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This study aims to compare two broiler pre-slaughter chain methods: (i) the automatic pre-slaughter chain (APC) and (ii) manual pre-slaughter chain (MPC). The comparison is based on the evaluation of the trailer microclimate, number of injuries, and breast muscle (m. pectoralis major) quality. Transportation lasts 3.5 h, unloading 1 h. The selection of two hundred 39-day-old broilers (Ross 308 and Cobb 500 breeds) is random for each type of method. After slaughter, the pH value, electrical conductivity (EC), and color (lightness) of breast muscle tissues are determined at different post-mortem intervals. The MPC negatively affects the microclimate (p < 0.001), meat qualitative characteristics (p < 0.001), and places a greater strain on the body of chickens compared with APC. The average pH15min value of MPC broiler breast muscle tissue, generally used as the main meat quality parameter, is 5.97 ± 0.12, in contrast to 6.36 ± 0.16 for APC. Higher pH15min value of APC indicates better welfare and pre-slaughter handling. Values of EC and L* of breast tissues also confirms a difference between the methods of broiler handling (p < 0.001). No difference is found between the breed lines (p > 0.05).