# L-Glutamine Supplementation Improves the In Vitro Qualitative Parameters of Cryopreserved Qinchuan Bull Sperm

## TL;DR

Adding L-glutamine to frozen bull semen improves its quality after thawing by reducing damage and boosting antioxidants.

## Contribution

L-glutamine is identified as a novel endogenous cryoprotectant that enhances sperm freezability in Qinchuan bulls.

## Key findings

- L-glutamine supplementation improves post-thaw sperm motility and membrane integrity.
- L-glutamine enhances antioxidant activity and reduces oxidative stress in cryopreserved sperm.
- L-glutamine supports mitochondrial function and energy metabolism in thawed sperm.

## Abstract

For ease of storage and transportation, semen is typically preserved by freezing. However, cryopreservation inevitably exposes sperm to oxidative stress and other damages, leading to reduced sperm quality. Thus, exploring the mechanisms underlying sperm freezability and developing novel sperm cryoprotectants are urgent priorities. In this study, metabolomic analysis was used to characterize metabolite changes in semen with high and low freezability. The results revealed significant differences in metabolite composition between semen with high freezability and that with low freezability, with differential metabolites primarily enriched in amino acid metabolism and lipid metabolism pathways. Notably, among these differential metabolites, the abundance of L-glutamine (L-Gln) in the high freezability group was significantly higher than that in the low freezability group. Based on this, we further investigated the role of L-Gln in sperm freezability and found that supplementation with L-Gln significantly improved freeze–thaw sperm motility, acrosome and plasma membrane integrity, as well as enhanced antioxidant capacity and energy metabolism. This suggests that L-Gln holds promise as an endogenous cryoprotectant to alleviate sperm damage during cryopreservation. These findings provide a theoretical basis for improving semen freezability and contribute to enhancing reproductive efficiency in livestock production.

Semen cryopreservation is a crucial technology for enhancing reproductive efficiency in livestock production; however, oxidative stress-induced sperm damage during the freeze–thaw process remains a significant challenge. In this study, metabolomics was used to analyze the differences in metabolites in semen from Qinchuan cattle with different freezing tolerance, and to screen out the candidate markers of sperm freezing tolerance. The metabolomics results indicate that a total of 264 differential metabolites were identified, and KEGG pathway annotation revealed that amino acid metabolism (15.07%) were prominently represented, and L-glutamine (L-Gln) showed a particularly high abundance in high freezability group (HFG) compared to the low freezability group (LFG). Further experiments demonstrated that L-glutamine supplementation significantly improved post-thaw sperm motility, plasma membrane integrity, and acrosomal integrity (p < 0.05). It also enhanced sperm antioxidant capacity by increasing the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), while reducing malondialdehyde (MDA) content (p < 0.05). Additionally, L-Gln maintained mitochondrial function and energy homeostasis by elevating mitochondrial membrane potential (MMP) and promoting AMPK phosphorylation (p < 0.05). These results indicate that L-glutamine alleviates oxidative damage during cryopreservation and enhances semen freeze tolerance.