples cryopreserved with glycerol (75.1±2.2 vs. 53.5±2.2) and DMSO (32.1±1.9 vs. 22.0±1.8) in hot months. The results also demonstrated that CASA parameters (VAP and VCL) were significantly increased in low compared to high melatonin doses in glycerol based extender during cold and hot months. The activity of total antioxidant capacity (TAC) was significantly higher in samples supplemented with low melatonin dose (0.49 mM/L ±0.09) than high melatonin dose (0.16 mM/L±0.09) in DMSO extender. Transcript abundance of CPT2, ATP5F1A and SOD2 genes was increased significantly in glycerol based extender groups and this was more apparent in low melatonin dose compared with all other glycerol based extender groups in cold months. On the other hand, NFE2L2 gene was up-regulated in groups cryopreserved with DMSO compared with those cryopreserved in glycerol based extender in both cold and hot months. It could be concluded that the type of extender and season of collection represent the main factors affecting semen quality, antioxidant defense and molecular activities. Furthermore, melatonin supplementation to extender enhances antioxidant enzymes and genes regulating mitochondrial activity during cold period, which may maintain the post-thaw fertilizing ability of buck semen. Key words: Buck semen, melatonin, cryoprotectants, enzymes activities, genes profile, cold and hot months