# Differential Metabolomic Signatures in Boar Sperm with Varying Liquid Preservation Capacities at 17 °C

## TL;DR

This study identifies unique metabolomic signatures in boar sperm that can predict how well the sperm will survive storage, helping improve artificial insemination success in pig farming.

## Contribution

The study identifies glyceric acid and lysoPC(20:3) as potential biomarkers for predicting boar sperm storage tolerance.

## Key findings

- Sperm with different storage tolerances show distinct metabolomic profiles.
- Glyceric acid and lysoPC(20:3) are key metabolites associated with sperm preservation capacity.
- Differentially expressed metabolites are enriched in amino acid and lipid metabolism pathways.

## Abstract

In modern pig farming, reproduction is predominantly achieved through artificial insemination, which consists of inserting spermatozoa into the female reproductive tract. The success of this technique relies on several factors, especially the quality of the sperm used. Following collection, fresh ejaculates are processed into seminal doses that can be stored for days. However, during storage, the sperm quality declines at different rates among ejaculates, leading to inconsistent fertility outcomes. To identify those with varying storage tolerances early and thus improve reproductive outcomes, we investigated the metabolome—the complete set of small molecules that mirror cell activity—of spermatozoa with differential abilities to withstand storage. The findings revealed that the spermatozoa possess distinct metabolome profiles, which could facilitate early diagnosis upon semen collection. Specifically, the prevalence of two metabolites, glyceric acid and lysoPC(20:3), emerges as a practical indicator for the timely characterization of semen with differential abilities to sustain storage. These insights have the potential to improve semen dose management, boosting reproductive efficiency and profitability.

In the swine industry, artificial insemination (AI) primarily uses chill-stored semen, making sperm preservation crucial for reproductive success. However, sperm quality declines at varying rates during chilled storage at 17 °C, distinguishing high-survival semen from low-survival semen. This study investigates the metabolomic profiles of boar sperm with different abilities to survive liquid storage. We analyzed sperm motility, kinematics, and morphology in freshly extended (Day 0) and 7-day stored AI semen doses. The AI semen doses were classified as high-motile (HM) or low-motile (LM) based on sperm motility after 7 days of storage (Day 7). Metabolomic data were collected in positive (ESI+) and negative (ESI−) ion modes using a Vanquish Flex UPLC coupled with a Q Extractive Plus. We consistently detected 442 metabolites (251 in ESI+, 167 in ESI−, and 24 in both) across samples and storage durations. In freshly extended and 7-day stored AI doses, we identified 42 and 56 differentially expressed metabolites (DEMs), respectively. A clustering analysis showed significant changes in DEMs between the HM and LM samples. These DEMs were mainly enriched in amino acid metabolism, the pentose phosphate pathway, glycerolipid metabolism, glyoxylate and dicarboxylate metabolism, terpenoid backbone biosynthesis, etc. In summary, this study highlights the metabolomic differences between semen doses with varying abilities to survive liquid storage. Glyceric acid and lysoPC(20:3) emerged as potential markers for sperm preservation.