AbstractThe effect of pre-slaughter handling conditions and the RYR1 gene on blood Cortisol, lactate and creatine Phosphokinase (CPK) levels at exsanguination were assessed using 2,923 surveyed pigs from 106 deliveries to five Spanish abattoirs across two seasons. The relationship between blood parameters, carcase skin damage and pork quality traits was also assessed. The season influenced blood cortisol, lactate and CPK values. Females always showed higher concentrations of cortisol, lactate, and CPK than males. Pigs carrying the recessive allele of the RYR1 gene exhibited increased lactate and CPK concentrations but not cortisol. The cortisol concentration decreased in lean pigs that were slaughtered in winter after short lairage periods. The lactate concentration decreased with loading time and increased in summer with lairage time and carcase lean content. The CPK concentration increased with lairage time, carcase weight, and carcase lean content, and with the duration of winter transports. Each truck delivery only explained approximately 10% of the variance in blood parameters. Lairage time is the most influential pre-slaughter handling practice on the assessed welfare indicators. In addition, different optimal lairage times might be appropriate depending on season. Blood cortisol, lactate, and CPK concentrations increased concomitantly with skin damage score. Blood parameters were weakly correlated and they also showed low association with pork quality traits.