Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production
Oliveira, Viviane Purri deMarques, Mariana GrokeSimões, RenataAssumpção, Mayra Elena Ortiz DÁvilaVisintin, José Antonio
Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel- free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10 8 cells/mL. The sperm suspension was incubated for 2 h at 25°C, refrigerated and maintained for 1 h at 15-18°C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 μL saline was prepared. In the dark, 40 μL PI/CFDA final solution was added to 10 μL semen and after 8 min, slides were analyze d on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount ® , diluted 2:1 in xilol to avoid staining oxidation. (...)(AU)
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