approach for rapid sexing embryo

The ability to determine animal gender could improve efficiency of animal production and animal selection. In dairy systems, sex determination would permit the specific production of either male or female embryos. In commercial dairy operations, female embryos of high genetic merit would be preferred for use as replacement stock. Artificial insemination organizations making planned matings would prefer males for their potential value as sires. A reliable method of sex determination is required for such a system.

Several methods have now been reported for mammalian sex determination as following: (i) Using polymerase chain reaction (PCR) to amplify a Y-chromosome-specific repeat sequence, DYZI, that is present 500-8000 times on the Y chromosome. The presence of this amplified target sequence indicates the presence of the Y-specific DNA sequence in the sample DNA and, hence, indicates that the animal is male. A disadvantage of this technique is that the absence of the amplified target sequence may be due either to the absence of the sequence in the sample DNA or to a failed reaction, (ii) Second method involves PCR-based genotyping. In brief, a pseudo-autosomal region (common to both X and Y chromosomes) present in both X and Y chromosomes is amplified. These homologous regions are called ZFY (in the Y chromosome) and ZFX (in the X chromosome). The amplified product is then digested with restriction enzymes that take advantage of restriction enzyme fragment length polymorphisms between ZFY and ZFX. The digested DNA is separated via electrophoresis. The ZFX and the ZFY DNA are identified by their different digestion patterns, (iii) Another PCR-mediated approach uses three sets of primers for sex determination of pre-implantation bovine embryos. As a positive control, the first set of primers amplifies a bovine-specific satellite DNA sequence. Thus, both male and female samples have this amplified target sequence. Two pairs of primers that recognize repetitive bovine Y-chromosome-specific sequences are then used in a PCR amplification reaction and (iv) Lastly and recently, novel technique was reported and developed. The main objective of our study is to develop improve in rapid sexing method for preimplantation embryos of bovine using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. This rapid and simple detection system was improved for bovine embryo sexing by adding ethidium bromide (EB) or CuSO4 at different concentrations to the product of LAMP reaction. The result of these additions was a colour change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. This study showed that the present method can be applied in breeding programs to facilitate manipulation of the sex ratio of offspring. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing both the bovine and buffalo embryos.

The sex determination assay developed in this study will be a useful adjunct to the technologies of embryo transfer and cloning. A sex determination assay of this kind will also facilitate scientific studies of gender differences in embryonic and fetal gene expression and development.