Simple SummaryWith the increase of protease types and products, it is time-consuming and laborious to evaluate the effect of protease on feed protein utilization with animal experiments, and it is not conducive to evaluate a large number of samples in a short time. The purpose of this study was to quickly evaluate the effects of four proteases (acidic, neutral, alkaline and keratinase) on feed ingredients (corn gluten meal, corn and soybean meal) using an in vitro method to determine the optimal dosage of each protease for corn gluten meal and corn and soybean meal, and to explore the factors affecting the effect of proteases. In addition, this research also carried out animal experiments to verify the effect of protease on the corn–soybean meal diet of 31-day-old broilers.AbstractThis study was conducted to investigate the effects of different proteases alone or in combination on protein digestibility of broilers. In vitro, the properties of four proteases in broilers, including acidic protease (AcP), alkaline protease (AlP), neutral protease (NeP) and keratinase (Ker), on endogenous protease activity and their effects on protein digestibility of common ingredients in broiler diets were investigated using a gut-mimicking model. In vivo, 640 1-day-old male broilers were randomly divided into 8 groups of 10 with 8 replicates of 10 birds per replicate cage. Eight dietary treatments included a corn–soybean meal basal diet (control), and the basal diet with 1.6 U AcP/g, 0.8 U NeP/g, 0.8 U AlP/g, 0.4 U Ker/g, 1.6 U AcP/g + 0.8 U NeP/g, 1.6 U AcP/g + 0.8 U AlP/g, or 1.6 U AcP/g + 0.4 U Ker/g added. The experiment lasted for 31 days. The results showed that the optimum pH values of AcP, NeP, AlP and Ker were 3.0, 9.0, 11.0 and 11.0 in vitro, respectively. Ker recovery proportion was 37.68% at pH 3.3–6.2. AcP alone or in combination with NeP, AlP or Ker increased in vitro crude protein digestibility (IVCPD) and decreased ileal apparent digestibility of crude protein in 31-day-old broilers (p< 0.05). All protease supplementation reduced the ileal apparent digestibility of amino acids compared to the control (p< 0.05). Acidic protease had a positive effect on trypsin and chymotrypsin activities, while AlP and Ker showed a negative effect. In vivo, average daily gain and average daily feed intake were significantly (p< 0.05) increased in broiler diets supplemented with AcP compared to the control group. When adding exogenous proteases to broiler diets, their sensitivity to digestive pH and their negative effects on endogenous protease activity, dosage and combination effects should be taken into account. In addition, the properties and dosage of proteases and the protein level in the feed should be considered.