A drop of HTF medium (human tubal fluid medium (Irvine Scientific, CA) containing 12.5 mM Hepes and 0.4% BSA) was made in a dish, covered with mineral oil and maintained at 37 °C. Cauda epididymis was put in the oil and a sperm clump was gently taken out from incisions made with scissors and moved to the buffer with a needle. After incubating for 60 min, sperm that swam up around the buffer wall was transferred to ice-cold PBS-0.5% BSA and collected by centrifugation for 2 min at 2000g at 4 °C. The pellet was gently broken, treated with lysis buffer (0.1% SDS, 0.5% TritonX100, [64 (link)]) for 10 min on ice to lyse somatic cells, and washed with PBS-0.5% BSA.
Five sperm samples (1 × 106 sperm/sample) were obtained from five F1 males of different litters for the control and arsenic groups, respectively. Sperm was lysed in lysis buffer (7.3 mM Tris–HCl (pH 7.5), 7.3 mM NaCl, 18.2 mM EDTA, 1.8% SDS, 1.8% 2-ME, 1.4 mg/ml proteinase K) and DNA was extracted with a phenol–chloroform mixture, precipitated with ethanol, dried and dissolved in 10 mM Tris (pH 7.4).
Five sperm samples (1 × 106 sperm/sample) were obtained from five F1 males of different litters for the control and arsenic groups, respectively. Sperm was lysed in lysis buffer (7.3 mM Tris–HCl (pH 7.5), 7.3 mM NaCl, 18.2 mM EDTA, 1.8% SDS, 1.8% 2-ME, 1.4 mg/ml proteinase K) and DNA was extracted with a phenol–chloroform mixture, precipitated with ethanol, dried and dissolved in 10 mM Tris (pH 7.4).