Pysical Characteristics of Freemartins

Normal (left) vs. Freemartin (right) External Genetalia

Parrish, 2008

Normal (left) vs. Freemartin (right) Internal Genetalia

Parrish, 2008

Freemartins lack an anterior vagina and have an enlarged clitoris.

Additional abnormalities seen on freemartins: small seminal vesicles, absent cervix, and disconnected uterin horns and vagina

(Cavalieri and Farin, 1999)

Lab Detection of Freemartins

Blood typing

• Performed on both the male and female twins to display 2 blood group populations through the detection of different red cell surface antigens (Long, 1990)
• Expensive procedure and requires twice as many blood samples (Ennis, 1999)

Karyotyping

• Provides definite detection of freemartins
• Involves staining of cells after cell arrest during cell division
• Tedious, time-consuming, and expensive (Ennis, 1999)

Cytogenetic testing

• Analysis of tissue cultured peripheral blood lymphocytes to identify XY cells in suspected freemartins (Long, 1990)
• Process
  • Culture heparinized whole blood
  • Addition of material, such as poleweed mitogen, to stimulate lumphocyte proliferation
  • Mitotic nuclei harvested, stained, and examined under light microscopy for sex chromosomes in metaphase spreads
• 95-99% accurate
• Requires greater presence of male cells for detection than some of the other tests
• Requires extremely careful collection and handling of samples, is labor intensive, and requires cytogenetics expertise (McNeil, 2006)

Polymerase Chain Reaction (PCR) based testing

• Process
  • Obtain blood samples
  • Select sequence to amplify
    • Example: AMX/Y because AMX/Y allele on Y chromosome contains a base pair deletion that the AMX/Y allele on the X chromsome does not
  • Products evaluated using gel electrophoresis and visualized under UV light
    • Look for bands associated with each type of chromosome, X and/or Y
• Can detect existance of XY cells when as little as 0.1% in female blood (McNeil, 2006)

(Fujishiro, 1995)

Loop-mediated isothermal amplification (LAMP)

• Process
  • Peripheral blood samples are obtained from female calves twin to bull calves
  • Peripheral blood mixed with Loopamp Bovine Embryo Sexing Kit
  • Male-specific DNA amplification of target sequence at constant temperature between 60-65 degrees F for 15-60 minutes
  • During amplification of target sequence, white precipitate is derived as a by-product of the LAMP Reactio
  • Turbidity measurement of reaction allows for rapid detection of a positive reaction without electrophoresis
• Can detect existance of XY leukocytes when as little as 0.01% in female blood (Hirayama, 2007)

(Hirayama, 2007)

Fluorescence in situ hybridization

• Process
  • Peripheral blood samples taken by jugular venipuncture
  • Lymphocytes from whole blood cells directly prepared by hypotonic treatment
  • Synthesis of 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence
  • Labeling of probe with digoxigenin by PCR
  • FISH performance on metaphase spreads and interphase nuclei of blood lymphocytes
  • Probe binds to nucleus of male cell (Sohn, 2007)

Probes bind to nuclei of male cells

(Sohn, 2007)

Next page: Uses for Freemartins

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