This study aimed to investigate the effects of replacement different levels and sources of methionine (Met) with betaine on jejunal morphology, duodenal mitochondrial respiration, and lipid peroxidation in heat-stressed broiler chickens. A total of 1,200 one-day-old Ross 308 broilers were randomly assigned to two similar poultry houses. The experiment was designed as a 2 (ambient temperatures) * 2 (Met sources) * 3 (Met levels) * 2 (betaine amounts) split-plot factorial arrangement. Basal diets (Low-Met) were formulated with DL-or L-Met to meet Ross 308 nutrient recommendations except for Met which was 30% lower than the recommendation. Met level in basal diets was increased to the recommendation and/or 30% more than recommendation (High-Met) by supplemental DL-or L-Met. Betaine was or was not partially substituted at a 30% equivalent level of supplemental DL- or L-Met. HS was induced by increasing ambient temperature to 32 C for 6 h daily in one house from 10 to 42 d. The highest feed conversion ratio (FCR) was observed in Low-Met diets. Low-L-Met diets showed greatest FCR than Low-DLMet diets groups. Breast muscle malondialdehyde (MDA) concentration was decreased by increasing dietary Met level under HS. Duodenal MDA concentration and complex (Cox) III activity was lower and higher in L-Met diets than DL-Met diets, respectively. Cox II activity was increased in High-Met diets, and also was improved by betaine replacement. Villus height (VH) and Villus surface (VS) was increased in L-Met diets compared to DL-Met diets. Generally, L-Met was more effective than DL-Met in jejunal morphology, reducing duodenal MDA, and increasing Cox III activity. Betaine had the potential to be a partial replacement for Met.

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