Machado Grazieli M, Ferreira Allice R, Guardieiro Monique M, Bastos Michele R, Carvalho José O, Lucci Carolina M, Diesel Tiago O, Sartori Roberto, Rumpf Rodolfo, Franco Maurício M, Dode Margot A N
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, Av. W5 Norte final, Caixa Postal 02372, Brasília, DF 70770-917, Brazil.
Reprod Fertil Dev. 2013;25(4):600-8. doi: 10.1071/RD11282.
The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo- and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down- and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality.
本研究旨在比较使用孵化后发育(PHD)系统完全在体外产生的第14天牛胚胎与第7天至第14天未进行或进行短暂PHD培养的体内来源胚胎。第14天的胚胎用于使用实时定量(q)聚合酶链反应(PCR)进行性别鉴定以及PLAC8、KRT8、CD9、SLC2A1、SLC2A3、PGK1、HSF1、MNSOD、HSP70和IFNT的基因表达分析。首先,将第7天体内和体外产生的胚胎置于PHD系统中。在第14天观察到体外胚胎的存活率更高。比较完全在体内或完全在体外产生的第14天胚胎发现,前一组的平均大小大于后一组(分别为10.29±1.83与2.68±0.33mm)。在体外胚胎中,HSP70和SLC2A1基因的表达分别下调和上调。本研究表明,在PHD系统中培养的体外胚胎比体内胚胎小,并且在分析的10个基因中,两组之间只有两个基因差异表达。这些发现表明,由于存活率低,PHD系统对于评估体外胚胎质量不可靠。