<p>Protease supplementation has been observed to increase crude protein and amino acid (AA) digestibility in broiler chickens. Positive effects on apparent metabolisable energy (AME) and net energy (NE) have also been observed. The magnitude of these improvements in AME are typically larger than the sum of energy contributed by the improvement in AA digestibility, which indicates that protease supplementation may have energy-sparing effects. A five-part study was conducted to explore the energy-sparing effects of protease supplementation in broiler diets. The primary objective of this project was to quantify these energy responses and establish a basic understanding of the mechanisms by which proteases influence energy metabolism in growing broilers. Moreover, protease supplementation has traditionally been examined as part of enzyme admixtures or in isolation within poultry diets, which may lead to nebulous results, as mono-component phytase and carbohydrase enzymes are typically included in commercial poultry diets. Therefore, the effects of monocomponent protease were considered in tandem with a mono-component phytase and xylanase enzymes, to enhance understanding of protease efficacy within commercial poultry diets. The first experiment evaluated the effects of supplemental protease and diet type (maize- or wheat -based) on jejunal and ileal digestibility and total tract metabolisability of nitrogen, starch, and energy in broilers from 6 to 31 d of age. The effect of protease was more pronounced in broilers offered the maize-based diet, with protease increasing nutrient digestibility in both the jejunum and ileum. Diet type affected nutrient utilisation and growth performance in a consistent manner, with broilers receiving maize-based diets having a higher nutrient utilisation, but less efficient FCR, compared with those receiving wheatbased diets. Protease supplementation in the wheat-based diet inconsistently affected individual AME. The second experiment further evaluated the effects of supplemental protease and cereal grain source (maize- or wheat-based) on nutrient digestibility and performance in broiler chicks fed diets with reduced amino acid concentrations. Protease inclusion improved ileal starch digestibility, but the efficacy appeared to be dependent on the source of cereal-grain used and formulation strategy. For example, cereal grain type led to a significant difference in performance, with broilers offered maize-based diets exhibiting better performance than those offered wheat-based diets. The third experiment assessed the effects of protease supplementation in an all plant-based protein maize-based diet formulated with reduced concentrations of digestible AA (dAA) on nutrient digestibility and net energy utilisation in broilers. Protease supplementation increased ileal N digestibility and NE of the diet. The fourth experiment evaluated the effects of varying AME concentrations (low-, moderate-, or high-AME) and protease supplementation (without or with) on broiler growth performance and jejunal and ileal digestibility of broilers during a 5-week production period. Both AME concentrations and supplemental protease independently affected broiler performance. Feeding adequate or higher-AME concentrations resulted in optimal early growth performance and cumulative FCR. Protease supplementation positively influenced broiler performance during the starter and grower phases. The minimal interactive and absent main effects of supplemental protease on nutrient digestibility, in conjunction with its positive effects on performance, indicated that benefits of supplemental protease extended beyond direct digestibility effects, to additional net effects. Finally, the fifth experiment evaluated the effects of supplemental protease (without or with) and dAA concentrations (standard or reduced) on broiler live performance and energy partitioning. Protease supplementation improved FCR and energy and N partitioning in broilers, and responses were more pronounced in broilers fed diets with reduced-dAA concentrations. Protease did not affect AME, but numerically increased NE and significantly reduced the heat increment of feed. These results demonstrated that diet type, AME and dAA concentrations can affect broiler live performance. Additionally, the inconsistent effects of protease on nutrient digestibility, in conjunction with the positive effects on live performance and energy partitioning, indicate that nutrient digestibility was not limiting in these experiments, and that the benefits of protease supplementation extend beyond direct digestibility to additional net effects. Moreover, protease supplementation reduced the heat increment of feed by 0.19 MJ/kg, suggesting that protease likely reduced maintenance energy requirements in growing broilers by altering digestive physiology. Additional mechanistic research investigating the specific sources of these energy-sparing effects is warranted to improve understanding and fully capitalize on these responses.</p>