Background: Methionine (Met) and cystine (Cys) play a critical role in protein synthesis, and overall health of laying hens. The source and concentration of Met and Cys in diets can significantly influence productive performance. Objectives: This experiment aimed to compare the effects of DL-methionine (DL-Met) with methionine hydroxy analogue (MHA) and to evaluate the impact of dietary level of digestible Met + Cys on productive performance, egg quality traits, blood metabolites and immune response of laying hens. Methods: A total of 324 Shaver White laying hens were used in a randomized complete block design as a 2 × 3 factorial arrangement, comprising six treatments, six replicates and nine birds per replicate, during 34–46 weeks of age. The main effects included two Met sources: MHA and DL-Met, along with three dietary levels of Met + Cys: lower (0.555%), recommended (0.742%) and higher than the strain recommendation (0.929%). Results: Hens fed diets with Met + Cys levels equal to or exceeding the strain recommendation exhibited higher egg production compared to 0.555% Met + Cys level, which was below the strain recommendation (p < 0.05). Increased levels of Met + Cys enhanced egg mass, egg weight and daily feed intake, while the feed conversion ratio (FCR) was decreased. The levels of triglycerides and creatinine in the blood of hens with lower or equal Met + Cys levels were higher than those at the recommended level, while blood albumin level was lower at high Met + Cys levels (p < 0.05). Conclusions: The level of 0.929% Met + Cys in the diet increased egg size and egg mass while improving FCR. MHA can serve as a suitable substitute for DL-Met without negatively affecting performance in the diets of laying hens.

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