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

Reproductive efficiency is fundamental to sustainable aquaculture and the maintenance of biomedical model organisms. Short-term storage of zebrafish (Danio rerio) sperm is critical for routine breeding and spermatology research, as existing extenders typically preserve usable sperm quality for ≤ 24 h. Here, we combined seminal plasma characterization with stepwise extender screening and optimization to improve cold storage outcomes. Zebrafish seminal plasma showed a mean osmolality of 251.4 ± 5.2 mOsmol kg⁻¹, a NaCl-dominated ionic profile (Na⁺ 98 mM, Cl⁻ 122 mM), moderate K⁺ (29.4 mM), low Ca²⁺ (0.66 mM), and pH 7.85, providing a physiological basis for extender formulation. Male zebrafish produce a very small amount of milt; therefore, we first validated pooled sperm as an experimental unit. Five independent pools did not differ in motility or velocity across 36 h of storage (P > 0.05). In extender screening (E1-E20), formulations with NaCl ≥ 120 mM and 40 mM KCl best preserved function; at 36 h, E13 and E15 maintained 40.67% and 49.0% motility with measurable velocities, whereas controls were immotile. Further optimization (E21-E27) identified E27 (150 mM NaCl, 40 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM Tris, pH 7.5; ∼385 mOsmol kg⁻¹) as superior, retaining ∼52% motility at 36 h and sustaining velocity (VCL and VSL) significantly above all other treatments (P < 0.0001). Delaying extender addition by 2-4 h resulted in irreversible loss of motility and velocity, indicating that immediate ionic/osmotic stabilization is critical.