Seminal plasma MicroRNA dynamics in stallion semen due to progressive motility and conception success.

Overview

• This study investigates the relationship between specific microRNAs (miR-34b, miR-122, and let-7a) in stallion seminal plasma with sperm motility and conception success, aiming to assess their potential as biomarkers for fertility.

Study Objective

• To analyze the expression patterns of three microRNAs (miR-34b, miR-122, and let-7a) in seminal plasma of stallions.
• To examine how these microRNAs correlate with progressive sperm motility and conception outcomes after artificial insemination.
• To evaluate the potential of these microRNAs as biomarkers for stallion semen quality and fertility prediction.

Study Design and Methods

• Fifteen adult stallions were enrolled, each providing semen samples.
• Samples collected included 1 ml of seminal plasma stored at -80 °C, 2 ml of fresh semen for sperm analysis, and additional semen used for artificial insemination of one mare per stallion.
• Stallions were grouped based on progressive motility into:
  • High Progressive Motility (HPM; n=8)
  • Low Progressive Motility (LPM; n=7)
• Pregnancy outcomes at day 14 were used to classify into:
  • Positive Pregnancy (n=8)
  • Negative Pregnancy (n=7)
• Sperm motility was measured using a computer-assisted semen analyzer (CASA).
• Flow cytometry assessed various sperm parameters including:
  • Viability
  • High Mitochondrial Membrane Potential (HMMP)
  • Plasma Membrane Integrity (PMAI)
  • Non-capacitated sperm rate
  • Lipid Peroxidation (LPO) parameters
• MicroRNA expression levels quantified by quantitative PCR (qPCR).
• Bioinformatics analyses conducted for:
  • Target gene prediction of miRNAs
  • Protein-protein interaction (PPI) network construction
  • Functional enrichment analysis to identify biological pathways regulated by the target genes

Key Findings

• Sperm motility measurements:
  • Total motility was significantly higher in the HPM group (82.13% ± 3.04) compared to LPM (p < 0.01).
  • Progressive motility was also significantly greater in HPM (45.88% ± 6.03, p < 0.001).
  • Non-capacitated sperm rate was higher in HPM (45.76% ± 3.92; p < 0.05).
• MicroRNA expression differences:
  • miR-34b and miR-122 were significantly upregulated in HPM seminal plasma compared to LPM:
    • miR-34b showed approximately 11-fold increase (p < 0.05)
    • miR-122 showed about 6-fold increase (p < 0.05)
  • A positive correlation was observed between miR-34b expression and total motility (correlation coefficient 0.674; p < 0.01).
• Bioinformatics and network analysis:
  • Target gene prediction revealed 102 genes and 421 interaction edges forming a regulatory network influenced by the studied miRNAs.
  • Significant protein-protein interaction networks were identified involving 19 key proteins.
  • Functional enrichment analysis indicated these miRNAs regulate critical biological pathways potentially linked to sperm function and fertility.

Interpretation and Implications

• The elevated levels of miR-34b and miR-122 in seminal plasma are associated with improved progressive motility of stallion sperm, suggesting these miRNAs play roles in regulating sperm quality.
• The positive correlation between miR-34b and total motility supports its function as a biomarker candidate for semen quality assessment.
• Bioinformatics findings suggest these miRNAs modulate key genes and protein networks important for sperm viability, motility, and fertilization capability.
• These miRNAs have potential use as non-invasive biomarkers to predict stallion fertility and improve breeding management decisions by selecting samples with higher conception success likelihood.
• Further research is warranted to validate these miRNAs in larger populations and to explore their mechanistic roles in sperm physiology.

Conclusions

• miR-34b and miR-122 levels in stallion seminal plasma vary with progressive sperm motility and correlate with fertility outcomes.
• They present promising molecular markers for semen quality evaluation and conception success prediction in stallion breeding programs.
• Understanding miRNA-mediated regulatory mechanisms may aid in developing improved reproductive biotechnologies for horses.