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

Abstract

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.

Molecular Sexing of the White-Winged Guan (<b><i>Penelope albipennis</i></b>) and Other Wild Birds of the North of Peru

“…We determined that the sexing marker HPF/HPR is the most accurate and reliable, since it showed a consistency of 100% in the determination of sex by PCR of 24 cracids, 5 falconids, and 8 accipitrids that had been previously sexed ( Table 3 ). Our results are similar to those reported by Li et al [2015], who used these primers for the sexing of Chinese chickens and ducks. The second marker evaluated (CHD1Wr/NP/CHD1Zr) showed a low capacity for sex resolution, with an error rate of 50% in previously sexed cracids ( Table 3 ) and 41.07% in cracids of unknown sex ( Table 4 ).…”

“…4 B, 5 B). Similar results have been documented in the sexing of Chinese ducks and domestic chickens, as well as in specimens of the families Phasianidae and Anatidae in comparison with the primer sets P2/P8, 1237L/1272H, and 2550F/2718R [Li et al, 2015].…”

“…In the present work, we tested the HPF/HPR primers developed by Li et al [2015] and found that they have a high capacity for sex discrimination in P. albipennis and other 8 species of cracids. Likewise, we studied the applicability of the technique in 4 species of falconids, 4 species of accipitrids, and 3 species of psittacines, verifying their effectiveness and reliability in previously sexed birds.…”

Characterization of microRNAs during Embryonic Skeletal Muscle Development in the Shan Ma Duck

“…Almost all authors checked the morphology of the embryos to ensure the correct developmental timing. Embryonic sex was determined by PCR amplification of the Chromo-helicase-DNA-binding 1 (CHD1) gene with DNA from the liver as a template [19]. Three breast muscle samples of E13 and E19 female embryos, respectively, were selected for RNA extraction.…”

Long‐term stability of sex chromosome gene content allows accurate qPCR‐based molecular sexing across birds

“…Despite the popularity and apparent ease of the PCR‐based methods, molecular sexing using the CHD1 gene can be inaccurate in some avian species due to (i) the preferential amplification of CHD1 ‐ W or CHD1 ‐ Z allele in females leading to pseudomale identification due to a single visible band in the electrophoretic gel (Medeiros et al, 2012), (ii) polymorphisms in size of the CHD1 ‐ Z allele resulting to pseudofemale identification (Casey et al, 2009; Dawson et al, 2001), or (iii) small size variation between the two gametologues below the detection efficiency of the electrophoresis (Zhang et al, 2013). In several avian species, PCR with the standard CHD1 primers results in poor or a lack of amplification (Chang, Gu, et al, 2008; Li et al, 2015; Reddy et al, 2007; Sulandart & Zein, 2012; Wang & Zhang, 2009), and/or variation in the pattern of bands in the electrophoresis gel (Çakmak et al, 2017). In addition, the PCR conditions, especially the annealing temperature and the selection of suitable primers need to be adjusted for a given species, which makes the method time consuming (Faux et al, 2014).…”