258 FACTORS AFFECTING COMMERCIAL SEXING PROGRAM AND MULTIPLE GENETIC ANALYSIS PERSPECTIVES OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

The present study aimed to evaluate the interactions among different factors on the viability and sex ratio of in vitro-produced (IVP) bovine embryos, submitted to a large scale sexing program. Additionally, whole genome amplification (WGA) technology was used to amplify genomic DNA from IVP bovine embryo biopsies, in order to perform multiple genetic analyses. The survey was performed in a 4650 IVP bovine sexed embryo database. Embryos were biopsied by a microaspiration technique and sex was determined by PCR of DNA from the biopsy. Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified in accordance with embryo sex (male, female, and indeterminate), five laboratories (A, B, C, D, and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein, and Jersey), embryo stage (MO, EB, BL, XB, and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “nonstandard”). The statistical analysis was carried out by association chi-square test, chi-square for a 1:1 ratio, and logistic regression analysis (PROC LOGISTIC) of SAS. PCR showed 93.3% efficiency, 93.2% accuracy, and male and female rates of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Significant differences were not observed between male and female viability, although indeterminate embryos resulted in more death after micromanipulation. For quality 2 and 3 embryos, the mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryos. For embryos whose biopsies were classified as nonstandard, the embryonic mortality rate was 3.6-fold higher than standard ones. Mortality rate was not affected by embryo stage at biopsy (P > 0.05). Although sex ratio was significantly skewed to male embryos, differences were not observed among laboratories (P > 0.05) and breeds (P > 0.05) on the sex ratio of IVP bovine embryos. To test the feasibility of using WGA method for multiple genetic analysis, biopsies from 28 IVP embryos were submitted to the GenomePlex Single Cell System (Sigma-Aldrich, St. Louis, MO, USA). Aliquots from each DNA sample were purified using column chromatography and submitted to PCR using sexing primers BRY4a, SRY, UMN0920, and S4B. PCR was successful and in agreement among tested DNA aliquots from each single biopsy. The WGA strategy used herein was a useful tool for applications involving restricted amounts of starting genetic material (DNA), such as in preimplantation genetic diagnosis using IVP bovine embryos.

To FAPESP and UNESP.