Effect of a Multi-Protease Supplementation in Soybean Meal on Apparent Metabolizable Energy and Amino Acid Digestibility in Broilers

Soybean meal (SBM) is the most important plant protein source in broiler diets. Although most of the proteins in SBM are highly digestible, some proteins including glycinin, protease inhibitors, and antigenic proteins are indigestible and can cause intestinal damage and impair immune functions resulting in sub-optimal growth performance (Pan et al., 2016). The supplementation of SBM with appropriate commercially available proteases provides a potential strategy to enhance the utilization of SBM proteins. The benefits and efficacy of exogenous microbial protease have also been well-reported to improve performance of animals through the control of protein and amino acid (AA) digestibility (Angel et al., 2011; Cowieson et al., 2017; Cowieson et al., 2019).

Research on exogenous proteases in poultry diets has focused on protein and AA digestibility but the effect on apparent metabolizable energy (AME) is not well-studied. Although an increase in AME is expected with the increase in protein and AA digestibility, the improvements in AME are typically greater than the sum of energy contributed by AA digestibility, indicating an improvement in energy partitioning (Cowieson et al., 2019). Therefore, this study was undertaken to determine whether the supplementation of a multiprotease could deliver better energy utilization and AA digestibility in soybean meal.

Two experiments were carried out at Monogastric Research Centre, Massey University, New Zealand. In the first experiment to determine the AME, a total of 256 one-day-old male Ross 308 broiler chicks were allocated into four treatments with eight replicates per treatment using a completely randomized design. A basal diet based on corn-soybean meal was formulated and the test diet was developed by replacing 300 g/kg (w/w) of the basal diet with SBM sample using the substitution method (Table 1). From the basal and test diets, four experimental diets were developed using different doses (0 and 300 g/t) of KemzymeTM Protease and no phytase was used in both experiments. The AME values were determined using the classical total collection method. Each diet was fed to eight replicate cages (eight 14-day old broilers/cage) for 7 days (from day 14 to 21) with the first 3 days serving as an adaptation period. During the last 4 days (day 17 to 21), feed intake was monitored, and the excreta was collected daily, weighed, and pooled within a cage. Pooled excreta were mixed well (using a blender) and representative samples were obtained and freeze-dried for dry matter (DM) determination. Dried excreta samples were ground to pass through a 0.5 mm sieve and stored in airtight plastic containers at 4°C for chemical analyses. The DM, gross energy (GE) and nitrogen (N) of the diets, excreta samples, and soybean meal sample were determined.

In the second experiment to determine apparent ileal amino acid digestibility of soybean meal with or without protease supplementation, a total of 128 one-day-old male Ross 308 broiler chicks were allocated into two treatments with eight replicates per treatment using a completely randomized design. The AA digestibility coefficients were determined by the direct method. An assay diet, based on corn starch and test soybean meal as the only source of protein was formulated to supply 18% crude protein in the diet (Table 2). The assay diets contained titanium dioxide at 5 g/kg as an indigestible marker. Each diet was offered ad libitum to eight cages (eight birds/cage) of male broilers from 17 to 21 days of age. On day 21, all birds were euthanized by an intravenous injection of sodium pentobarbitone solution, and the contents of the lower half of the ileum were collected by gently flushing with distilled water into plastic containers. The ileum was defined as that portion of the small intestine extending from Meckel's diverticulum to a point 40 mm proximal to the ileocaecal junction. Samples from all birds within a cage were pooled, frozen immediately after collection and subsequently freezedried. Soybean meal sample, diets and ileal digesta samples were ground to pass through a 0.5 mm sieve and stored in airtight containers at 4°C for chemical analyses (DM, titanium, N and amino acids, including sulphur-containing amino acids, but not tryptophan).

All analyses were conducted in an ISO17025 accredited laboratory (Nutrition Laboratory, Massey University). Dry matter content was determined in a convection oven at 105°C (AOAC 930.15; AOAC 925.10). Nitrogen content was determined by the combustion method using a CNS-2000 carbon, N and sulphur analyser (LECO® Corporation, St. Joseph, Michigan, USA). Gross energy was determined using an adiabatic bomb calorimeter (Gallenkamp Autobomb, UK) standardised with benzoic acid. Crude fat was measured using a Soxhlet extraction procedure (Method 2003.06; AOAC, 2005). Titanium content was measured on a UV spectrophotometer following the method of Short et al. (1996).

Amino acids were determined by hydrolysing the samples with 6 N HCl (containing phenol) for 24 h at 110 ± 2°C in glass tubes sealed under vacuum. Amino acids were detected on a Waters ion-exchange HPLC system, and the chromatograms were integrated by using dedicated software (Millenium, version 3.05.01, Waters, Millipore, Milford, MA) with the amino acids identified and quantified by using a standard amino acid mixture (product no. A2908, Sigma, St. Louis, MO). Amino acids were eluted by a gradient of pH 3.3 sodium citrate eluent to pH 9.8 sodium borate eluent at a flow rate of 0.4 mL/min and a column temperature of 60°C. Cysteine and methionine were analysed as cysteic acid and methionine sulfone, respectively, by oxidation with performic acid for 16 h at 0°C and neutralisation with hydrobromic acid before hydrolysis (Ravindran et al., 2009).

Protease supplementation (300 g/t) did not significantly increase the AME as compared to the diet without protease (P > 0.05), but numerically increased AME by 0.29 MJ/kg (~69 kcal/kg) and AMEn by 0.27 MJ/kg (~64.5 kcal/kg).

The effect of supplementation of protease on AA digestibility is presented in Figure 1. Significant improvements were observed in the apparent ileal digestibility of nitrogen and twelve amino acids (particularly Aspartic Acid (Asp), Threonine (Thr), Serine (Ser), Proline (Pro), Glycine (Gly), Valine (Val), Leucine (Leu), Histidine (His), Lysine (Lys), Arginine (Arg) and Cysteine (Cys)) with supplementation of protease (P < 0.05).

In both experiments, it was evident that the addition of protease had significant benefits to the digestibility of nitrogen and amino acids. While there was no significant increase in AME, a positive numerical improvement of AME was observed. Taken together, the addition of exogenous protease in the diet formulation complemented that of the endogenous protease to increase AME and to improve protein digestibility in diets.