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.

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