Animal Biotechnology

METHODS

Sexing the sperm

This is done by using flow cytometer attached with a sorter. In this method, the sperm is stained with a stain – Hoechst 33342 and then sex specific sperms are separated by using the flow cytometer’s sorter. This method has an accuracy of 90 % sexing sperms. Commercially it is used in cattle.

Embryo sexing

Y-specific DNA probes

• Sexual differentiation of mammalian embryos is determined by the presence or absence of genetic elements located on the Y-chromosome. Molecular genetic techniques allow the detection of specific DNA sequences in interphase cells.
• Sex determination with Y-specific probes involves the identification and cloning of a probe (a DNA fragment with a nucleic acid sequence found only on the Y-chromosome) and the use of this probe to diagnose the presence or absence of Y-chromosomal DNA in a sample of embryonic cells.

PCR

• 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,

PCR-RFLP

• 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.