Prior to Simulated Transport

Six hours before treatment exposure, hens were placed in 1 of 4 feed withdrawal pens (18 hens/F resulting in 6 hens/d; 1.2 m × 1.3 m pen) with wheat straw bedding and an aluminum drinker pail (30 cm diameter). Hens (6 WF and 6 PF) were then randomly assigned to one of the T/RH and D combinations. Hen preparation involved taking initial body weights (n = 6/replicate), then hens were orally administered a miniature data logger (iButton Thermochron DS1922 L; Maxim Integrated, San Jose, CA), which travelled to the crop/gizzard and recorded CBT every minute. Baseline readings were obtained after hen preparation, by allowing hens to rest in the holding crates for 15 min. Hens received 2 wing bands for identifications, and blood samples were also taken via the brachial vein into a dipotassium EDTA tube (n = 5/replicate). Blood smears were prepared (n = 5/replicate) for analysis of the H/L ratio at a later date. Blood slides were stained using PROTOCOLTM Hema 3 (Fisher Scientific, Ottawa, Canada) and were stored in a slide box until read. Slides were read under 100X oil magnification (microscope B-290 TB; Optika, Bergamo, Italy), and the H/L ratio was determined by counting the number of heterophils and lymphocytes in the field of view until a total of 100 cells was reached. Blood samples were also analyzed immediately after collection (n = 3/replicate) for pH, partial pressure of carbon dioxide (pCO₂; mmHg), partial pressure of oxygen (pO₂; mmHg), base excess in the extracellular fluid compartment (BE; mmol/L), bicarbonate (HCO₃₋; mmol/L), total carbon dioxide (tCO₂; mmol/L), oxygen saturation (sO₂; %), sodium (mmol/L), glucose (mmol/L), hematocrit (% packed cell volume), and hemoglobin (mmol/L) using a CG8+ cartridge in an i-STAT1 handheld analyzer (Abbott Point of Care Inc, Princeton, NJ). Hens were then transported 750 m in a van to the environmental chambers (College of Engineering, University of Saskatchewan, Saskatoon, Canada) and placed into one half of the crate (0.56 m × 0.39 m) at a target crate density of 54.5 kg/m² resulting in 6 WF hens on one side and 6 PF hens on the other side of each crate.