Efeitos da redução ou substituição do soro fetal bovino por outros compostos na maturação in vitro de oócitos bovinos / Effects of reduction or replacement of fetal calf serum by other compounds during in vitro maturation of bovine oocytes

A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). [...] Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9por cento, 43,2 por cento, 43,1 por cento e 36,5 por cento, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4 por cento. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3 por cento, 51,3 por cento e 50,8 por cento dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.(AU)

The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). [...] Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9 percent, 43.2 percent, 43.1 percent and 36.5 percent in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4 percent). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3 percent, 51.3 percent and 50.8 percent of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5 percent without affecting nuclear and cytoplasmic maturation rates.(AU)

Suplementação proteica no meio de cultivo in vitro de folículos pré-antrais / Protein supplementation on In Vitro Preantral Follicle Medium Culture

The in vitro culture of preantral follicles is an important tool to elucidate the mechanisms involved in controlling early folliculogenesis as well as to improve reproductive efficiency by providing a large number of oocytes for in vitro production of embryos. To this end, improvements in the performance of current culture systems are needed, including changes in the composition of the medium. Supplementation of culture medium with proteins is a great strategy to improve survival and in vitro development of preantral follicles. This review highlights the importance of in vitro culture of preantral follicles with emphasis on the main protein sources used to supplement the culture media.

Suplementação proteica no meio de cultivo in vitro de folículos pré-antrais / Protein supplementation on In Vitro Preantral Follicle Medium Culture

The in vitro culture of preantral follicles is an important tool to elucidate the mechanisms involved in controlling early folliculogenesis as well as to improve reproductive efficiency by providing a large number of oocytes for in vitro production of embryos. To this end, improvements in the performance of current culture systems are needed, including changes in the composition of the medium. Supplementation of culture medium with proteins is a great strategy to improve survival and in vitro development of preantral follicles. This review highlights the importance of in vitro culture of preantral follicles with emphasis on the main protein sources used to supplement the culture media.(AU)

Is the early reduction of fetal calf serum concentration in bovine in vitro embryo culture beneficial?

This study was designed to investigate the effect of supplementation with 5 or 10% fetal calf serum (FCS) on bovine embryo development in three types of culture media added with amino acids (aa): Charles Rosenkrans 2 medium (CR2aa), potassium simplex optimized medium (KSOMaa) and synthetic oviduct fluid (SOFaa). In vitro matured (IVM) and fertilized (IVF) oocytes were in vitro cultured (IVC) from Day 1to 10 of development. The increase in FCS in CR2aa media did not affect embryo development and hatching. However, the increase from 5 to 10% FCS in KSOMaa and SOFaa induced a decrease in the number of blastocysts. Embryo hatching was similar for embryos cultured with 5 or 10% FCS in both media. We concluded that a lower concentration of FCS increased embryo development rate in SOFaa and KSOMaa media and that there was no detrimental effect of high FCS concentration in CR2aa.(AU)

Is the early reduction of fetal calf serum concentration in bovine in vitro embryo culture beneficial?

This study was designed to investigate the effect of supplementation with 5 or 10% fetal calf serum (FCS) on bovine embryo development in three types of culture media added with amino acids (aa): Charles Rosenkrans 2 medium (CR2aa), potassium simplex optimized medium (KSOMaa) and synthetic oviduct fluid (SOFaa). In vitro matured (IVM) and fertilized (IVF) oocytes were in vitro cultured (IVC) from Day 1to 10 of development. The increase in FCS in CR2aa media did not affect embryo development and hatching. However, the increase from 5 to 10% FCS in KSOMaa and SOFaa induced a decrease in the number of blastocysts. Embryo hatching was similar for embryos cultured with 5 or 10% FCS in both media. We concluded that a lower concentration of FCS increased embryo development rate in SOFaa and KSOMaa media and that there was no detrimental effect of high FCS concentration in CR2aa.

Efeito de sistema de cultivo, célula somática e soro em co-cultura sobre o desenvolvimento de embriões bovinos fecundados in vitro / Effects of culture system, somatic cells and serum in co-culture on the development of in vitro fertilized bovine embryos

O objetivo deste experimento foi o de avaliar o efeito de sistemas de cultivo e de diferentes células somáticas e soro bovino na co-cultura sobre a produção de embriões bovinos fecundados in vitro. No experimento um avaliou-se o efeito do sistema de co-cultura com células da tuba uterina e do sistema "definido". No experimento dois utilizaram-se células da granulosa ou da tuba uterina para a co-cultura em meio CR1aa (Charles Rosenkrans). No experimento três utilizou-se soro de vaca em cio (SVC) ou soro fetal bovino (SFB), ambos em co-cultura com células da granulosa em CR1aa. Os ovócitos utilizados foram obtidos de ovários colhidos em matadouro e maturados in vitro em meio 199 com soro de vaca em cio e FSH por 24h. Após a maturação, os ovócitos foram fecundados in vitro por 22h e posteriormente divididos aleatoriamente nos tratamentos. Avaliaram-se a taxa de clivagem no dia três do cultivo, a produção de blastocistos nos dias sete e oito, e de blastocistos eclodidos nos dias nove e dez. Não houve diferença entre os sistemas em co-cultura e "definido" quanto à taxa de clivagem (80,7% e 75,4%) e de produção de blastocisto (19,4% e 17,7%). Entretanto, a taxa de blastocistos eclodidos foi superior para o sistema em co-cultura (37,5%) quando comparado com o sistema "definido" (8,7%). O cultivo embrionário em células da tuba uterina ou da granulosa resultaram em taxas de clivagem, produção de blastocisto e blastocistos eclodidos semelhantes entre si (65,5% e 66,7% de clivagem, 11,6% e 13,7% de blastocistos e 23,1% e 50,0% de blastocistos eclodidos; P>0,05), bem como o cultivo com SFB ou SVC (63,9% e 70,2% de clivagem, 14,3% e 8,7% de blastocisto e 41,2% e 33,3% de blastocistos eclodidos; P>0,05). Conclui-se que o sistema de cultivo "definido" pode ser utilizado para estudos com cultivo de embriões in vitro, no entanto, os resultados quanto à taxa de eclosão ainda são inferiores ao sistema em co-cultura... (AU)

The aims of this study were to evaluate the effects of culture system and different somatic cells and bovine serum in co-culture on in vitro production of bovine embryos. On the first experiment oviduct epithelial cells for co-culture system in CR1aa (Charles Rosenkrans) medium for "defined" system were studied. On the second experiment granulosa or oviduct epithelial cells, both in CR1aa medium were used. On the third experiment estrous cow serum (ECS) or fetal calf serum (FCS), both in granulosa cells co-culture in CR1aa medium was used. Bovine cumulus-oocytes complexes, obtained from ovaries collected at slaughterhouse, were matured in vitro in medium 199 added with ECS and FSH for 24h. Right after, the oocytes were fertilized in vitro for 22h and randomly divided in the treatments of the experiments. The cleavage rate was evaluated on day three, the blastocyst production on seventh and eighth days and the hatched blastocysts on ninth and tenth days. No differences were obtained (P>0.05) between co-culture and "defined" systems on the cleavage rate (80.7% and 75.4%) and on the blastocysts production (19.4% and 17.7%). However, the hatched blastocyst rate was higher (P<0.05) in the co-culture than in the "defined" system (37.5 and 8,7%, respectively). Similar results of embryo co-culture in oviduct epithelial and granulosa cell cultures were obtained regarding to similar cleavages, blastocyst and hatched blastocyst rates (65.5% and 66.7% of cleavage, 11.6% and 13.7% of blastocyst and 23.1% and 50.0% of hatched blastocyst; P>0.05), as well as with FCS and ECS (63.9% and 70.2% of cleavage, 14.3% and 8.7% of blastocyst and 41.2% and 33.3% of hatched blastocyst; P>0,05). The results show that the "defined" system can be used for in vitro embryo culture studies. Nevertheless, the hatched blastocyst rate was higher in co-culture system. Granulosa and oviduct epithelial cells had similar effects on the in vitro embryonic development... (AU)