Abstract
This study aimed to scientifically assess cold stress in dairy calves and optimize winter rearing protocols. A combined approach of feeding trials, expert surveys, and multidimensional data analysis was used to evaluate the effects of outdoor (-5~-28 °C) and indoor (5 °C) environments on Holstein dairy calves. A 60-day controlled trial was conducted with 20 healthy 5-day-old calves. In parallel, an interdisciplinary panel of 20 experts and 8 farmers established a cold stress evaluation system via the analytic hierarchy process (AHP), with cold stress levels quantified through fuzzy comprehensive evaluation. Environmental (weight = 0.62), physiological (weight = 0.22), and behavioral (weight = 0.16) factors contributed differentially to cold stress assessment, with data showing that outdoor calves were under mild cold stress (maximum membership degree = 0.64). The temperature-humidity index (THI) showed significant correlations with multiple physiological and biochemical parameters. Generalized linear mixed model (GLMM) analysis confirmed that THI variation significantly influenced calf standing time, respiratory rate (RR), malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC). In feeding trials, indoor calves exhibited marginally higher average daily gain and body weight in early stages, whereas outdoor calves demonstrated significantly better growth performance by day 60. The outdoor group displayed increased lying and defecation behaviors, along with reduced locomotor/standing time and respiratory frequency. No significant intergroup differences were observed in serum immune or antioxidant indicators. Metabolomic analysis identified 20 differentially expressed metabolites, indicating an enhancement in the activity of energy metabolism pathways in calves. This study establishes a quantitative methodology for cold stress evaluation, clarifies environment-physiology-behavior interactions, and provides a theoretical basis for winter calf management. The results confirm that outdoor cold exposure did not hinder calf growth without compromising health, offering scientific support for optimizing outdoor rearing strategies in cold regions.