Sperm Collection and Preparation for IVF

Cauda epididymides were collected from both groups, cut using micro-spring scissors, squeezed, and transferred to 1 mL of human tubal fluid (HTF) medium [25 (link)]. The medium consisted of 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM K₂PO₄, 0.2 mM MgSO₄∙7H₂O, 2 mM CaCl₂, 25 mM NaHCO₃, 2.78 mM glucose, 0.33 mM sodium pyruvate, 21.4 mM, sodium lactate, 286 mg/L penicillin G, 228 mg/L streptomycin, and 5 mg/mL bovine serum albumin. After incubation for 60 min at 37.5°C in 5% CO₂ in humidified air, the upper layer of the medium containing motile and capacitated sperm was collected. Sperm from 10 animals in each group were used for subsequent IVF experiments, and sperm from 3 animals in each group were used for DNA extraction.