# Effects of Adding Punicalagin or Oleuropein to TRIS Diluent on Quality of Frozen–Thawed Semen from Rams

## TL;DR

Adding punicalagin or oleuropein to a TRIS diluent improves the quality of frozen-thawed ram semen by enhancing sperm motility and viability.

## Contribution

This study demonstrates that specific concentrations of punicalagin or oleuropein in TRIS diluent enhance post-thaw ram sperm quality.

## Key findings

- Punicalagin at 0.1 mg/100 mL improved sperm motility, vitality, and motility patterns in frozen-thawed rams.
- Oleuropein at 1 mg/100 mL enhanced progressive motility and sperm vitality, with higher concentrations affecting acrosome integrity.
- Both antioxidants increased HOST-positive sperm percentages compared to the control group.

## Abstract

The sensitivity of ram sperm to reactive oxygen species is high. The supplementation of antioxidant-rich diluents may help to protect sperm against oxidative damage. This study aimed to evaluate the role of adding natural antioxidants (punicalagin or oleuropein) as a supplement to the basic TRIS extender on the post-thaw quality parameters of spermatozoa from Najdi rams. The results exhibited that the addition of punicalagin or oleuropein to the diluent could boost the quality of ram sperm through improving the viability, total motility and progressive motility, and patterns of motility in the considered spermatozoa.

This study aimed to investigate the effects of adding different levels of punicalagin or oleuropein to TRIS diluent on the quality of frozen–thawed semen from Najdi rams. Semen was diluted using TRIS-based diluter with 15% egg yolk (control group); supplemented with 0.1, 0.5, or 1 mg/100 mL punicalagin (in Experiment 1); or supplemented with 1, 2.5, or 5 mg/100 mL oleuropein (in Experiment 2). The collected semen was evaluated and cryopreserved, with the motility and concentration of sperm assessed using a CASA system. The results showed that the total motile spermatozoa (TMS), percentage of progressive motile spermatozoa (PMS), curvilinear velocity (VCL), rectilinear velocity, average path velocity (VAP), linearity coefficient, straightness index, minor defects, and sperm vitality were higher in the 0.1 mg/100 mL punicalagin group (p < 0.05) than in other groups. HOST% post-thawing was significantly higher in all punicalagin groups compared to the control group (p < 0.001). The percentages of PMS, TMS, VCL, minor defects, and sperm vitality were higher in the 1 mg/100 mL oleuropein group (p < 0.05) than in other groups. Oleuropein supplementation at 5 mg/100 mL decreased VAP in cooled sperms, while all levels increased VAP post-thawing. HOST-positive sperms% post-thawing was higher in all oleuropein-treated groups than the control group (p < 0.001). Moreover, oleuropein nonsignificantly increased the acrosome integrity in cooled sperms, while higher studied concentrations of oleuropein (2.5 and 5 mg/100 mL) decreased the acrosome integrity in frozen sperms. In conclusion, adding punicalagin (0.1 mg/100 mL) or oleuropein (1 mg/100 mL) to TRIS diluent improved the quality of frozen–thawed semen from rams.