Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse.
Authors: Ortiz-Rodriguez José M, Ortega-Ferrusola Cristina, Gil María C, Martín-Cano Francisco E, Gaitskell-Phillips Gemma, Rodríguez-Martínez Heriberto, Hinrichs Katrin, Álvarez-Barrientos Alberto, Román Ángel, Peña Fernando J
Journal: PloS one
Summary
# Editorial Summary: Gene Expression Changes in Embryos from Cryopreserved Equine Sperm Cryopreservation remains central to equine reproductive programmes, yet its impact on early embryonic development has received little direct investigation despite consistently lower pregnancy rates compared with fresh semen. Researchers compared gene expression profiles in embryos collected at days 8, 10, and 12 post-ovulation from matched pairs of mares inseminated with either fresh or frozen-thawed semen from the same stallion, using RNA sequencing and pathway analysis to identify transcriptional differences. Embryos derived from cryopreserved sperm showed significant downregulation of genes across multiple critical pathways—particularly oxidative phosphorylation, DNA replication, DNA binding, and immune response—with consistent changes observed throughout the early developmental window examined. Notably, many of the suppressed genes are orthologs of genes that cause embryonic lethality in mice, suggesting the altered transcriptional profile may compromise developmental competence and survival rates. These findings have important implications for practitioners relying on frozen semen: whilst cryopreservation remains invaluable for genetic preservation and logistical management, the demonstrated molecular compromise warrants consideration of alternative strategies (such as fresh semen protocols where feasible) for critical breeding programmes, and may prompt investigation of semen handling protocols that could mitigate these transcriptional effects.
Read the full abstract on PubMed
Practical Takeaways
- •Cryopreserved sperm may compromise early embryo development at the molecular level, which could explain the lower pregnancy rates observed clinically with frozen sperm compared to fresh sperm in mares
- •Consider using fresh sperm when available for artificial insemination programs, as cryopreservation appears to negatively impact critical developmental gene expression pathways
- •Further research into methods to minimize cryopreservation damage or improve cryoprotectant protocols may be warranted to preserve embryo viability
Key Findings
- •Embryos from cryopreserved sperm showed significantly altered transcriptional profiles compared to fresh sperm embryos across all timepoints (8, 10, and 12 days post-ovulation)
- •Genes involved in oxidative phosphorylation, DNA binding, DNA replication, and immune response were significantly downregulated in cryopreserved sperm-derived embryos
- •Many downregulated genes in cryopreserved sperm embryos are known to be embryonic lethal in mice, suggesting developmental compromise
- •This is the first direct evidence of altered gene expression in embryos resulting from cryopreserved sperm fertilization in any species