39 Nanowater enhances cryoprotective effects of glycerol during ram semen freezing

Abstract

It was suggested that unique physicochemical properties of nanowater (NW, water declustered in the cold plasma generator), such as a low dielectric constant and high diffusivity, might ameliorate ram semen freezing by improving bioavailability of extender constituents. This study was undertaken to evaluate the effects of NW added to glycerol-containing semen extenders on the characteristics of frozen-thawed ram semen. A total of 18 ejaculates collected from 6 Olkuska breed rams during the breeding season were divided into six equal portions. The ejaculates were frozen in the modified fructose-skimmed milk-egg yolk Kareta extender containing 3 or 7% glycerol and diluted in deionized water (control extenders; Aqua Purificata, Prolab; C3% and C7%, respectively) or NW (declustered for 15 min (NW15’) or 30 min (NW30’); Nanotechnology Systems) to a final concentration of 800 × 10⁶ spermatozoa/mL. They were placed in 0.25-mL plastic straws and frozen in liquid nitrogen. The two declusterization times were chosen on the basis of previous laboratory tests and fertility trials yielding the best results in terms of semen quality post-thawing and pregnancy rates after AI. All semen samples were evaluated for sperm concentration, progressive motility (Sperm Class Analyzer), and morphological defect rates (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of alanine transferase, alkaline phosphatase, and aspartate aminotransferase were measured, along with the proportions of apoptotic, necrotic, and live spermatozoa determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson) at 0 and 1 h post-thawing. Data were analysed by one-way analysis of variance and Holm-Sidak method using SigmaPlot statistical software (Systat Software Inc.). The proportion of spermatozoa with mid-piece defects was significantly (P < 0.05) reduced in NW15’-3% compared with C3% (1.3 ± 0.3% vs. 4.0 ± 06%; mean ± s.e.m.). The proportion of necrotic spermatozoa 1 h after thawing was greater (P < 0.05) in C7% compared with NW30’-7% (20.7 ± 0.4% vs. 17.6 ± 1.0%), whereas the proportions of live cells detected immediately (0 h) and 1 h after thawing were greater (P < 0.05) in NW30’-7% than in C30’ (0 h: 54.0 ± 0.6% vs. 50.4 ± 1.2%; 1 h: 54.4 ± 1.2% vs. 59.2 ± 0.4%, respectively). The mean survival time of spermatozoa was greater (P < 0.05) in extenders dissolved in NW30’compared with their respective controls (NW30’-3% vs. C3%: 215 ± 4 min vs. 189 ± 6 min; and NW30’-7% vs. C7%: 242 ± 8 min vs. 217 ± 4 min, respectively). Alkaline phosphatase concentrations in extenders prepared with NW30’ were lower (P < 0.05) compared with the control groups (NW30’-3% vs. C3%: 3105 ± 231 IU vs. 4490 ± 458 IU; and NW30’-7% vs. C7%: 2516 ± 128 IU vs. 3956 ± 263 IU, respectively). Our results indicate that NW significantly improves the effects of glycerol on ram sperm viability post-thawing, with the reduction in sperm necrosis/overall enhancement of sperm survivability being greater with 7% compared with 3% of glycerol in semen extender.