Chris Shaw and Dr. Robert Park, Zoology
In recent years, ostriches have been used more frequently by ranches to fill the market demand for low fat meat, exotic leather and different oils for lubrication. As the numbers of ostriches have increased, the demand for a reliable way to determine the gender of young ostriches has risen as well. The problem with sexing comes from the fact that the cloacas of young ostriches don’t develop until they are nearly six months old. Without the developed cloaca, it is impossible to differentiate the males birds from the females. Often, the birds are sold as breeding pairs when they are three months old, but not knowing the sex of them creates obvious problems. There is another methods for determining sex in ostriches using a technique called micro satellite mapping. This project provided an equally effective alternative to it.
Random Amplified Polymorphic DNA (RAPD) is a powerful, but somewhat temperamental technique for finding genetic differences in two groups of animals. It has been used in the past for differentiating between groups of pigs with desirable but unobservable characteristics, determining the genealogy of an individual animal, and distinguishing between the sexes in various types of animals. This project focused on finding a sex linked marker for ostriches that could eventually be used to determine the sexes of young ostriches through an alternative technique.
Ostrich blood samples were taken from a group of fifteen males and fifteen females. The red blood cells were broken down by enzymes and the DNA was extracted from them. The extraction method used repeated washings with phenol chloroform to separate the proteins and purify the DNA. Once free from protein, the DNA was diluted and screened using RAPDs.
RAPDs is an application of the PCR and was used for this project because the arbitrarily sequenced tenmer primers were inexpensive and the polymorphic DNA fragments it produces in amplification are easily separable by size in an agarose gel. After amplification, the DNA fragments were subsequently dyed with ethydium bromide and illuminated under UV light. Nearly 2500 reactions were done, but only five primers produced DNA bands that were different in the females and the males. These bands were called markers. The markers were found in every one of the females and it were absent in all but one of the males. Fortunately, the male that showed the female marker was positive for all five markers. This could mean that the sample was mislabeled, the adult ostrich was incorrectly identified as a male or he was genetically aberrant.
Hopefully this work will be used to create a longer nucleotide probe and make a better, more specific PCR test for gender determination in ostriches.
Figures 1-3 show three of the five polynucleotide products of the primers. The arrows point to the female polymorphisms which will be refined and used as markers. The arrow on the male side is pointing to the aberrant male #111M.