Seasonal dynamics in acrosome reaction induction in ram spermatozoa

Two longitudinal studies were conducted to evaluate the seasonal dynamics in acrosome reaction induction and osmotic tolerance limit of ram sperm. Semen was collected using an electroejaculator once a month from 6 mature rams (three ejaculates per each ram per season) throughout study period during winter (Dec-Feb), spring (Mar-May), summer (Jun-Aug) and autumn (Sep-Nov). In study 1 acrosome reaction was induced in vitro by using different inducing agents; LPC and CI. The samples were then incubated in water bath at 37°C for 3 hrs. At various intervals (15, 30, 60, 120 and 180 min) after stimulation with LPC or CI, the acrosomal status of the samples was monitored by using fluorescent staining (FITC-PNA). The AR induced in the presence of LPC or CI showed different pattern in different seasons. The percentage of acrosome reacted sperm was higher (P<0.05) in LPC and control during winter season compared to other seasons at all incubation time intervals (15-180 min). However, CI induced the AR in a time dependant manner (reacted sperm rate gradually increased over incubation time of 180 min) and it varied dramatically between seasons. During winter and summer seasons, the CI-induced AR remained higher (P<0.05) at all incubation time points. In study 2 the sperm samples were exposed to fructose-sodium citrate solutions of different osmolalities (50,100, 290, 500 and 1000 mOsm/L). The samples were then incubated at 35°C for 15 min. The isosmotic and hyperosmotic incubation samples (290, 500, 750 and 1000 mOsm/L) were returned to hypo-osmotic condition. After osmotic challenges, the sperm viability and membrane integrity were estimated using a combined hypo-osmotic swelling and eosin test (HE test). The live sperm percentage was recorded lowest (P<0.05) at all hypo, iso and hyperosmotic conditions during winter season. Similarly lowest (P<0.05) intact and live-intact sperm percentages were observed in 50, 100, 290, 500 and 100 mOsm/L conditions during winter season. The highest membrane integrity rate in terms of live, intact and live-intact sperm proportions after exposure to anisoomotic media were recorded in autumn season whereas, these parameters were lowest during winter season under the same osmotic conditions in this studyBest membrane integrity was recorded during autumn season. In conclusion both LPC and CI are use different pathways for AR induction and responsible to provoke signaling transduction pathway. It can be hypothesized that the extra receptivity of ram sperm to undergo AR in fresh, LPC or CI treated groups during winter season as observed in the present study, might be due to an expected increase in melatonin during this season. Moreover, season has significant influence on osmotic tolerance limit of ram sperm. The best osmotic tolerance during autumn might be the reason of better fertility in breeding season in ram.