Evaluation of bull semen frozen with different additives

This study, it was aimed to determine the effects of adding melatonin and liposome to Tris-based extender on post-thaw spermatological parameters in freezing bull semen. For this purpose, ejaculates obtained from three normospermic bulls via artificial vagina were combined to form experimental groups. Different doses of melatonin and liposomes were added to the tris-based extenders containing glycerol 5% (G5) and glycerol 3% + 60 mM trehalose (G3T). The combined ejaculates were divided into 10 equal volumes and diluted with the following diluents: G5, G5+ 0,25 mM melatonin (G5M0.25), G5+ 0,75 mM melatonin (G5M0.75), G5+ 1,25 µl/ml liposome (G5L1,25), G5+3,75 µl/ml liposome (G5L3.75), G3T, G3T+0,25 mM melatonin (G3TM0.25), G3T+0,75 mM melatonin (G3TM0.75), G3T+1,25 µl/ml liposome (G3TL1.25), G3T+3,75 µl/ml liposome (G3TL3,75). The dilution process was carried out at 37o C and the diluted sperm samples were frozen in liquid nitrogen vapor after equilibration into straws and stored in liquid nitrogen. After freezing and thawing, motility in sperm samples was evaluated by looking at the parameters of live-dead ratio (SYBR+), plasma membrane and acrosome integrity (MPAI), mitochondrial oxidative stress (MITOSOX+) and spermatozoon apoptosis rate (Annexin V) in flow cytometry analysis. Post-thaw sperm motility rates in G5, G5M0.25, G5M0.75, G5L1.25, G5L3.75, G3T, G3TM0.25, G3TM0.75, G3TL1.25, G3TL3.75 groups were determined as 61,7±0,01%; 69,20±0,02%; 60,80±0,01%; 65,80±0,02%; 60,00±0,01%; 63,30±0,01%; 66,70±0,02%; 60,80±0,01%; 67,50±0,02%; 59,20±0,01%; respectively. G5M0.25 group provided a higher improvement in post-thaw motility compared to G5 (61,7±0,01%), G5M0.75 (60,80±0,01%), G5L3.75 (60,00±0,01%), G3T (63,30±0,01%), G3TM0.75 (60,80±0,01%), G3TL3.75 (59,20±0,01%) groups (p<0,001). G3TM0.25 and G3TL1.25 groups significantly improved post-thaw motility value compared to the G5 group (p<0,001). In the flow cytometry analysis results, no statistically significant difference was observed between the groups regarding dead-alive ratio, plasma membrane and acrosome integrity, mitochondrial oxidative stress level and apoptosis rates (p>0,05). It was concluded that melatonin added to a Tris-based extender during freezing of bull semen improved post-thaw spermatological parameters. It was determined that melatonin added to G3T and G5 extenders at 0,25 mM and 0,75 mM doses gave similar results in both extenders. As a result, it was observed that antioxidant addition to the G3T extender containing low glycerol protected and improved post-thaw spermatological parameters as much as the G5 extender; it could be used instead of the G5 extender (p<0,001). It was concluded that the toxic effect of glycerol on fertility and especially the low results in spermatological parameters that may arise from the use of low glycerol can be eliminated by adding Trehalose to the extender containing low glycerol. Keywords: Freezing-Thawing; Liposome; Melatonin; Spermatological parameters; Spermatozoon;