A New Primer for Sex Identification of Ducks and a Minimally Invasive Technique for Sampling of Allantoic Fluid to Detect Sex during Bird Embryo Development.

During the early incubation period of the duck, from embryonic day 1 to 13, a precise identification of the sex may be difficult. In a preliminary test, we found a defect in the use of the classical P2/P8, 1237L/1272H, and 2550F/2718R primers for chromo-helicase-DNA-binding 1 gene (CHD1) as a PCR-based test to identify sex in ducks. Therefore, universal PCR primers HPF/HPR for sexing ducks were designed. The PCR product was cloned, sequenced, and analyzed using GenBank. The effectiveness of the primers was compared using samples of blood and feathers from adult birds and chorioallantoic membranes and allantoic fluid (AF) of embryos as a source of DNA. The 495-bp CHD1-Z and the 351-bp CHD1-W PCR amplicons could be easily distinguished on a 3% agarose gel, and females (ZW) displayed 2 visible bands whereas only a single band was found in males (ZZ). The results indicated that HPF/HPR primers were highly efficient and more reliable than the classical primers used for sexing ducks. During the design of the new primers, an AF sampling technique was established to collect a very small amount of AF from free-living birds. This technique, which was minimally invasive, had no adverse effects on either embryos or the post-hatching survival of young ducks and could be used in developmental biology research in birds.