Avaliação da produção, qualidade e criotolerância de embriões cultivados em sofaaci tradicional e sequencial em três atmosferas de oxigênio
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Data
2021-12-16
Autores
Pastorio, Rafaela Faresin
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Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo avalia a produção, qualidade, criotolerância e estresse oxidativo de embriões bovinos desenvolvidos em dois meios de cultivo, SOFaaci tradicional e SOFaaci sequencial, em diferentes atmosferas de cultivo. Um total de 11640 cumulus oócitos foram maturados em meio de maturação (TCM 199) por 24h. Após os oócitos foram co-incubados com sêmen em meio Talp-FIV por aproximadamente 20h. Logo, os presumíveis zigotos foram cultivados em meio SOFaaci tradicional ou SOFaaci sequencial. Seis grupos experimentais foram formados, MT20= meio tradicional a 20% de O2; MT12= meio tradicional a 12% de O2; MT5= meio tradicional a 5% de O2; MS20= meio sequencial a 20% de O2; MS12 = meio sequencial em 12% de O2 e MS5= meio sequencial em 5% de O2. O cultivo dos embriões com o meio sequencial foi iniciado com SOFaaci A, às 96 e 120hpf era feito a substituição de 75% do volume das gotas de CIV com o meio SOFaaci B. Nos grupos em que foi feito o CIV com meio SOFaaci tradicional, a renovação era realizada da mesma maneira, porém com o próprio SOFaaci tradicional. Ao se completarem sete dias de CIV os embriões foram vitrificados em hastes de vitrificação. Os resultados foram analisados com ANOVA e posterior Teste de Tukey e Teste de X2 com nível de significância à 5%. Na produção embrionária, os grupos em que foi utilizado o meio SOFaaci tradicional foram superiores ao meio SOFaaci sequencial (P=0.028) e a redução na atmosfera de O2 também teve efeito benéfico na taxa de embriões produzidos MT20= 45,3%; MT12= 49,7%; MT5= 53,9%; MS20= 40,2%; MS12= 41,6%; MS5= 45,0%, o número de células totais foi maior nos grupos cultivados em meio tradicional e atmosfera controlada (P<0.05) (MT20=114; MT12= 128; MT5=131; MS20=98; MS12=110; MS5= 112;), tendo, também um maior estresse oxidativo em embriões produzidos em atmosfera controlada (P<0.01) (MT20=4,10; MT12=5,30; MT5=7,46; MS20=4,23; MS12=8,27; MS5=5,93). Os embriões vitrificados e reaquecidos que foram cultivados em SOFaaci tradicional não diferiram entre si na taxa de recuperação nas três atmosferas utilizadas MT20= 60,2%; MT12= 66,6%; MT5= 60,6%. Por outro lado, os embriões cultivados em SOFaaci sequencial apresentaram maior taxa de recuperação na atmosfera de 5% de O2 (P<0,05) MS20= 42,3%, MS12= 53,0%; MS5= 80,1%. Entretanto, a taxa de eclosão dos embriões vitrificados e reaquecidos cultivados em meio SOFaaci tradicional nas atmosferas de 20% e 12% de O2 apresentaram as maiores taxas de eclosão (P<0,05) MT20= 70,7%; MT12= 81,2% e semelhante aos embriões cultivados em SOFaaci sequencial na atmosfera de 12% de O2 MT12= 70,9%. O estresse oxidativo de embriões vitrificados, não diferiu entre os grupos. Concluímos que embriões cultivados em SOFaaci tradicional em atmosferas controladas, apresentam maior produção, número de células e concentrações de espécies reativas de oxigênio. A recuperação após o reaquecimento foi melhor no SOFaaci sequencial na atmosfera de 5% de O2, mas as maiores taxas de eclosão foram obtidas em SOFaaci tradicional e sequencial na atmosfera de 12% de O2.
This study evaluates bovine embryos production, quality, cryotolerance and oxidative stress when developed in two culture medias, traditional SOFaaci and sequential SOFaaci in different culture atmospheres. A total of 11.640 cumulus oocytes were matured in maturation medium (TCM 199) for 24h. Afterwards, the oocytes were co-incubated with semen in Talp-FIV medium for approximately 20h. Therefore, presumptive zygotes were cultured in traditional SOFaa or sequential SOFaaci medium. Six experimental groups were formed, TM20= traditional medium at 20% O2; TM12= traditional medium at 12% O2; TM5= traditional medium at 5% O2; SM20= Sequential medium at 20% O2; SM12 = sequential medium in 12% O2 and SM5= sequential medium in 5% O2. The cultivation of embryos with the sequential medium was started with SOFaaci A, at 96 and 120hpf, 75% of the volume of the CIV drops was replaced with SOFaaci B medium. In groups where the CIV was carried out with traditional SOFaa, the renovation was carried out in the same way, but with the traditional SOFaa itself. At the end of seven days of CIV, the embryos were vitrified in vitrification rods. The results were analyzed with ANOVA and subsequent Tukey Test, and X2 Test with a 5% significance level. In embryo production, the groups in which the traditional medium was used were superior to the sequential medium (P=0.028) and the reduction in the O2 atmosphere also had a beneficial effect on the rate of embryos produced (TM20=45%; TM12=49%; TM5=53%; SM20= 40%; SM12=41%; SM5: 45%), the number of total cells was higher in the groups cultivated in traditional medium and controlled atmosphere (P=0.5) (TM20=114; TM12= 128; TM5=131; SM20= 98; SM12= 110; SM5= 112;), also having a higher oxidative stress in sequential medium and atmosphere embryos controlled (P=0.001) (TM20=4,10; TM12=5.30; TM5=7.46; SM20=4.23; SM12=8.27; SM5=5.93). The vitrified and reheated embryos that were cultivated in traditional SOFaaci did not differ from each other in the recovery rate in the three atmospheres used TM20 = 60.2%; TM12 = 66.6%; TM5 = 60.6%. On the other hand, embryos cultured in sequential SOFaaci higher rate of recovery in atmosphere 5% O2 (P < 0.05) SM20 = 42.3%, SM12 = 53.0%; SM5 = 80.1%. However, the hatch rate of vitrified and rewarmed embryos cultured in traditional SOFaaci medium in 20% and 12% O2 atmospheres outside the highest hatch rates (P <0.05) TM20 = 70.7%; TM12 = 81.2% and similar to embryos cultured in sequential SOFaaci in the atmosphere of 12% O2 SM12 = 70.9%. Oxidative stress of vitrified embryos did not differ between groups. We concluded that embryos cultivated in traditional SOFaaci in controlled atmospheres present greater production, number of cells and groups of reactive oxygen species. Recovery after reheat was better in sequential SOFaaci in 5% O2 atmosphere, but higher hatch rates were upgraded in traditional and sequential SOFaaci in 12% O2 atmosphere.
This study evaluates bovine embryos production, quality, cryotolerance and oxidative stress when developed in two culture medias, traditional SOFaaci and sequential SOFaaci in different culture atmospheres. A total of 11.640 cumulus oocytes were matured in maturation medium (TCM 199) for 24h. Afterwards, the oocytes were co-incubated with semen in Talp-FIV medium for approximately 20h. Therefore, presumptive zygotes were cultured in traditional SOFaa or sequential SOFaaci medium. Six experimental groups were formed, TM20= traditional medium at 20% O2; TM12= traditional medium at 12% O2; TM5= traditional medium at 5% O2; SM20= Sequential medium at 20% O2; SM12 = sequential medium in 12% O2 and SM5= sequential medium in 5% O2. The cultivation of embryos with the sequential medium was started with SOFaaci A, at 96 and 120hpf, 75% of the volume of the CIV drops was replaced with SOFaaci B medium. In groups where the CIV was carried out with traditional SOFaa, the renovation was carried out in the same way, but with the traditional SOFaa itself. At the end of seven days of CIV, the embryos were vitrified in vitrification rods. The results were analyzed with ANOVA and subsequent Tukey Test, and X2 Test with a 5% significance level. In embryo production, the groups in which the traditional medium was used were superior to the sequential medium (P=0.028) and the reduction in the O2 atmosphere also had a beneficial effect on the rate of embryos produced (TM20=45%; TM12=49%; TM5=53%; SM20= 40%; SM12=41%; SM5: 45%), the number of total cells was higher in the groups cultivated in traditional medium and controlled atmosphere (P=0.5) (TM20=114; TM12= 128; TM5=131; SM20= 98; SM12= 110; SM5= 112;), also having a higher oxidative stress in sequential medium and atmosphere embryos controlled (P=0.001) (TM20=4,10; TM12=5.30; TM5=7.46; SM20=4.23; SM12=8.27; SM5=5.93). The vitrified and reheated embryos that were cultivated in traditional SOFaaci did not differ from each other in the recovery rate in the three atmospheres used TM20 = 60.2%; TM12 = 66.6%; TM5 = 60.6%. On the other hand, embryos cultured in sequential SOFaaci higher rate of recovery in atmosphere 5% O2 (P < 0.05) SM20 = 42.3%, SM12 = 53.0%; SM5 = 80.1%. However, the hatch rate of vitrified and rewarmed embryos cultured in traditional SOFaaci medium in 20% and 12% O2 atmospheres outside the highest hatch rates (P <0.05) TM20 = 70.7%; TM12 = 81.2% and similar to embryos cultured in sequential SOFaaci in the atmosphere of 12% O2 SM12 = 70.9%. Oxidative stress of vitrified embryos did not differ between groups. We concluded that embryos cultivated in traditional SOFaaci in controlled atmospheres present greater production, number of cells and groups of reactive oxygen species. Recovery after reheat was better in sequential SOFaaci in 5% O2 atmosphere, but higher hatch rates were upgraded in traditional and sequential SOFaaci in 12% O2 atmosphere.
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Palavras-chave
Estresse oxidativo, Metabolismo, Oxigênio