PCR-SEXING OF BOVINE EMBRYOS - A SIMPLIFIED PROTOCOL

To make bovine embryo sexing under farm conditions more feasible we de veloped a simplified protocol utilizing manual biopsy and detection of the Y chromosome directly from polymerase chain reaction (PCR) reacti on tubes. Twenty-four embryos (morulae and blastocysts) were biopsied manually into 2 to 4 samples. One sample of each original embryo was d iagnosed for sex, based on restriction fragment length polymorphism of PCR-amplified DNA of the ZFX/ZFY locus. The remaining 44 samples were diagnosed using the tube detection assay. In this assay the biopsies were pipetted into 0.5 -ml reaction tubes containing lysis mixture, in cubated 10 to 60 min at 37 degrees C and inactivated 10 min at 98 degr ees C. Then the PCR mixture was added containing buffer, DNA polymeras e, ethidium bromide and primers designed to amplify the highly repeate d btDYZ-1 region of the bovine Y chromosome. After 50 cycles of PCR, t he reaction tubes were examined under UV illumination for pink fluores cence indicating the presence of Y-chromosomal DNA. All sexing results from the replicates were in agreement with the ZFX/ZFY assay, with 12 of the original embryos diagnosed as females and 12 as males. We conc lude that highly efficient and accurate PCR-sexing of embryos can be a ccomplished without the use of micromanipulators, control primers and electrophoresis. The 2 reaction mixtures needed for sex diagnosis can be stored at -20 degrees C and -196 degrees C, respectively. The tube detection assay minimizes the risk of carryover contamination by previ ously amplified products as there is no need to open the tubes followi ng PCR.