Cook medium

Sperm from cauda epididymides were released into Cook medium (Cook Medical) and squeezed from vas deferens.

To count sperm, cauda epididymides and vas deferens were harvested in pre-warmed (37°C) Cook medium (Cook Medical). 20 μl of a sperm suspension was diluted in 500 μl of Cook medium and counted in a hemocytometer using an AxioPlan 2 (Carl Zeiss) microscope. Isolated sperm motility was determined by computer assisted sperm analysis (CASA) of path velocity (VAP), straight velocity (VSL), curvilinear velocity (VCL) using HTM-IVOS (Version 12.3) motility analyzer (Hamilton Thorne). Sperm were further observed for morphological changes by light microscopy after staining for DNA with Hoechst 33342. In addition, acrosome exocytosis of the isolated sperm was induced by 20 μM calcium ionophore (A23187, Sigma Aldrich) in pre-warmed HTF media followed by incubation (37°C, 5% CO₂, 90 min).

Sperm was collected from the cauda epididymis of 12-week-old male mice. Mature MII oocytes in HTF containing 10% KnockOut^TM serum replacement (Thermo Fisher Scientific, Fair Lawn, USA) were inseminated with capacitated spermatozoa (1–2 × 10⁶/mL) and incubated for 6 h. Fertilized oocytes were transferred to COOK medium (COOK, Queensland, Australia), covered with mineral oil, and incubated at 37 °C in a humidified 6% CO₂ atmosphere. The cleavage rate was recorded on day 2, and the number of embryos that reached the blastocyst stage (hatching and/or hatched blastocyst) was assessed on day 5.