Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear

Abstract

RESUMO -Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2) com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO 2 em ar, durante sete dias, e transferidos para receptoras sincronizadas. As taxas de clivagem e desenvolvimento a blastocisto de embriões reconstruídos com células cultivadas por tempo maior foram inferiores às obtidas com os demais tempos de cultivo. Além disso, os blastocistos produzidos não resultaram no desenvolvimento de uma gestação a termo. Embora a taxa de clivagem em embriões fêmeas tenha sido maior, o número de embriões que atingiram o estádio de blastocisto foi maior nos embriões machos. No período gestacional, fêmeas apresentaram maior taxa de aborto entre 90 e 120 dias de gestação. Esses resultados indicam que células doadoras de núcleos cultivados por longos períodos dificultam a produção de blastocistos e aumentam as chances de perdas durante a gestação. Embriões clonados machos têm maior competência para se desenvolver a blastocisto e resultam em menor taxa de perda gestacional.Palavras-chave: bovino, fibroblastos, passagem celular, sexo, transferência nuclear Effect of culture time and gender of nuclei donor cells on bovine development produced by nuclear transferABSTRACT -The objective of this study was to evaluate the effects of culture time and sex of nuclei donor cells on embryo and fetal development after nuclear transfer. Thus, bovine oocytes were matured, enucleated and reconstructed with somatic cells from an adult animal. After fusion and chemical activation, the reconstituted zygotes were cultured in Charles Rosenkranz 2 (CR2) on a granular monolayer cell at 38.8ºC in a humidified atmosphere 5% CO 2 in air for seven days, and transferred to synchronized receptors. Cleavage rates and development to blastocyst of embryo reconstructed with cells cultured for a longer time were lower than rates obtained with other culture times. Moreover, these produced blastocysts did not result in the development of full term pregnancy. Although cleavage rates were higher in female embryos, the number of embryos that reached blastocyst stage was higher in male embryos. During gestation period, females showed higher abortion rates from 90 to 120 days of gestation. These results indicate that cells donnors of nuclei cultured for long periods make the production of blastocysts difficult and increase the chances of losses during pregnancy. Cloned male embryos are more succesful in becoming blastocysts and result in lower gestational loss rate.

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

“…We observed that the developmental potential of SCNT-♀ preimplantation embryos was greater than that of SCNT-♂ embryos. Similar to that, Merighe et al demonstrated that the rates of cleavage and the blastocyst were higher in female bovine SCNT embryos than that of male cloned embryos (Merighe et al, 2010). The epigenetic status was affected by the sex of cloned embryos (Sandhu et al, 2016).…”

“…However, Liu et al reported that the donor cells' sex had significant effects on nuclear transfer efficiency of cloned goats (Liu et al, 2016a). What is more, Merighe et al demonstrated that the rates of cleavage and the blastocyst were higher in bovine SCNT-♀ embryos than that of SCNT-♂ embryos (Merighe, Miranda, Bem, Watanabe, & Meirelles, 2010). Furthermore, the incomplete epigenetic reprogramming of donor that associated with abnormal methylation patterns results in the developmental failure of bovine cloned embryos (Han, Kang, Koo, & Lee, 2003;Kang et al, 2001).…”

Effects of Prolonged in vitro Culture and Cryopreservation on Viability, DNA Fragmentation, Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

“…According Merighe et al. (), the culture period is one of the bottlenecks in NT and transgenesis, because it can destabilize biological events subsequent to the process, compromising embryo development.…”