Quality of deconserved bull sperm for the action of nanosuccinates Zn, Cu and Mn in the diluents

Abstract

The purpose of this work was to compare effect of different doses of trace elements such as Cu2+, Zn2+ and Mn2+ that have been included as nano succinates into lactose-yolk-glycerol medium for cryopreservation of bull sperm and some physiological and biochemical sperm parameters assessment before and after cryopreservation. In this research each fresh ejaculate obtained from 4 bulls has been divided into parties consisting a control sample and its experimental counterparts. Control samples were diluted with industrial lactose-yolk-glycerin diluent only but their experimental counterparts were diluted and supplemented with nano acquacuccinates of Cu, Mn and Zn as solutions at concentration 2–5 g/l but different doses of 0.005, 0.01 and 0.05 mg/ml. When ejaculates were taken, the following physiological parametres of ejaculate quality were established: volume (ml), sperm concentration (billion/ml), live sperm count (%) and dynamic sperm count (CASA) and survival (h); content of total protein, respiratory activity of sperm, activity of enzyme markers of fertilizing ability — succinate dehydrogenase (SDH, units) and cytochrome oxidase (CHO, units) in diluted ejaculates with introduced minerals. After the ejaculates were diluted, semen was equilibrated for three hours at 4°C and frozen in a container (7 min over nitrogen vapor followed by immersion in liquid nitrogen). The semen was thawed in a water bath at 38°C for 20 seconds. The above physiological and biochemical parameters of the sperm of the bulls were redetermined immediately after thawing. Spermatozoa concentration in diluted bull sperm was 8.3% of the initial or ejaculate diluted 12-fold according to technological requirements (P<0.001). The number of live sperm decreased by 12.6% compared to fresh sperm (P<0.05), and the survival of sperm during incubation decreased by 6.8% for 7.4 hours. Total protein content in 100 ml of sperm decreased by 41.3% after dilution compared to fresh ejaculate (P<0.001). Respiratory activity decreased by 11.8% after the ejaculates was diluted. Succinate dehydrogenase activity decreased by 10.7% and cytochrome oxidase activity by 13.0%. In thawed bull sperm the respiratory sperm activity is higher in counterparts when 0.05 mg/l Zn2+, 0.05 mg/l Cu2+ and 0.05 mg/l Mn2+ are added to the medium. Enzyme activity at the same doses was higher. The highest activity among these groups of succinate dehydrogenase was at 0.05 mg/l Zn2+ (P<0.05) added to the cryopreservation medium, and the lowest at 0.01 mg/l Mn2+. Cytochrome oxidase activity was highest when 0.05 mg/l Cu2+ was added to the cryopreservation medium. The optimum concentrations of nanosuccinates that ensure the normalization of oxidation processes in the diluted bull sperm are: 0.05 mg/l Mn2+, 0.05 mg/l Cu2+ and 0.05 mg/l Zn2+. The higher concentration of metal nano succinates in the diluent inhibits the respiratory sperm activity and reduces the activity of succinate dehydrogenase and cytochrome oxidase. Similar effect has been estimated in dynamic performance of spermatozoa after thawing.