Desenvolvimento de embriões bovinos e de ratas cultivados in vitro em meios utilizados em embriões humanos

Abstract

The objective was to evaluate culture systems proposed to use in human embryos: single culture system and sequential culture system, compared with culture used in experimental model bovine and Wistar rats. Specifically we evaluated the cleavage and blastocyst rate, embryo kinetics, quantification of gene expression in bovine blastocysts and evaluated the viability and quality by morphology and Tunel of embryos derived from female rats. For bovine cumulus-oocyte complexes were matured, fertilized in vitro. In experiment 1 zygotes were distributed in culture systems: CR2aa, SOFaa, sequential culture system ECM®/Multiblast® and single culture system Global®. We evaluated cleavage and blastocyst rate and gene expression PRDX, HSP70.1, GLUT1 and GLUT5 in blastocysts. In experiment 2, the embryo development was evaluated on the development of bovine embryos biopsied. The zygotes were distributed in culture systems: CR2aa, sequential culture system G1e/GZ) and ECM®/Multiblast®, single culture system Global®. For all groups, the 8-16 cells embryos were biopsied and returned to culture. We evaluated cleavage and blastocyst rate. For Wistar rats, we used animals of the Center for Biology of Reproduction (CBR/UFJF), with twelve weeks of life and superovulated with 150IU/kg of PMSG and 75 IU/kg hCG intraperitoneally. In experiment 1 the embryo collection proceeded 24 hours after hCG and we evaluated embryo viability In experiment 2 rats were distributed between the control and superovulated and embryos were collected at 48 and 72 h after hCG administration. Weight of ovaries, the total number of embryonic structures and quality of embryos were analyzed. In experiment 3 embryo collection proceeded to 72 h after hCG administration. We evaluated the quality of embryos cultured in vitro from superovulated rat through the blastocyst rate and apoptotic index. Already in experiment 4 embryo collection was performed 48 h after hCG administration. Embryos recovered were randomized according to the groups: sequential culture system ECM®/Multiblast®, single culture system Global®, KSOM. We evaluated the rate of blastocyst. Statistical analysis was performed using the chi-square test, Student's test, ANOVA and gene expression was analyzed with REST®. In experiment 1 bovine cleavage rate was similar between groups, but the total blastocyst rate was lower in the sequential system and expanded blastocyst rate was higher in single culture system. Furthermore, the embryo cultured in single culture resulted in higher expression of GLUT1 and GLUT5 (P <0.01) and similar expression levels of HSP70.1 and PRDX1 compared to embryos cultured in sequential media. In experiment 2 both cleavage andblastocyst rate was similar between bovine groups. For rats in experiment 1, 98.2% collected embryos were were considered viable. In experiments 2 superovulated group showed ovaries with greater weight and greater number of embryos, compared with control group. However, embryos from superovulated group showed smaller number of cells 72 hours after hCG administration. In experiment 3 the blastocyst rate was 83.17%, with 8.91% ± 10:32 apoptotic rate of. In experiment 4 the total blastocyst rate was lower in sequential system (13.6%) compared to KSOM (24.0%) and single culture system (32.9%) (p <0.05). It was concluded that the culture of embryos in single medium is more favorable than sequential media, superovulation was successful in a rat model and the TUNEL technique proved feasible in evaluating the quality of embryos from embryonic Wistar rats.