Combined {"type":"entrez-nucleotide","attrs":{"text":"H33258","term_id":"978675","term_text":"H33258"}}H33258/chlortetracycline fluorescence ({"type":"entrez-nucleotide","attrs":{"text":"H33258","term_id":"978675","term_text":"H33258"}}H33258/CTC) assessment of spermatozoa

{"type":"entrez-nucleotide","attrs":{"text":"H33258","term_id":"978675","term_text":"H33258"}}H33258/CTC dual staining assays were conducted to examine the capacitation status of spermatozoa between HFS and LFS using a dual-staining method^46,57. Shortly, 15 μL of {"type":"entrez-nucleotide","attrs":{"text":"H33258","term_id":"978675","term_text":"H33258"}}H33258 solution were added to 135 μL of samples, and incubated for 10 min at room temperature. 250 μL of 2% polyvinylpyrrolidone in Dulbecco’s phosphate-buffered saline (DPBS) was added, and the mixture was centrifuged at 100 × g for 2.5 min. The supernatant was removed and 100 μL of DPBS and CTC solution were added into pellet. Capacitation status was detected using a Microphot-FXA microscope with ultraviolet BP 340–380/LP 425 and BP 450–490/LP 515 excitation/emission filters for {"type":"entrez-nucleotide","attrs":{"text":"H33258","term_id":"978675","term_text":"H33258"}}H33258 and CTC, respectively. Four different type of patterns were observed; dead (D pattern, blue fluorescence), non-capacitated (F pattern, bright yellow fluorescence presented evenly over the entire sperm head), capacitated (B pattern, bright yellow fluorescence presented over the acrosomal region and a dark post-acrosomal region), or acrosome-reacted (AR pattern, no fluorescence over the head, or yellow fluorescence only in the post-acrosomal region) as previously reported^29,58. For each of the three independent replicate experiments, three samples were used.