Effect of early chorioallantoic membrane manipulation on chicken embryo viability

The chorioallantoic membrane (CAM), a vascularized extraembryonic membrane of the developing chick embryo, is widely utilized in biomedical research. There is a growing interest in pre-hatch embryo sex determination to eliminate the current practice of culling 1-day-old male chicks in the layer industry, and CAM interrogation may provide this information. The objective of the current study was to assess the effect of early CAM manipulation on pre-hatching embryo viability at embryonic day 16 (ED16), since reliable access to CAM without compromising embryo survival is essential for a strategy of sex determination during early embryogenesis. Early sexing techniques based on CAM-derived biomarkers could offer an ethically acceptable alternative to post-hatch sexing determination. This study assessed the effects of early CAM manipulation (ED6, 8, and 10) in chicken embryos, focusing on comparisons of egg disinfection, eggshell (ES) drilling, eggshell membrane (ESM) puncture, CAM sampling and ES sealing strategies. Our results showed that manipulation of CAM at ED6 was associated with reduced viability compared to ED8 and ED10 under various conditions. CAM manipulation at ED6 resulted in 0% viability, while viability was partially retained at ED8 (44%) or ED10 (42%). CAM sampling was associated with elevated mortality on ED6 (0% viability), ED8 (11%) and ED10 (30%). Our research demonstrates that early manipulation or sampling of CAM has a deleterious impact on embryo viability, especially at the earliest time points. This study outlines limitations for the development of CAM sampling-based sex determination methods.