Semen Sexing - Anatomy & Physiology

Principle

X and Y chromosomes contain different quantities of DNA.
X-bearing sperm contain 2.8-4.2% more DNA (species dependent) than Y-bearing sperm.
This allows them to be sorted into two subpopulations.
Separation requires the uptake of DNA stain/dye called Fluorochrome into living and dead sperm.
X-bearing sperm take up more dye than Y-bearing sperm.
Vital dyes emit light at a specific wavelength when excited or activated.
- X-bearing sperm will emit a larger signal than Y-bearing sperm.
This allows the sperm to be sorted by flow cytometry.

Schematic Diagram to show Semen Sexing by Flow Cytometry

^{©Nottingham University 2008}

Ejaculated spermatozoa are treated with a DNA dye (fluorochrome).
X-bearing sperm absorb more dye than Y-bearing sperm.
X-bearing sperm thus emit more intense light when excited by a laser.
Sperm are also treated with a dye that greatly suppresses the signal from dead sperm.
- Dead sperm are therefore identified and rejected.

Once spermatozoa enter the flow cytometer chamber, they pass single-file through a small nozzle.
At a region just outside the nozzle, an excitation laser beam activates the fluorescent dye in each sperm.
Each live sperm produces an emission with an intensity that is directly related to the quantity of DNA within the sperm head.
- X-bearing live sperm produce more intensity.
A light sensing device is coupled to a computer that determines the intensity of light emission by each sperm and the order of passage of each sperm through a column below the nozzle.
When the sperm pass through charged plates, they are assigned either a positive or negative charge depending on their DNA content (X or Y chromosome).
When the microdroplet containing a single sperm passes through an electromagnetic field the computer applies an appropriate charge and directs the droplet (and sperm) to one side or the other.
Dead sperm are discarded into the centre tube.
At the end there are three vessels that contain sperm:
- One contains a high proportion of X-bearing sperm.
- One contains a high proportion of Y-bearing sperm.
- One contains dead sperm