Embryo Sexing
Embryo sexing technique is used to predetermine the sex of offspring. Widespread use of embryo transfer technique has created the scope to control the sex of an offspring. The technique is of high demand for the conservation of endangered species and also in various laboratories and pet animals.
Embryo sexing is an alternative to sexed semen technology as sorted semen is expensive. There are two commonly used procedures, invasive and noninvasive methods.24.7.1 Invasive method
24.7.1.1 Karyotyping/Cytogenetic Analysis
A limited number of cells are taken from a biopsy and grown in a medium containing colcemid, a mitotic cell division stopping drug. The cells are then made to swell, causing the chromosomes to scatter. After being fixed and stained with a permanent DNA dye, such as Giemsa, the slides are viewed under a microscope. In metaphase, cells produce a spread of chromosomes that may be distinguished by their distinct banding patterns. The tiny size of the Y chromosome makes it easy to spot. The accuracy of sexing is 100% in this procedure but the process is labor-intensive and timeconsuming.
24.7.1.2 Y-Specific DNA Probe
In this technique, a tiny number of cells from the embryo are biopsied, and perfect cellular DNA from these cells is hybridized to a tagged sequence of DNA that is unique to the Y chromosome and hence the embryo’s male sex. Probes against several Y chromosomal genes like SRY, ZFY, and DDX3Y have been generated in livestock species like cattle, sheep, and embryonic sex has been determined.
24.7.1.3 Fluorescent In Situ Hybridization (FISH)
Y chromosome-specific DNA probes are labeled with DNA labeling materials like digoxigenin or avidin. The probe is hybridized to prefixed embryonic biopsy material on a slide with a suitable hybridization buffer and condition. Finally, washing is done with appropriate buffer and bound digoxigenin- or avidins-labeled probes are detected with fluorescin-conjugated anti-digoxigenin or anti-avidin antibody (Fig. 24.6).
24.7.2 Noninvasive method
Circulating cell-free fetal DNA (ccff DNA): Analysis of free fetal DNA analysis in maternal circulation is a noninvasive and effective method for determining fetal sex in animals.
24.8