Htm ivos version 12.3 motility analyzer

Sperm were collected from cauda epididymides of B6D2_F1 mice, capacitated in HTF medium supplemented with 4 mg/ml BSA for 1.5 hr. To assay the effect of Zn²⁺ on zonae pellucidae, Astl^Null eggs were collected, treated with hyaluronidase and pre-incubated in HTF medium supplemented with 50 μM MgSO₄ or ZnSO₄ for 1 hr. Alternatively, to mimic zinc sparks released from eggs, M2 medium with 2 mM of MgSO₄ or ZnSO₄ was injected into the perivitelline space of Astl^Null eggs using a FemtoJet 4i (Eppendorf) until the zona pellucida was swollen. Under each experimental condition, eggs were then thrice washed in 30 μl droplets of HTF medium, inseminated with capacitated sperm (3×10⁵ ml⁻¹ final concentration) in 100 μl of HTF and incubated for 6 hr. To determine the effect of Zn²⁺ on mouse sperm motility, Capacitated sperm were added in 100 μl of HTF with 25–50 μM MgSO₄ or ZnSO₄ at a final concentration of 5×10⁵ ml⁻¹. Sperm motility were determined by a HTM-IVOS (Version 12.3) motility analyzer (Hamilton Thorne).