# Effects of Selenium Nanoparticles and Sodium Selenite Supplementation on Cryopreserved Ram Sperm Quality, Oxidative Status, and PRDX5 Gene Expression

## TL;DR

Adding low-dose selenium nanoparticles to ram semen extenders improves frozen-thawed sperm quality better than conventional selenium.

## Contribution

Low-dose selenium nanoparticles improve cryopreserved ram sperm quality more effectively than sodium selenite.

## Key findings

- Low-dose selenium nanoparticles (1 µg/mL) significantly improved sperm motility, viability, and morphology compared to controls.
- Chromatin condensation abnormalities were lowest in the selenium nanoparticle-treated group.
- Selenium nanoparticle supplementation did not significantly affect total oxidant status or PRDX5 gene expression.

## Abstract

The freezing and thawing process of ram semen can damage sperm cells and reduce their fertility. Antioxidants are added to semen extenders to help protect sperm during this process. In this study, we compared the effects of different doses of conventional selenium and selenium nanoparticles on semen freezing. Our results showed that a low dose of selenium nanoparticles provided the best protection after thawing. These findings suggest that adding a low dose of selenium nanoparticles to semen extenders is a simple and effective way to improve the quality of frozen–thawed ram semen.

Cryopreservation of ram semen is an essential tool in assisted reproductive technology; however, oxidative stress generated during the freezing process may compromise sperm quality. This study evaluated the effects of Se and SeNPs on post-thaw sperm quality, PRDX5 expression, and oxidative status in cryopreserved ram semen. In this study, semen samples collected from five mature rams (three collections at 2-week intervals, yielding a total of 15 ejaculates) were frozen in liquid nitrogen using extenders supplemented with selenium (1 μg/mL, S1; 10 μg/mL, S2) or selenium nanoparticles (SeNPs; 1 μg/mL, N1; 2 μg/mL, N2) alongside a nonsupplemented control extender. Post-thaw sperm quality was evaluated using computer-assisted sperm analysis (CASA) for motility, kinematic parameters, viability, membrane integrity (HOST) assays, chromatin condensation assessment, and morphological analysis. Total oxidant status (TOS) measurements and PRDX5 gene expression analysis were performed separately. Low-dose SeNPs (1 µg/mL) significantly improved total motility (55.73 ± 19.01%), progressive motility (25.05 ± 15.34%), viability (57.27 ± 19.30%), HOST-positive spermatozoa (50.87 ± 18.91%), and morphologically normal spermatozoa (88.27 ± 4.10%) compared with the control and high-dose sodium selenite groups (p < 0.05). Chromatin condensation abnormalities were lowest in the SeNP-treated group. S1 and N2 also improved motility and morphology compared with the control; however, the increases were numerically smaller than those observed in the N1 group. In contrast, S2 supplementation showed limited benefit, with values that were similar to those of the control. Morphologically normal spermatozoa were highest in N1, followed by S1 and N2, while S2 and the control exhibited the lowest values (p < 0.05). In contrast, no significant differences were detected in TOS or PRDX5 gene expression among the experimental groups (p > 0.05). These findings indicate that low-dose SeNPs enhance post-thaw sperm functional integrity and cryotolerance without inducing measurable changes in bulk oxidative markers or gene transcription.