Estudo da viabilidade de embriões bovinos pós-biópsia e da amplificação de todo o genoma: modelo experimental para a realização do diagnóstico pré-implantação (PGD)

Abstract

The impact of biopsy technique on posterior embryo development is still poorly studied and there is no single universally practiced protocol for implementing PGD. Another thing to consider is the small amount of genomic DNA available to perform the genetic study, due to the reduced number of cells obtained from the biopsy. Alternatively, methodologies using whole genome amplification (WGA) have been developed. Using these methods, it is possible to generate microgram quantities of DNA starting with a little genomic DNA. The aim of this study was to evaluate the effect of biopsy at 8-to 16-cell bovine embryos on subsequent development and WGA on removed blastomeres. A group of 706 complexes cumulus oocytes (CCOs) obtained from local slaughterhouse ovaries were matured and fertilized in vitro, in incubator at 38.8°C with 95% humidified air and 5% of CO2. The zygotes were semidenuded and cultured in CR2aa medium under the same conditions as with in vitro fertilization. On the fourth day after fertilization the 8-to 16-cell embryos were distributed randomly across two groups: control (n=103) and biopsy (n=92). The amount of cells removed cells was one-fourth the embryo of blastomeres. The removed blastomeres were submitted to whole genome amplification (WGA) followed by PCR. The embryos were returned to culture for development evaluation. The chisquare test was used for statistic evaluation of the results of blastocyst rate on the eight day after fertilization and hatching rate on tenth day between biopsy and control group, to test the WGA efficiency and to determine the sex proportion of biopsied samples submitted to PCR. The number of cell per embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The blastocyst proportion was evaluated with Wilcoxon test. Differences were considered significant if P<0.05. A total of 92 embryos were submitted to biopsy technique. The blastocyst production was 53.3%, with 44.9% of hatching rate. This rate was similar to control group (66.0% and 42.6%) found on 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. The proportion of blastocyst in different stages of development on the 8th day did not differ among groups (P>0.05). Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of samples were sexed by PCR. In conclusion biopsy of 8-to 16-cell bovine embryos did not affect subsequent development. WGA was successful in removed blastomeres and was possible to determinate the sex in the samples.