Cloning and sequencing of buffalo male-specific repetitive DNA: Sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequen ced in order to develop a sensitive method for sexing of buffalo preimplant ation stage embryos using polymerase chain reaction(PCR). A highly sensitiv e and reliable sex determination assay using a primary (BRY.I), nested (BuR YN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuR YN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZF Y loci was amplified to serve as a positive control for both male and femal e samples. Accuracy of the sex determination assay was initially verified w ith genomic DNA obtained from blood of known gender. Further sensitivity an d reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging fro m the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from a ll embryo samples. Accuracy of assays was determined by comparing results f rom a single cell with those of blastocyst stage embryos, thereby indicatin g that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficie nt for sex determination by PCR. No misidentification was observed within t he embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuR YN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable met hod for sexing buffalo embryos. (C) 1999 by Elsevier Science Inc.