Combination of fetuin and trehalose in presence of low glycerol has beneficial effects on freeze-thawed ram spermatozoa

Andrology. 2021 May;9(3):1000-1009. doi: 10.1111/andr.12974. Epub 2021 Feb 2.

Abstract

Background: Freeze-thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze-thawing is suggested to improve sperm parameters.

Objectives: The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant-related gene expressions and DNA integrity during the freeze-thawing process, in low glycerol concentration.

Methods: Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris-based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15).

Results: G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05).

Conclusions: Co-supplementation of tris-based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze-thawing process is recommended.

Keywords: fetuin; freeze-thawing; glycerol; ram spermatozoa; trehalose.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cryopreservation*
  • Cryoprotective Agents*
  • Fetuins
  • Glutamate-Cysteine Ligase / metabolism
  • Glutathione S-Transferase pi / metabolism
  • Glycerol
  • Male
  • NAD(P)H Dehydrogenase (Quinone) / metabolism
  • Oxidative Stress
  • Sheep
  • Spermatozoa / enzymology*
  • Trehalose

Substances

  • Cryoprotective Agents
  • Fetuins
  • Trehalose
  • NAD(P)H Dehydrogenase (Quinone)
  • Glutathione S-Transferase pi
  • Glutamate-Cysteine Ligase
  • Glycerol