Avoiding handling-induced stress in poultry: Use of uniform parameters to accurately determine physiological stress

Due to increase in awareness of poultry welfare and concomitant legislation, it has become necessary to determine poultry's response to stress, with minimal harm and maximum reliability. Several methods to determine the response to physiological stress were developed throughout the years to identify stressors and to measure stress in poultry. The most commonly used are plasma corticosterone levels and peripheral blood heterophil/lymphocyte ratio (H/L ratio). However, the value of these responses to determine a state of stress has been questioned in several instances, as these parameters are increased during the process of bird handling and blood sampling irrespective of the general state of stress. Due to these limitations, it appears that the classic stress markers might be sub-optimal in evaluating stress in poultry, particularly those encountered in high-stress environments. Thus, there is a continuing need for stress indicators, preferably indicators that are quantitative, highly repeatable, not influenced by handling and sampling, determined in peripheral blood, represent an initial response to the stressor, and do not daily fluctuate. As the immune system has been shown to rapidly respond to stress, we assessed pro-inflammatory gene expression in peripheral blood cells as an indicator for stress. We initially show that while corticosterone plasma levels and the H/L ratio were responsive to handling and blood sampling, pro-inflammatory gene expression (lysozyme, IL-1?, IL-6, and HSP-70) was not. We then determined the expression of the same pro-inflammatory genes during acute stress (transit) in layer pullets (hen and Turkey) and during chronic stress (different caging densities of layers utilizing 2, 3, and 4 hens/cage). While gene expression was significantly and highly elevated during transit, the effect of differing caging densities on gene expression was minimal; collectively, this might indicate that expression of pro-inflammatory genes is more responsive to acute stress than to chronic stressors. We propose to use pro-inflammatory gene expression in peripheral blood cells to measure responses to stress in poultry.