Large scale sex typing of ostriches using DNA extracted from feathers
© Malagó et al; licensee BioMed Central Ltd. 2002
Received: 19 June 2002
Accepted: 1 October 2002
Published: 1 October 2002
Ostrich (Struthio camelus) breeds have been gaining increasing significance around the world. The large-scale sex determination of chicks is an important task in the development of these breeds. To date, two PCR-based methods have been established for ostrich sex typing, neither of them intended for large-scale analyses. Here, we report on a protocol adapted to carry out large-scale gender scoring using DNA obtained from chick feathers.
The DNA was extracted using a fast and simple alkaline extraction protocol that provided sufficient template for an early diagnosis. Tests with several primer sets enabled us to determine the best internal control primers associated with the sex-specific primers, avoiding spurious bands. Using DNA extracted from a single bulb and the best set of primers, we applied this protocol to simultaneously sex-type 96 individuals accurately.
We have established a fast, safe, accurate and inexpensive procedure for large-scale sex typing of ostriches using DNA extracted from feathers. This procedure is useful for the gender identification of chicks in the first days of nestling life.
In many bird species, it is very difficult to distinguish between males and females based on an analysis of their external morphology, especially young birds. Moreover, every discriminating procedure employed should be safe, accurate, fast and inexpensive. Karyotyping may be a good choice for the sex typing of birds but, unfortunately, the low divergence of Z and W chromosomes precludes this approach in ostriches [1, 2]. On the other hand, conventional techniques to identify the sex of ostriches present significant problems: the animals may suffer stress, since they are subjected to invasive procedures such as endoscopy and cloaca touch. Furthermore, the bird's sex can be identified only in the adult stage, and even then only with a low rate of efficiency (in juvenile birds the rate of error may reach up to around 40%). In the last few years, two reports described PCR-based methods to sex-type ostriches [3, 4]. A method by which small PCR bands are produced, was established by . Another method uses sex-specific primers for a larger female fragment associated with internal standard primers in amplification reactions . Both these methods involved the use of DNA extracted from blood and they were not yet adapted for large-scale analysis. This paper presents an improved protocol, beginning with DNA extracted from feathers bulbs, which can provide sex-identification in the first days of nestling life. Furthermore, we used different control primers to determine the best sets for sex typing and to avoid any kind of spurious band. Finally, we adapted the procedure for use with 96 wells PCR plates, providing a large-scale method.
Results and Discussion
In this report, we have established a fast, safe, accurate and inexpensive procedure for large-scale sex typing of ostriches using DNA extracted from feathers. The method described here begins with a quick and easy DNA extraction step that dispenses with the use of phenol chloroform. Furthermore, the use of chick feathers provides an early diagnosis that can be adapted for large-scale analyses being useful for the sex identification of chicks in the first days of nestling life.
A group of 5-day-old chicks was subjected to sex typing according to . One male and one female individual were selected for the analysis described herein. They were also subsequently sex identified by conventional techniques at 3 months of age. Additional analyses of about 100 individuals were made to verify the reliability and accuracy of the procedure reported herein.
Sequences of primer pairs used in multiplex reactions and expected sizes of PCR products
Length of PCR product
We are indebted to Mr. Ariovaldo de Freitas from Fazenda Três Irmãs (Samua Comercial Ltda.), Araraquara, SP, Brazil, for providing the feathers of the animals. DNA Consult Genetics and Biotechnology S/C Ltda., São Carlos, SP, Brazil and the PIPE program from FAPESP supported this work.
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