Abstract

Environmental pollution remains a significant challenge in animal production. The "ideal protein" concept refers to an amino acid profile that precisely meets the animal's nutritional requirements, optimizing nutrient utilization and minimizing waste excretion. This study applied untargeted metabolomics to explore metabolic changes induced by limiting AA. Two experimental diets were used in 47-day-old growing rabbits: Met+ (with a methionine level balanced to its optimal utilization) and Met- (with a methionine level that was clearly limiting). A total of 68 blood samples were taken for untargeted metabolomics analysis and 88 were taken for targeted plasmatic urea nitrogen analysis, collected at 08:00 (in ad libitum feeding animals) and 21:00 (after a feeding event in 10 h fasting animals). Our results revealed that both sampling time and diet (at each time point) exerted a significant modulatory influence on the metabolome. Interestingly, the difference between the metabolomes obtained with the different diets was less pronounced at 08:00, likely due to the caecotrophy effect, compared to 21:00, when higher intake and lower caecotrophy frequency were observed. This study identifies pseudourine, citric acid, pantothenic acid, and enterolactone sulfate as promising metabolites that could be targeted in order to refine the ideal protein concept, thus improving nutrient efficiency and reducing the environmental impact of animal production.