# Supplementation of Trimethylamine N-Oxide or Betaine in Semen Improves Quality of Boar Spermatozoa Stored at 17 °C Following Hydrostatic Pressure Stress

## TL;DR

Adding TMAO or betaine to boar semen helps protect sperm quality under high pressure when stored at 17°C.

## Contribution

This study shows that TMAO and betaine, alone or combined, reduce high-pressure-induced sperm damage in boars.

## Key findings

- Optimal concentrations of TMAO (8 mmol/L) and betaine (20 mmol/L) significantly improved sperm viability and membrane integrity.
- Betaine increased total antioxidant capacity and reduced oxidative stress markers in boar sperm under high pressure.
- Lipidomic analysis revealed that betaine and the combination treatment modulated sphingolipid and phosphatidylcholine signaling pathways.

## Abstract

HP, as an isotropic physical stress, has been widely applied in cell biology and reproductive research to simulate the effects of environmental pressure on cellular functions. In this study, the elastic silicone membrane of a novel bionic insemination catheter was employed as the pressure medium, with semen perfused into a sealed silicone chamber. As the silicone membrane underwent controlled deformation, the liquid inside the chamber generated a nearly uniform isotropic pressure, thereby maintaining spermatozoa in a stable HP environment. Boar sperm are susceptible to physiological and functional damage under HP stress, which can impair fertilization capacity. This study aimed to investigate the effects of TMAO, BET, or their combination on the quality of semen from eight Landrace boars under HP during storage at 17 °C (experiment repeated three times). Semen was collected using the manual collection method and treated with different concentrations of TMAO or BET. Sperm motility parameters were assessed using a CASA system to determine the optimal concentrations. Subsequently, experimental groups were established: the fresh group, HP control group, T group (optimal TMAO), B group (optimal BET), and H group (optimal TMAO + BET). The results showed that the optimal concentrations were 8 mmol/L for TMAO and 20 mmol/L for BET. Compared with the HP control group, the T, B, and H groups showed significantly improved sperm viability, mitochondrial membrane potential (MMP), and plasma membrane integrity (p < 0.05), and significantly reduced DFI, ROS, MDA, and NO contents (p < 0.05), while acrosome integrity showed no significant differences (p > 0.05). Additionally, the B group showed significantly increased T-AOC (p < 0.05). Non-targeted lipidomic analysis revealed 49 differential lipids in the T group, 262 in the B group, and 269 in the H group compared with the HP control. These differential lipids were mainly associated with PC, AcCa, and sphingolipid signaling pathways, with key sphingolipid pathway lipids including Cer, SM, and DG. These findings indicate that BET and TMAO + BET improve HP-induced sperm damage by modulating the sphingolipid signaling pathway and maintaining PC and AcCa levels, whereas TMAO alone may exert protective effects through additional mechanisms. In conclusion, TMAO, BET, or their combination effectively mitigates the detrimental effects of HP on boar sperm.