Short-term preservation techniques for zebrafish (Danio rerio) sperm.

Area of Science:

• Aquatic biology
• Reproductive biology
• Animal science

Background:

• Sustainable aquaculture and biomedical research rely on reproductive efficiency.
• Current zebrafish sperm extenders preserve quality for only 24 hours, limiting breeding and research.
• Optimizing sperm storage is crucial for zebrafish (Danio rerio) model organism maintenance.

Purpose of the Study:

• To develop and optimize a novel extender for improved short-term cold storage of zebrafish sperm.
• To characterize zebrafish seminal plasma to inform extender formulation.
• To extend the usable quality of zebrafish sperm beyond the current 24-hour limitation.

Main Methods:

• Characterized zebrafish seminal plasma osmolality, ion concentrations (Na⁺, Cl⁻, K⁺, Ca²⁺), and pH.
• Validated pooled sperm as a reliable experimental unit for motility and velocity assessments.
• Screened and optimized various extender formulations (E1-E27) based on seminal plasma composition and sperm function.

Main Results:

• Zebrafish seminal plasma has a mean osmolality of 251.4 mOsmol kg⁻¹, NaCl-dominant ions, and pH 7.85.
• Formulations with ≥120 mM NaCl and 40 mM KCl best preserved sperm function.
• Optimized extender E27 (150 mM NaCl, 40 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM Tris, pH 7.5) retained ~52% motility and sustained velocity at 36 hours.
• Immediate extender addition was critical to prevent irreversible loss of sperm motility and velocity.

Conclusions:

• A novel extender formulation (E27) significantly improves short-term cold storage of zebrafish sperm up to 36 hours.
• Understanding seminal plasma composition is key to developing effective sperm extenders.
• This advancement supports sustainable aquaculture and enhances the utility of zebrafish as a biomedical model.