Metabolomics of stallion seminal plasma: Identification of potential fertility biomarkers based on differences between high and low pregnancy stallions.

Overview

• This study analyzed the metabolites in stallion seminal plasma to identify differences between stallions with high and low pregnancy rates and to find biomarkers predictive of fertility.

Background and Rationale

• Seminal plasma (SP) constitutes approximately 98% of stallion ejaculate volume and is secreted by the testes, epididymis, and accessory sex glands.
• Metabolomics is the study of small molecules (metabolites) that represent the endpoints of cellular metabolic pathways, offering insights into physiology, including reproductive health.
• Understanding metabolomic profiles of SP can reveal biochemical markers correlated with fertility status, an area that could improve stallion reproductive management.

Objective

• To characterize the metabolomic profile of stallion seminal plasma.
• To investigate differences in metabolites between stallions with high and low pregnancy rates.
• To identify specific metabolites that serve as potential biomarkers associated with fertility.

Study Design and Methods

• Subjects: 24 Criollo breed stallions sampled once during the breeding season.
• Grouping: Stallions were categorized based on pregnancy rates at day 16 post-insemination into High Pregnancy (HP, ≥60%) and Low Pregnancy (LP, ≤50%) groups.
• Semen Analysis: Evaluations included sperm concentration, motility/kinetics, morphology, and membrane integrity to correlate sperm quality with metabolomic data.
• Metabolomic Profiling: UHPLC-QTOF-MS (Ultra-High Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Mass Spectrometry) was used to identify and quantify metabolites in the SP samples.

Key Findings

• A total of 18 different metabolites were detected in seminal plasma, including amino acids, fatty acids, and other metabolic compounds such as:
  • Acetyl carnitine, carnitine, isoleucine, methylbutanoyl carnitine
  • Ornithine, phenylalanine, tryptophan, taurine
  • Palmitate, lactate, phosphate, oleate
  • Stearate, citrate, fructose, hippurate
  • 1,3-dioxan-5-ol and melatonin
• Metabolite Differences Between Groups:
  • Oleate was found in higher concentrations in the Low Pregnancy (LP) group (statistically significant, P < 0.05).
  • Taurine and phosphate were enriched in the High Pregnancy (HP) group (statistically significant, P < 0.05).
• Correlation Analysis:
  • Pearson correlation analysis showed that 13 metabolites had significant associations with reproductive outcomes.
  • Phosphate and 1,3-dioxan-5-ol were the most strongly correlated metabolites related to fertility parameters.
• Phosphate’s Role:
  • Phosphate plays an essential role in sperm energy metabolism, particularly involving pyrophosphate breakdown, which is critical for generating the energy required for sperm motility and function.
  • This role underpins phosphate’s potential as a biomarker for stallion fertility.

Conclusions and Implications

• The study identifies specific metabolites in seminal plasma that differentiate stallions with high and low fertility, advancing understanding of molecular mechanisms involved in subfertility.
• Phosphate emerges as a promising biomarker for predicting stallion fertility, potentially aiding in early identification of subfertile males.
• Findings provide valuable information for reproductive management and optimization strategies in breeding programs by targeting metabolite profiles associated with better fertility outcomes.
• The use of metabolomic profiling adds a novel dimension to fertility assessment beyond traditional semen analysis.