Assessment of pupillometry across different commercial systems of laying hens to validate its potential as an objective indicator of welfare
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Background
Reliable and non-invasive methods for assessing welfare in poultry are essential for improving evidence-based welfare monitoring and advancing management practices in commercial production systems. The iris-to-pupil (IP) ratio, previously validated by our group in primates and cattle, reflects autonomic nervous system balance and may serve as a physiological indicator of stress in laying hens. This study evaluated the utility of the IP ratio under field conditions across diverse commercial layer housing systems.
Materials and Methods
296 laying hens (Lohmann Brown, n = 269; White Leghorn, n = 27) were studied across four locations in Canada housed under different systems: Guelph (indoor; pen), Spring Island (outdoor and scratch; organic), Ottawa (outdoor, indoor and scratch; free-range), and Toronto (outdoor and hobby; free-range). High-resolution photographs of the eye were taken under ambient lighting. Light intensity was measured using the light meter app. The IP ratio was calculated using NIH ImageJ software. Statistical analysis included one-way ANOVA and linear regression using GraphPad Prism.
Results
Birds housed outdoors had the highest IP ratios, followed by those in scratch systems, while indoor and pen-housed birds had the lowest IP ratios (p < 0.001). Subgroup analyses of birds in Ottawa and Spring Island farms confirmed significantly higher IP ratios in outdoor environments compared to indoor and scratch systems (p < 0.001). IP ratio correlated weakly with ambient light intensity (r = 0.25) and age (r = 0.05), indicating minimal influence of these variables. Although White Leghorn hens showed lower IP ratios than Lohmann Browns, this difference was confounded by housing type; all White Leghorns were housed in pens. Thus, housing system but not breed was the primary driver of IP variation.
Conclusion
IP ratio is a robust, non-invasive physiological marker of welfare assessment in laying hens, sensitive to housing environment but minimally influenced by light or age. Its potential for integration with digital imaging technologies supports its use in scalable welfare assessment protocols.
