The quality of ram spermatozoa after thawing with the addition of Mn2+, Zn2+ and Cu2+ nanocitrate to cryopreservation diluent

Abstract

The aim of the study was to find out the effect of adding nanocitrate of Mn, Zn and Cu to the diluent for ram spermatozoa cryopreservation on its quality and ability for fertilizing. The experiment was carried out on six clinically healthy breeder 2–4-year-old rams of the Texel breed. The received ejaculates of the rams were evaluated for the volume, sperm concentration and motility and then divided into control and experimental groups. Control sperm samples were diluted with lactose-yolk-tris-citrate-glycerin medium (LYTCGM). Nanocitrates of microelements were added to the medium in experimental samples of ram sperm in the following doses: Zn2+ and Mn2+ — 2.5, 5.0 and 7.5 μg/l, Cu2+ — 1.25, 2.5 and 3.75 μg/l. The diluted sperm was packaged in straws, equilibrated for 2.5 h and frozen. After thawing of sperm we determined motility, survival of sperm, activity of succinate dehydrogenase (SDH) and cytochrome oxidase (CO), activity of antioxidant protection enzymes superoxide dismutase (SOD), glutathione peroxidase (HPO) and catalase (CAT). A dose- dependent effect of Mn, Zn, and Cu nanocitrates upon their addition to LYTCGM was established. Addition of nanocitrates of Mn, Zn to LYTCGM at a dose of 5.0 μg/l increased sperm motility by 22.2% (P<0.05) and 26.0% (P<0.01), and sperm survival, respectively, by 12.6% on (P<0.01) and 5.9% (P<0.05) compared to the control. Nanocitrates of Mn, Zn at a dose of 5.0 μg/l as part of LYTCGM caused a probable increase in SDH (P<0.001) and CO (P<0.05–0.01), which indicates a high fertilizing ability of ram spermatozoa. Similarly, when Mn, Zn nanocitrates were added to LYTCGM at a dose of 5.0 μg/l, SOD activity decreased by 29.6% (P<0.01) and 38.8% (P<0.01) and HPO activity increased by 43.5% (P<0.01) and 39.1% (P<0.01), and CAT — by 40.0% (P<0.05) and 37.5% (P<0.05), respectively. At the same time, the addition of Cu nanocitrate to LYTCGM with an increase in the dose significantly reduces the activity, survival and fertilizing capacity of thawed ram spermatozoa, and also worsens their antioxidant protection.