Today, ostrich breeding has been widely developed in Iran and other countries due to the ability of this animal to produce quality meat, leather, and oil. However, one of the main problems in breeding them is sex determination using aggressive techniques with low accuracy. This study aimed to determine the sex of immature ostriches using specific primers in a multiplex PCR reaction. This study considered 20 specimens of unspecified immature and six specimens (three adult males and females) of known-sex African ostriches as controls. SS and OSFES primers were used to amplify part of the female-specific sequence and 18S primer was used as a control in a PCR reaction. The presence of SS and OSFES bands in gel electrophoresis indicated the amplification of the desired parts related to the female sex and the absence of these bands indicates the male sex of the species. In total, out of 20 African ostriches studied, 50% of them belonged to females and 50% of them belonged to males. Later, with the growth of immature individuals, the results of this experiment were confirmed. In this study, it was found that the use of feather samples for DNA extraction and multiplex PCR is a suitable, accurate, and cost-effective method in identifying and determining the sex of young ostrich and leads to more real and reliable results, avoiding stress in birds.

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