Sexing of mid-incubation avian embryos as a management tool for zoological breeding programs

Skewed sex ratios in zoo breeding programs may require housing single birds of an overrepresented gender, increasing demands on limited resources that could otherwise be diverted to breeding pairs or other important species. The ability to selectively incubate and hatch eggs of a desired sex represents a significant improvement in the long-term management of avian species. This study describes a successful method for in ovo sexing of embryos from stage 30 through 42 of incubation (Hamburger and Hamilton [1951] J Morphol 88:49-92). A 0.01-1 µl blood sample was collected from either the vitelline vessel (VV) or the blood vessels of the chorio-allantoic membrane (CAM) of embryos at stages 14-18 or 30-42, respectively. DNA was isolated from whole blood using the Chelex method (Walsh et al. [1991] Biotechniques 10:506-513; Jensen et al., [2003] Zoo Biol 22:561-571). Sex was determined by PCR amplification using the previously described P2/P8 (Griffiths et al. [1998] Mol Ecol 7:1071-1075) and 1237L/1272H (Kahn et al. [1998] Auk 115:1074-1078) primers or by commercial vendor. Success rate was calculated as the percent of sampled embryos surviving to hatch. Embryos of the undesired sex were not incubated, thus not included in the calculation. There was a considerable difference in success rate when blood was collected from the stage 14-18 VV (0-25%, average 12%) vs. stage 30-42 CAM (33-100%, average 76%). In conclusion, in ovo sexing of embryos between stages 30 and 42 yields acceptable embryo survival rates while providing enough blood for genetic testing.