Pre-Hatch Sexing for Chicken Embryo Based on Immuno-Interrogation

Abstract

Intensive selection for commercial poultry production has resulted in highly divergent layer and broiler pedigree lines. However, the sex ratio after hatching remains approximately 1:1, resulting in a large-scale culling of male chicks in the industrial egg-laying sector, which raises concerns about animal welfare and ethics. Therefore, early (pre-hatch) sex identification is considered to be essential to address ethical / animal welfare issues, and the Canadian egg production industry aims to discontinue the practice of killing male chicks after hatching. Determining the sex of the chick embryo using non-invasive or minimally invasive approaches before hatching is the focus of intensive research. In birds, males possess the ZZ genotype, and females the ZW genotype; therefore, sex is determined by the presence of the W sex chromosome. The detection of W chromosome–encoded protein(s) in embryonic tissues may allow for sex prediction. We hypothesized that W-encoded proteins may be detectable in the chorioallantoic membrane (CAM), an extraembryonic tissue, using immunochemical approaches. The objective of this research project was to develop and validate an innovative immunochemical approach for sex determination in-ovo by detecting the presence of CAM protein(s) encoded on the W chromosome at an early embryonic day (ED 6, 8, or 10). In this study, various CAM manipulation techniques were identified and evaluated, including the use of eggshell (ES) surface disinfectants, ES drilling, various surgical tools and sealant methods. My results indicated that CAM at ED 6 is more challenging to sample, with lower retention of embryo viability (assessed at ED 16) than at ED 8 and 10, which are more compatible with embryo viability. LC/MS/MS-based proteomics was utilized to evaluate the protein profile of CAM tissue harvested at embryonic days (ED) 6, 8, 10, and 12, in order to highlight the expression of distinct CAM functionalities during embryonic development and to gain insight into possible sex-based differences. A total of 2,688 CAM proteins were identified, with 2,347, 2,265, 2,351, and 1,267 proteins detected at ED 6, 8, 10, and 12, respectively. Notably, 1,191 common proteins were identified across all stages, while 124, 47, 86, and 2 proteins were uniquely expressed at ED 6, 8, 10, and 12, respectively. Notably, a sex-specific analysis identified 614, 320, 314, and 212 proteins that were uniquely expressed in female embryos, and 212, 273, 144, and 56 proteins only in male embryos at ED 6, 8, 10, and 12, respectively. The project's future focus will be to develop and utilize sensitive, specific polyclonal antibodies against synthetic peptides for W-encoded protein(s), with the goal of optimizing an immunoassay for one or more candidate W chromosome-linked proteins in CAM tissue. These findings highlight the potential of CAM in both research and biomedical applications, as well as providing a basis for the development of in-ovo sexing technology.