Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

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)

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

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. (...)

Influência dos tempos de maturação e fecundação e da ativação oocitária com cálcio ionóforo sobre os índices de zigotos monospérmicos produzidos in vitro em suínos

A produção in vitro de embriões suínos tem alcançado altos índices de embriões polispérmicos, os quais atingem o estádio de blastocisto, porém têm qualidade inferior quando comparado aos monospérmicos, apresentado menor massa celular interna, o que pode ser a causa do atraso ou mesmo da paralisação do desenvolvimento embrionário in vitro em suínos. Este trabalho teve como objetivo avaliar os índices de zigotos monospérmicos, observando os efeitos de três tempos de fecundação (2, 4 e 6 horas) e espermatozóides capacitados previamente ou simultaneamente à fecundação in vitro com 2mM de cafeína ou dois tempos de maturação oocitária (36 ou 44 horas) e ativação oocitária com cálcio ionóforo (0 ou 50?M) durante 4 horas de fecundação in vitro. Foram utilizados oócitos de fêmeas suínas pré-púberes maturados no meio TCM 199 enriquecido com 10 UI de hCG, 10 UI de eCG, 50 UI de eGF e 90?L de pFF nas primeiras 20 horas e sem hormônios nas 22 horas subseqüentes. Para a fecundação in vitro foi utilizado o meio m TBM. Os presumíveis zigotos foram cultivados no meio NCSU 23 acrescido de 4 mg/ml de BSA por 12 horas. Na classificação das estruturas em oócitos não fecundados e zigotos monospérmicos ou polispérmicos foi empregado o Hoechst 33342 para visualização dos pró-núcleos. Os índices de oócitos não fecundados foram de 58,9; 38,6 e 30,1%, de zigotos monospérmicos de 23,3; 40,3 e 41,6% e de zigotos polispérmicos de 17,8; 21,1 e 28,3% para o sêmen capacitado previamente, respectivamente, nos tempos 2, 4 e 6 horas. Para o sêmen capacitado simultaneamente, os índices de oócitos não fecundados foram de 51,6; 49,4 e 41,3%, os de zigotos monospérmicos de 23,6; 30,5 e 32.6% e de zigotos polispérmicos de 24,8; 20,0 e 26,1%, respectivamente, nos tempos de 2, 4 e 6 horas. Para o tempo de 36 de maturação com 0 ou 50?M de CaA, as médias dos quadrados mínimos e desvios padrão, para os oócitos não fecundados foram, respectivamente, de 31,5 ? 7,3 e 43,5 ? 2,5 e para zigotos monospérmicos de 39,1 ? 4,3 e 29,7 ? 4,9 e para zigotos polispérmicos de 32,3 ? 10,7 e 38,6 ? 9,4. Para os oócitos maturados por 44 horas com 0?M e 50?M de CaA, as médias dos quadrados mínimos e desvios padrão para os oócitos não fecundados foram, respectivamente, de 21,7 ? 2,5 e 43,1 ? 2,5 e para zigotos monospérmicos de 20,0 ? 3,9 e 47,4 ? 7,2 e para zigotos polispérmicos de 17,1 ? 5,1 e 30,8 ? 7,0. Concluiu-se que os índices de zigotos monospérmicos foram melhores nos tempos de 4 e 6 horas de fecundação in vitro e que não houve influência da capacitação espermática prévia ou simultânea, assim como do tempo de maturação oocitária, sendo maior às 36 e às 44 horas, respectivamente, na ausência e presença de cálcio ionóforo