Immunofluorescence and biochemical investigation of the protective effects of naringin and diosmin on the freezability of merino ram semen

Cryo Letters. 2024 Sep-Oct;45(5):309-319.

Abstract

Background: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage.

Objective: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen.

Materials and methods: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 10 7 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37 degree C for 25 s and analyzed in terms of spermatological parameters.

Results: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p < 0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p < 0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p < 0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells.

Conclusion: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process. Doi.org/10.54680/fr24510110412.

MeSH terms

  • Acrosome / drug effects
  • Animals
  • Antioxidants / pharmacology
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cryopreservation* / methods
  • Cryopreservation* / veterinary
  • Cryoprotective Agents / pharmacology
  • Diosmin* / pharmacology
  • Flavanones* / pharmacology
  • Male
  • Oxidative Stress / drug effects
  • Semen / drug effects
  • Semen Analysis
  • Semen Preservation* / methods
  • Semen Preservation* / veterinary
  • Sheep
  • Sperm Motility / drug effects
  • Spermatozoa* / drug effects
  • Superoxide Dismutase / metabolism

Substances

  • Diosmin
  • naringin
  • Flavanones
  • Antioxidants
  • Cryoprotective Agents
  • Superoxide Dismutase