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

Abstract

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.

Keywords: chilled storage; metabolites; metabolome; motility; pig; sperm.

Figure 1

Ranking of boar semen samples based on sperm motility on Day 7 post-storage. A total of 26 single-sire boar semen samples or doses (S1 to S26) were evaluated on Day 7, and semen were classified as low (LM), medium (MM), or high motile (HM).

Figure 2

Sperm motility of high- (HM) vs. low-motile (LM) freshly extended (Day 0) and 7-day stored (Day 7) boar semen. (A) Total motility and (B) progressive motility. N = 6 for each group; ** p ≤ 0.01.

Figure 3

Sperm morphology of high-(HM) vs. low-motile (LM) freshly extended (Day 0) and 7-day stored (Day 7) boar semen. (A) Normal morphology. (B) Sperm defects. N = 6 for each group; * p < 0.05.

Figure 4

Sperm velocities of freshly extended (Day 0) and 7-day stored (Day 7) boar semen. (A) Average path velocity (VAP); (B) curvilinear velocity (VCL); (C) straight-line velocity (VSL). N = 6 for each group; * p < 0.05.

Figure 5

Distribution of boar sperm metabolites in chemical classes.

Figure 6

OPLS-DA score plots showing the separation between high-motile (HM) and low-motile (LM) boar spermatozoa on Day 0 (A) and Day 7 (B) of storage based on metabolome. Data were acquired in positive (ESI+) and negative (ESI−) ion modes.

Figure 7

Distribution of differentially expressed metabolites between low- and high-motile semen in freshly extended (Day 0) and 7-day stored (Day 7) samples.

Figure 8

Selected differentially expressed metabolites candidate biomarkers detected in both freshly extended (Day 0) and 7-day stored (Day 7) boar semen.

Figure 9

Boxplots of selected differentially expressed metabolites between high- and low-motile semen common to Day 0 (Upper panel) and Day 7 (Lower panel) of storage. HMD0 and HMD7: high-motile semen on Day 0 and Day 7; LMD0 and LMD7: low-motile semen on Day 0 and Day 7. * p < 0.05 and ** p ≤ 0.01.

Figure 10

Mfuzz clusters of metabolites detected in the positive (A) and negative (B) ion modes. The clusters illustrate metabolite expression patterns between low- and high-motile spermatozoa on Day 0 and Day 7 of storage. Each individual line depicts the profile of a metabolite across groups and storage durations. Different colors depict different degrees of membership in the cluster. Violet, dark blue, light blue—high degree of membership; green—medium degree of membership; and yellow—poor degree of membership. Semen groups are represented by A for HMD0, B for LMD0, C HMD7, and D for LMD7.