Nanowater Enhances Cryoprotective Effects of Glycerol-Containing Extenders Used for Ram Semen Freezing

Nanowater (NW-water declusterized in the cold plasma generator) can potentially ameliorate ram semen quality after freezing. Eighteen ejaculates from six Olkuska rams were divided into six equal portions each, and then diluted (800 × 106 spermatozoa/ml) and frozen in the fructose-skimmed milk-egg yolk Kareta extender containing 3% or 7% of glycerol (C3% and C7%) and diluted in deionized water (DW) or NW declusterized for 15 min (NW15)’ or 30 min (NW30’). All frozen-thawed semen samples were subjected to standard evaluation. In addition, ex situ survival time of spermatozoa was measured, and the proportions of apoptotic, necrotic and live sperm were determined by flow cytometry. The percentage of spermatozoa with mid-piece defects was lower (p < 0.05) in NW15’-3% compared with C3%. The mean survival time of spermatozoa was greater (p < 0.05) in NW30’ extenders compared with their respective controls. The proportion of necrotic spermatozoa 1 h after thawing was greater (p < 0.05) in C7% compared with NW30’-7%, whereas the proportion of live cells detected immediately and 1 h after thawing were greater (p < 0.05) in NW30’-7% than in C7%. NW enhanced cryoprotective effects of glycerol-containing extenders with an overall increase in sperm survivability being greater with 7% than 3% of glycerol.