# L-Selenomethionine Alleviates Cryo-Induced Ferroptosis Through the NRF2–SLC7A11–GPX4 Pathway, Improving Post-Thaw In Vitro Quality of Dairy Goat Spermatozoa

## TL;DR

L-Selenomethionine improves frozen dairy goat sperm quality by preventing cell death through a specific antioxidant pathway.

## Contribution

L-SeMet is shown to alleviate cryo-induced sperm injury via the NRF2–SLC7A11–GPX4 pathway, offering a novel metabolic intervention for cryopreservation.

## Key findings

- L-SeMet at 6 μM improved sperm motility, membrane integrity, and mitochondrial function.
- L-SeMet reduced oxidative stress markers and activated the NRF2–SLC7A11–GPX4 antioxidant pathway.
- Proteomic and metabolomic analyses linked L-SeMet to ferroptosis inhibition and metabolic pathway enrichment.

## Abstract

Background: Cryopreservation induces oxidative stress, membrane disruption, and mitochondrial injury in spermatozoa, leading to impaired motility and fertility. Selenium, as an essential trace element, protects cells from oxidative damage through selenoproteins such as glutathione peroxidase 4 (GPX4), a critical enzyme that detoxifies lipid hydroperoxides and inhibits ferroptosis. This study investigated whether supplementation with L-selenomethionine (L-SeMet), an organic selenium source with superior bioavailability and lower toxicity than inorganic forms, could alleviate cryo-induced sperm injury by suppressing ferroptosis. Methods & Results: Dairy goat sperm were cryopreserved with 0, 2, 4, 6, 8, 10 μM L-SeMet. Supplementation with 6 μM L-SeMet significantly improved motility, membrane and acrosome integrity, and mitochondrial membrane potential. Biochemical assays showed reduced iron, ROS, and MDA levels, alongside increased ATP, SOD, and GSH contents. Proteomic analysis identified 148 differentially expressed proteins, including up-regulation of GPX4, FTH1, VDAC2, and VDAC3—core ferroptosis regulators. Metabolomic profiling further revealed enrichment in unsaturated fatty acid biosynthesis, amino acid metabolism, and the TCA cycle, pathways closely linked to ferroptosis regulation. Transmission electron microscopy confirmed that L-SeMet preserved mitochondrial ultrastructure. Mechanistically, L-SeMet mirrored the ferroptosis inhibitor N-acetyl-L-cysteine and reversed RSL3-induced oxidative damage. Western blotting verified activation of the NRF2–SLC7A11–GPX4 antioxidant axis and inhibition of KEAP1 expression. Conclusions: Collectively, these findings demonstrate that L-SeMet protects spermatozoa from cryo-induced injury by stabilizing redox homeostasis, maintaining mitochondrial function, and inhibiting ferroptosis. The results highlight ferroptosis as a critical mechanism of sperm cryodamage and identify L-SeMet as a promising metabolic intervention to enhance post-thaw sperm quality and fertility.