129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

Abstract

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO₂ in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality.

Financial support: Embrapa, CNPq, CAPES.