# Effects of a protease and essential oils on growth performance, blood cell profiles, nutrient retention, ileal microbiota, excreta gas emission, and breast meat quality in broiler chicks

Effects of a protease and essential oils on growth performance, blood cell profiles, nutrient... Abstract A total of 360 Ross male broiler chicks (39.8 ± 1.8 g) were used in a five week experiment to determine the effect of a protease and essential oils (EO) on growth performance, blood cell profile, nutrient retention, ileal microbiota, excreta gas emission, and breast meat quality in broiler chicks. Broiler chicks were randomly allotted to four dietary treatments with 15 birds/cage and six cages/treatment. Experimental treatments were arranged as a 2 × 2 factorial with two levels of protease (0 and 0.02%) and two levels of EO (0 and 0.03%). For days 8 to 21 and overall, body weight gain and the feed conversion ratio were better in broilers fed diets supplemented with protease (P < 0.05) than in those fed diets without protease supplementation. Protease and/or EO increased (P < 0.05) the total tract retention of dry matter, nitrogen, or gross energy, and decreased the excreta ammonia gas emission. In addition, there was a significant interaction between the protease and EO on total tract retention of nitrogen and excreta ammonia gas emission (P < 0.05). The density of ileal Lactobacillus increased and Escherichia coli decreased in broilers (P < 0.05) by the addition of EO to the diet. There were no significant differences in the measurements of breast meat quality and organ weight of broilers fed diets with protease or EO. In conclusion, diets with a combination of a protease and EO improved total tract retention of nitrogen and excreta ammonia gas emission in growing broiler chicks. INTRODUCTION Antimicrobial drugs have been extensively used at sub-therapeutic doses in diets to prevent poultry diseases and to improve production performance (Hume, 2011). However, current global trends in poultry production are to reduce the use of antimicrobial agents and increase use of natural feed additives. The use of herbs, spices, and essential oils in poultry diets as alternatives to antibiotics has attracted attention because of their growth-promoting potential. Essential oils (EO) have been extracted from various plant materials for use as feed additives, and many have shown potential benefits including performance improvement and antioxidant and antimicrobial activities for broiler chickens (Hoffman and Wu, 2010; Tiihonen et al., 2010; Du et al., 2015). In addition, dietary EO supplementation showed increased digestive enzyme activities of pancreas and intestine in broiler chicks (Jang et al., 2007). Endogenous proteases catalyze the hydrolysis of dietary proteins. Exogenous proteases are thought to not only complement the animals’ digestive enzymes, such as pepsin and pancreatic proteases, but to also destroy anti-nutrients, such as lectins and trypsin inhibitors (Ghazi et al., 2002). Numerous studies have shown that dietary protease supplementation has various effects, including improvements in growth performance and amino acid digestibility (Dozier et al., 2010; Angel et al., 2011; López‐Iglesias et al., 2011). We hypothesized that the addition of a combination of protease and EO would increase the growth performance by improving nutrient digestibility and intestinal environment. However, little is known about the combined effects of proteases and EO. Thus, the aim of the present study was to evaluate the effects of protease in combination with EO on growth performance, blood cell profiles, total tract retention, intestinal microbiota, noxious gas emissions, and breast meat quality in broilers. MATERIALS AND METHODS The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University, Republic of Korea. Birds and Housing One-day-old male broiler chicks (Ross × Ross; 39.8 ± 1.8 g) were obtained from a commercial hatchery and housed in stainless steel battery cages (124 cm wide × 64 cm long × 40 cm high) equipped with two drinker nipples and two open trough feeders. The temperature of the room was maintained at 33 ± 1°C for the first 3 d, and decreased to 24°C for the duration of the experiment. Diets (Table 1) were fed in mash form during three phases consisting of a starter phase from days 1 to 7, a grower phase from days 8 to 21, and a finisher phase from days 22 to 35. The chicks were given access ad libitum to water and feed. Table 1. Composition of the basal diets (on as-fed basis). Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 1Distiller's dried grains with solubles. 2Provided per kg of diet: 15,000 IU vitamin A; 3750 IU vitamin D3; 37.5 mg vitamin E; 2.55 mg vitamin K3; 3 mg thiamin; 7.5 mg riboflavin; 4.5 mg vitamin B6; 24 μg vitamin B12; 51 mg niacin; 1.5 mg folic acid; 0.2 mg biotin and 13.5 mg pantothenic acid. 3Provided per kg of diet: 37.5 mg Zn; 37.5 mg Mn; 37.5 mg Fe; 3.75 mg Cu; 0.83 mg I; 62.5 mg S and 0.23 mg Se. View Large Table 1. Composition of the basal diets (on as-fed basis). Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 1Distiller's dried grains with solubles. 2Provided per kg of diet: 15,000 IU vitamin A; 3750 IU vitamin D3; 37.5 mg vitamin E; 2.55 mg vitamin K3; 3 mg thiamin; 7.5 mg riboflavin; 4.5 mg vitamin B6; 24 μg vitamin B12; 51 mg niacin; 1.5 mg folic acid; 0.2 mg biotin and 13.5 mg pantothenic acid. 3Provided per kg of diet: 37.5 mg Zn; 37.5 mg Mn; 37.5 mg Fe; 3.75 mg Cu; 0.83 mg I; 62.5 mg S and 0.23 mg Se. View Large Experimental Design and Diets Broilers were randomly allotted to cages and fed diets with two levels of protease (0 or 0.02%) and two levels of EO (0 or 0.03%). There were six cages per treatment with 15 birds per cage. All diets were formulated to meet or exceed the suggested nutrient concentrations recommended by the NRC (1994). For the experiment, the EO was provided by DSM Nutritional Products Ltd. (CRINA® Poultry, DSM Nutritional Products Ltd., Basel, Switzerland). The concentration of EO was based on the manufacturer’ specification and the product contained 29% active ingredients, including thymol (Thymus vulgaris), eugenol (Cinnamomum spp.), and piperine (Piper spp.). The enzyme preparation used in this experiment was a commercial protease enzyme (Ronozyme ProActTM CT, DSM Nutritional Products, containing 75,000 protease units/g of product) produced through submerged fermentation of a Bacillus licheniformis strain. Sampling and Measurements The broilers were weighed by cage and feed intake (FI) was recorded on days 1, 7, 21, and 35 of the experiment to allow calculation of body weight gain (BWG) and feed conversion ratio (FCR). Blood samples were collected from the wing vein of five birds per cage on the last day of the growth assay. The samples were placed on ice, transported to the laboratory, and concentrations of white blood cells (WBC), red blood cells (RBC), and lymphocytes were determined using an automatic blood-cell analyzer (Hemavet 950 FS, Drew Scientific Inc., Miami Lakes, Florida, U.S.A). To allow determination of the apparent total tract retention (ATTR) of nutrients, all broiler chicks were fed diets with 0.2% Cr2O3 for days 32 to 35. Excreta from day 35 were collected, pooled by cage, homogenized, and a representative sample taken. The excreta samples were stored in a freezer at −20°C until analysis. Feed and excreta samples were thawed and dried for 72 h at 50°C in a forced-air oven (Model FC-610, Advantec, Toyo Seisakusho Co. Ltd., Tokyo, Japan), after which they were ground through a 1-mm screen. Then, feed and excreta samples were analyzed for dry matter (DM) (method 930.15), energy (using a bomb colorimeter, Parr 6100; Parr Instruments Co., Moline, IL), and nitrogen (method 990.03) using procedures outlined by the AOAC (2000). Chromium concentrations were determined with a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu, Kyoto, Japan) using the method of Williams et al. (1962). The formula for calculating the ATTR of nutrients was:$${\rm{ATTR}} = \, 1 - [ {\frac{{Nf \times Cd}}{{Nd \times Cf}}}] \times \, {100},$$ where Nf = concentration of nutrient in excreta (% DM), Nd = concentration of nutrient in the diet (% DM) Cd = concentration of chromium in the diet (% DM), and Cf = concentration of chromium in the excreta (% DM). For measurement of ileal microbiota, ileal digesta samples were collected at day 35 from 18 broilers per treatment. One gram of the composite excreta sample from each cage was diluted with 9 mL of 1% peptone broth and homogenized. Counts of viable Lactobacillus and E. coli in the ileal samples were conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) and MacConkey agar plates (Difco Laboratories, Detroit, USA), respectively. The lactobacilli medium III agar plates were incubated for 24 h at 39°C and the MacConkey agar plates were incubated for 24 h at 37°C, both under aerobic conditions. Colonies on each agar plate were counted using a colony counter and the results expressed as colony-forming units per gram (log10 CFU/g). At the end of the experiment, excreta samples were collected from each cage and analysis of noxious gas was measured according to the method described by Cho et al. (2008). A total of 100 g of excreta from each cage were placed in 2.6-L sealed plastic boxes. The samples were allowed to ferment for 30 h at 32°C and the gases evaluated using a gas sampling pump (model GV-100, Gastec Corp., Yokohama, Japan). To collect the gas sample, adhesive plaster on the box was punctured and 100 mL of the headspace air was sampled approximately 5 cm above the excreta. After blood collection, the broilers were weighed individually and killed by cervical dislocation. Breast muscle, abdominal fat, liver, bursa of Fabricius, spleen, and gizzard were removed and weighed so that organ weight could be expressed as a percentage of body weight. Duplicate pH values of each breast meat sample were measured via a glass-electrode pH meter (WTW pH 340-A, WTH Measurement Systems Inc., Ft. Myers, FL). Color values (L* = lightness, a* = redness, and b* = yellowness) of breast muscle were determined using a Minolta CR 410 Chroma Meter (Konica Minolta Sensing Inc., Osaka, Japan). Drip loss was determined as a percentage of original weight using 1 cm × 5 cm × 5 cm meat samples of breast meat suspended in a sealed plastic bag for 7 days at 4°C. Water-holding capacity (WHC) was determined by the method of Jin et al. (2011). Briefly, 5 g of meat sample was heated to 90°C in a water bath for 30 min. The samples were cooled with ice and centrifuged at 1000 × g and 5°C for 10 min. The WHC was calculated as the ratio (%) of liquid content in the sample after centrifugation, to liquid content in the sample before centrifugation. Statistical Analysis All data were analyzed as a 2 × 2 factorial using ANOVA (SAS Inst. Inc., Cary, NC, US) with cage as the experimental unit. Main effects of protease and EO and their interaction were included in the model. Where interactions (P < 0.05) were detected, treatment means were separated using the Tukey's test. Variability in the data is expressed as the standard error of the means (SEM). The alpha level used for the determination of significance was 0.05 and trends were discussed at P < 0.10. RESULTS Growth Performance During days 8 to 21 and 1 to 35, broilers fed the diets supplemented with protease had better BWG, FCR and/or FI (P < 0.05) than did those fed the diets without protease supplementation (Table 2). There was a trend (P < 0.10) for the addition of EO to improve BWG (days 1 to 7), FI (days 8 to 21), and FCR (days 1 to 35). Table 2. Effects of the protease and EO on body weight gain, feed intake and feed conversion ratio in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3body weight gain. 4feed intake. 5feed conversion ratio. View Large Table 2. Effects of the protease and EO on body weight gain, feed intake and feed conversion ratio in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3body weight gain. 4feed intake. 5feed conversion ratio. View Large Blood Cell Profiles Concentrations of white blood cell, red blood cell, and lymphocytes were not influenced by the addition of protease and/or EO (Table 3). Table 3. Effects of the protease and EO on red blood cell, white blood cell and lymphocyte in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3red blood cell. 4white blood cell. View Large Table 3. Effects of the protease and EO on red blood cell, white blood cell and lymphocyte in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3red blood cell. 4white blood cell. View Large Apparent Total Tract Retention The ATTR of DM, nitrogen and energy was greater in broiler chicks fed the diet with the protease than in those fed diets without protease (P ≤ 0.05; Table 4). The ATTR of energy for broilers fed diets with EO was higher than for broilers fed diets without EO (P < 0.05). In addition, there was an interaction among protease and EO addition for the ATTR of nitrogen (P < 0.05). Table 4. Effects of the protease and EO on total tract retention of dry matter, nitrogen, or energy in broilers.1 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,bMeans in the same row with different superscripts differ (P < 0.05). View Large Table 4. Effects of the protease and EO on total tract retention of dry matter, nitrogen, or energy in broilers.1 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,bMeans in the same row with different superscripts differ (P < 0.05). View Large Ileal Microbiota There were no differences in bacterial counts for Lactobacillus and E. coli for birds fed diets with protease, but Lactobacillus counts increased and E. coli counts decreased with the addition of EO to the diets (P < 0.05; Table 5). Table 5. Effects of the protease and EO on ileal Lactobacillus and E. coli microbiota in broilers.1 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. View Large Table 5. Effects of the protease and EO on ileal Lactobacillus and E. coli microbiota in broilers.1 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. View Large Excreta Noxious Gas Emissions Protease or EO supplementation did reduce (P < 0.05) the release of ammonia gas from excreta (Table 6). In addition, there was an interaction (P < 0.05) between protease and EO for excreta noxious gas emission. Hydrogen sulfide and total mercaptans were not influenced by the addition of the protease or EO. Table 6. Effects of the protease and EO on ammonia, hydrogen sulfide and methyl mercaptan gas emission in broilers.1 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,b,cMeans in the same row with different superscripts differ (P < 0.05). View Large Table 6. Effects of the protease and EO on ammonia, hydrogen sulfide and methyl mercaptan gas emission in broilers.1 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,b,cMeans in the same row with different superscripts differ (P < 0.05). View Large Meat Quality and Relative Organ Weight There were no differences in breast meat pH, color (L*, a*, and b*), drip loss and WHC between birds fed the diets with protease or EO and those fed diets with none added. Moreover, no differences were observed for the relative weight of breast muscle, abdominal fat, liver, bursa of Fabricius, spleen, and gizzard of between birds fed the treatments and controls (Table 7). Table 7. Effects of the protease and EO on chemical characteristics of breast meat and relative organ weight in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3water holding capacity. View Large Table 7. Effects of the protease and EO on chemical characteristics of breast meat and relative organ weight in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3water holding capacity. View Large DISCUSSION This experiment was designed to determine the effects of dietary combinations of a protease and EO on growth, nutrient utilization, blood cell profiles, ileal microbiota, and meat quality in broiler chicks. Results revealed that broilers fed diets supplemented with the protease had improved BWG and FI from day 7 to day 21 and overall (P < 0.05). Proteases play a critical role in several physiological processes, such as degradation of food proteins, protein turnover, cell division, blood coagulation, transport of polypeptide hormones, and activation of zymogens (Rao et al., 1998). In particular, proteases have been suggested to optimize feed protein utilization in poultry (Caine et al., 1998). Fru-Nji et al. (2011) reported that proteases used as feed additives could supplement the effects of endogenous pepsin and pancreatic enzymes by augmenting the hydrolysis and solubilization of protein in vitro and in vivo. These effects were also confirmed in other broiler studies, where improved protein and/or fat retention and greater growth performance were observed (Angel et al., 2011; Freitas et al., 2011). In the present study, EO supplementation showed a trend for increased BWG (day 1 to 7; P = 0.063) and FI (day 8 to 21; P = 0.088), and improved FCR (day 1 to 35; P = 0.067). Essential oils have been shown to exert antibacterial, antifungal, and antiviral activities in vitro (Windisch et al., 2008) and numerous studies have documented the benefits of EO on growth performance in poultry (Giannenas et al., 2003; Jang et al., 2007; Franz et al., 2010). Other effects of EO have been suggested to include enhanced voluntary feed intake and, thus, greater body weight gain (Zeng et al., 2015). Phytogenic compounds, which include EO, stimulate bile secretion (Lee et al., 2004; Platel and Srinivasan, 2004). Dietary EO blend including thymol, eugenol, and piperine have been shown to stimulate secretion of pancreatic digestive enzymes (e.g., α-amylase, maltase, and trypsin) in broilers (Jang et al., 2007). These results suggest that dietary EO could affect nutrient retention via quite different mechanisms from the direct effects of proteases. The present study indicates that supplementation with a combination of a protease and EO may synergistically improve nitrogen retention in broilers (protease x EO interaction, P = 0.011). We found no studies regarding the synergistic effects of protease and EO on nutrient retention, but the findings of our current study imply that improvement in growth performance of broilers fed protease and EO may result from higher nutrient retention. Aengwanich et al. (2009) suggest that changes in blood cell profiles can be used to determine the physiological, pathological, and nutritional status of an animal. White blood cells including lymphocytes can increase in response to infection or stress because they are one of the first lines of defense in the body (Wang et al., 2003). As far as we know, there are currently no reports of blood cell profiles including RBC, WBC and lymphocyte due to protease supplementation in broiler chicks, and whether proteases contributed to increase in blood cells is unknown. In contrast, the blood cell profiles of broilers fed EO derived from plants are inconsistent among researchers (Bardzardi et al., 2014; Torki et al., 2015; Attia et al., 2017). The results of the present study indicated that supplementation of protease and EO did not affect blood cell profiles (RBC, WBC and lymphocyte) in broilers. Intestinal microbiota of livestock and poultry plays an important role in supporting health and growth performance. The microbial community in a chicken's ileum is mainly dominated by Lactobacillus spp. (Bjerrum et al., 2006). Du et al. (2015) observed strong antibacterial activity of EO (thymol and carvacrol), in an in vitro minimum inhibitory concentration assay, against pathogenic E. coli, C. perfringens, and Salmonella strains. Other in vivo studies demonstrated inhibiting effects against pathogens such as C. perfringens, E. coli, or Eimeria species (Bozkurt et al., 2013). In the present study, Lactobacillus populations were increased and E. coli populations were decreased in ileal digesta following the addition of EO to broiler diets. Essential oils are a rich source of a variety of phytochemicals and bioactive components that could cause such changes in gut microflora and those changes may be responsible for the improved nutrient retention observed in our experiment. In the present study, emission of ammonia gas from excreta of broilers fed diets with EO was lower than that from birds fed diets without EO. In addition, these results suggest a synergistic effect of EO with a protease (protease × EO interaction, P = 0.043). The interaction between the protease and EO indicated that the reduction of ammonia gas emission from birds fed diets with EO was even more effective with the addition of the protease. There are no previous reports on the influence of EO on excreta ammonia gas emission in broilers. However, Cho et al. (2006) suggested that EO (from fenugreek, clove, and cinnamon) could reduce environmental pollutants, such as fecal ammonia and hydrogen sulfide, by improving feed efficiency and nutrient digestibility in pigs. Ammonia gas is mainly generated because of inefficient utilization of feed protein and the resulting excretion of nitrogen as uric acid. Considering that excreta mal odor and ammonia gas are related to the intestinal microbial ecosystem and nutrient utilization, the reduction in ammonia gas in excreta of broilers fed with protease or EO is easily understood. Meat pH is generally highly associated with meat color, WHC, and drip loss of chicken meat. Higher meat pH causes darker meat, whereas lower meat pH causes lighter meat (Livingston and Brown, 1981). Water holding capacity is the ability of meat to retain its water during processing, storage, and cooking. It is affected by pH and influences the drip loss and eating quality (Kim et al., 2014). Meat pH, color, drip loss, and the WHC of breast muscle were unaffected by protease or EO treatments in the present study. Therefore, the lack of change in meat quality characteristics can be explained by similar pH values among the treatments. Previous reports on the effects of enzyme and phytogenic feed additives on meat quality and organ weight in broilers have been inconsistent. Some authors reported advantages of administration of enzymes or EO (Omojola and Adesehinwa, 2007; Symeon et al., 2009), whereas others did not observe improvement when a protease or an EO was used (Tabook et al., 2006; Zakaria et al., 2010; Hassanein, 2011; Dalólio et al., 2015). Further study with more focus on meat quality associated with these additives is recommended. CONCLUSIONS Our results indicated that supplementation of protease and EO in diets for broiler chicks improved growth performance, increased ileal Lactobaicllus populations, and reduced ileal E. coli populations. Although blood profiles and breast meat quality were not affected, these additives reduced the concentrations of excreta ammonia emission and increased nitrogen retention. Additionally, supplementation with a combination of a protease and EO synergistically improved nitrogen retention and deceased excreta ammonia emission. 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# Effects of a protease and essential oils on growth performance, blood cell profiles, nutrient retention, ileal microbiota, excreta gas emission, and breast meat quality in broiler chicks

, Volume Advance Article (8) – Jul 11, 2018
7 pages

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Publisher
Oxford University Press
© 2018 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pey151
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### Abstract

Abstract A total of 360 Ross male broiler chicks (39.8 ± 1.8 g) were used in a five week experiment to determine the effect of a protease and essential oils (EO) on growth performance, blood cell profile, nutrient retention, ileal microbiota, excreta gas emission, and breast meat quality in broiler chicks. Broiler chicks were randomly allotted to four dietary treatments with 15 birds/cage and six cages/treatment. Experimental treatments were arranged as a 2 × 2 factorial with two levels of protease (0 and 0.02%) and two levels of EO (0 and 0.03%). For days 8 to 21 and overall, body weight gain and the feed conversion ratio were better in broilers fed diets supplemented with protease (P < 0.05) than in those fed diets without protease supplementation. Protease and/or EO increased (P < 0.05) the total tract retention of dry matter, nitrogen, or gross energy, and decreased the excreta ammonia gas emission. In addition, there was a significant interaction between the protease and EO on total tract retention of nitrogen and excreta ammonia gas emission (P < 0.05). The density of ileal Lactobacillus increased and Escherichia coli decreased in broilers (P < 0.05) by the addition of EO to the diet. There were no significant differences in the measurements of breast meat quality and organ weight of broilers fed diets with protease or EO. In conclusion, diets with a combination of a protease and EO improved total tract retention of nitrogen and excreta ammonia gas emission in growing broiler chicks. INTRODUCTION Antimicrobial drugs have been extensively used at sub-therapeutic doses in diets to prevent poultry diseases and to improve production performance (Hume, 2011). However, current global trends in poultry production are to reduce the use of antimicrobial agents and increase use of natural feed additives. The use of herbs, spices, and essential oils in poultry diets as alternatives to antibiotics has attracted attention because of their growth-promoting potential. Essential oils (EO) have been extracted from various plant materials for use as feed additives, and many have shown potential benefits including performance improvement and antioxidant and antimicrobial activities for broiler chickens (Hoffman and Wu, 2010; Tiihonen et al., 2010; Du et al., 2015). In addition, dietary EO supplementation showed increased digestive enzyme activities of pancreas and intestine in broiler chicks (Jang et al., 2007). Endogenous proteases catalyze the hydrolysis of dietary proteins. Exogenous proteases are thought to not only complement the animals’ digestive enzymes, such as pepsin and pancreatic proteases, but to also destroy anti-nutrients, such as lectins and trypsin inhibitors (Ghazi et al., 2002). Numerous studies have shown that dietary protease supplementation has various effects, including improvements in growth performance and amino acid digestibility (Dozier et al., 2010; Angel et al., 2011; López‐Iglesias et al., 2011). We hypothesized that the addition of a combination of protease and EO would increase the growth performance by improving nutrient digestibility and intestinal environment. However, little is known about the combined effects of proteases and EO. Thus, the aim of the present study was to evaluate the effects of protease in combination with EO on growth performance, blood cell profiles, total tract retention, intestinal microbiota, noxious gas emissions, and breast meat quality in broilers. MATERIALS AND METHODS The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University, Republic of Korea. Birds and Housing One-day-old male broiler chicks (Ross × Ross; 39.8 ± 1.8 g) were obtained from a commercial hatchery and housed in stainless steel battery cages (124 cm wide × 64 cm long × 40 cm high) equipped with two drinker nipples and two open trough feeders. The temperature of the room was maintained at 33 ± 1°C for the first 3 d, and decreased to 24°C for the duration of the experiment. Diets (Table 1) were fed in mash form during three phases consisting of a starter phase from days 1 to 7, a grower phase from days 8 to 21, and a finisher phase from days 22 to 35. The chicks were given access ad libitum to water and feed. Table 1. Composition of the basal diets (on as-fed basis). Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 1Distiller's dried grains with solubles. 2Provided per kg of diet: 15,000 IU vitamin A; 3750 IU vitamin D3; 37.5 mg vitamin E; 2.55 mg vitamin K3; 3 mg thiamin; 7.5 mg riboflavin; 4.5 mg vitamin B6; 24 μg vitamin B12; 51 mg niacin; 1.5 mg folic acid; 0.2 mg biotin and 13.5 mg pantothenic acid. 3Provided per kg of diet: 37.5 mg Zn; 37.5 mg Mn; 37.5 mg Fe; 3.75 mg Cu; 0.83 mg I; 62.5 mg S and 0.23 mg Se. View Large Table 1. Composition of the basal diets (on as-fed basis). Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 Ingredients, % Starter Grower Finisher (phase 1, d 1 - 7) (phase 2, d 8 - 21) (phase 3, d 22 - 35) Corn 42.63 49.33 48.99 Soybean meal 33.69 26.89 26.26 Wheat 10.00 10.00 10.00 Rapeseed meal 2.00 3.00 3.00 DDGS1 3.00 4.00 5.00 Limestone 1.17 0.94 0.86 Animal fat 4.70 3.24 3.52 Salt 0.25 0.25 0.25 Choline CL (50%) 0.10 0.10 0.10 L-lysine 0.22 0.11 0.03 Methionine (99%) 0.26 0.18 0.12 Dicalcium phosphate 1.72 1.70 1.62 Vitamin premix2 0.14 0.14 0.14 Mineral premix3 0.12 0.12 0.11 Total 100 100 100 Calculated composition  ME, kcal/kg 3300 3300 3300  CP, % 23.00 22.00 19.00  Methionine 0.50 0.45 0.38  Lysine, % 1.10 1.00 0.90  Ca, % 1.00 0.90 0.85  P, % 0.73 0.70 0.68 1Distiller's dried grains with solubles. 2Provided per kg of diet: 15,000 IU vitamin A; 3750 IU vitamin D3; 37.5 mg vitamin E; 2.55 mg vitamin K3; 3 mg thiamin; 7.5 mg riboflavin; 4.5 mg vitamin B6; 24 μg vitamin B12; 51 mg niacin; 1.5 mg folic acid; 0.2 mg biotin and 13.5 mg pantothenic acid. 3Provided per kg of diet: 37.5 mg Zn; 37.5 mg Mn; 37.5 mg Fe; 3.75 mg Cu; 0.83 mg I; 62.5 mg S and 0.23 mg Se. View Large Experimental Design and Diets Broilers were randomly allotted to cages and fed diets with two levels of protease (0 or 0.02%) and two levels of EO (0 or 0.03%). There were six cages per treatment with 15 birds per cage. All diets were formulated to meet or exceed the suggested nutrient concentrations recommended by the NRC (1994). For the experiment, the EO was provided by DSM Nutritional Products Ltd. (CRINA® Poultry, DSM Nutritional Products Ltd., Basel, Switzerland). The concentration of EO was based on the manufacturer’ specification and the product contained 29% active ingredients, including thymol (Thymus vulgaris), eugenol (Cinnamomum spp.), and piperine (Piper spp.). The enzyme preparation used in this experiment was a commercial protease enzyme (Ronozyme ProActTM CT, DSM Nutritional Products, containing 75,000 protease units/g of product) produced through submerged fermentation of a Bacillus licheniformis strain. Sampling and Measurements The broilers were weighed by cage and feed intake (FI) was recorded on days 1, 7, 21, and 35 of the experiment to allow calculation of body weight gain (BWG) and feed conversion ratio (FCR). Blood samples were collected from the wing vein of five birds per cage on the last day of the growth assay. The samples were placed on ice, transported to the laboratory, and concentrations of white blood cells (WBC), red blood cells (RBC), and lymphocytes were determined using an automatic blood-cell analyzer (Hemavet 950 FS, Drew Scientific Inc., Miami Lakes, Florida, U.S.A). To allow determination of the apparent total tract retention (ATTR) of nutrients, all broiler chicks were fed diets with 0.2% Cr2O3 for days 32 to 35. Excreta from day 35 were collected, pooled by cage, homogenized, and a representative sample taken. The excreta samples were stored in a freezer at −20°C until analysis. Feed and excreta samples were thawed and dried for 72 h at 50°C in a forced-air oven (Model FC-610, Advantec, Toyo Seisakusho Co. Ltd., Tokyo, Japan), after which they were ground through a 1-mm screen. Then, feed and excreta samples were analyzed for dry matter (DM) (method 930.15), energy (using a bomb colorimeter, Parr 6100; Parr Instruments Co., Moline, IL), and nitrogen (method 990.03) using procedures outlined by the AOAC (2000). Chromium concentrations were determined with a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu, Kyoto, Japan) using the method of Williams et al. (1962). The formula for calculating the ATTR of nutrients was:$${\rm{ATTR}} = \, 1 - [ {\frac{{Nf \times Cd}}{{Nd \times Cf}}}] \times \, {100},$$ where Nf = concentration of nutrient in excreta (% DM), Nd = concentration of nutrient in the diet (% DM) Cd = concentration of chromium in the diet (% DM), and Cf = concentration of chromium in the excreta (% DM). For measurement of ileal microbiota, ileal digesta samples were collected at day 35 from 18 broilers per treatment. One gram of the composite excreta sample from each cage was diluted with 9 mL of 1% peptone broth and homogenized. Counts of viable Lactobacillus and E. coli in the ileal samples were conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) and MacConkey agar plates (Difco Laboratories, Detroit, USA), respectively. The lactobacilli medium III agar plates were incubated for 24 h at 39°C and the MacConkey agar plates were incubated for 24 h at 37°C, both under aerobic conditions. Colonies on each agar plate were counted using a colony counter and the results expressed as colony-forming units per gram (log10 CFU/g). At the end of the experiment, excreta samples were collected from each cage and analysis of noxious gas was measured according to the method described by Cho et al. (2008). A total of 100 g of excreta from each cage were placed in 2.6-L sealed plastic boxes. The samples were allowed to ferment for 30 h at 32°C and the gases evaluated using a gas sampling pump (model GV-100, Gastec Corp., Yokohama, Japan). To collect the gas sample, adhesive plaster on the box was punctured and 100 mL of the headspace air was sampled approximately 5 cm above the excreta. After blood collection, the broilers were weighed individually and killed by cervical dislocation. Breast muscle, abdominal fat, liver, bursa of Fabricius, spleen, and gizzard were removed and weighed so that organ weight could be expressed as a percentage of body weight. Duplicate pH values of each breast meat sample were measured via a glass-electrode pH meter (WTW pH 340-A, WTH Measurement Systems Inc., Ft. Myers, FL). Color values (L* = lightness, a* = redness, and b* = yellowness) of breast muscle were determined using a Minolta CR 410 Chroma Meter (Konica Minolta Sensing Inc., Osaka, Japan). Drip loss was determined as a percentage of original weight using 1 cm × 5 cm × 5 cm meat samples of breast meat suspended in a sealed plastic bag for 7 days at 4°C. Water-holding capacity (WHC) was determined by the method of Jin et al. (2011). Briefly, 5 g of meat sample was heated to 90°C in a water bath for 30 min. The samples were cooled with ice and centrifuged at 1000 × g and 5°C for 10 min. The WHC was calculated as the ratio (%) of liquid content in the sample after centrifugation, to liquid content in the sample before centrifugation. Statistical Analysis All data were analyzed as a 2 × 2 factorial using ANOVA (SAS Inst. Inc., Cary, NC, US) with cage as the experimental unit. Main effects of protease and EO and their interaction were included in the model. Where interactions (P < 0.05) were detected, treatment means were separated using the Tukey's test. Variability in the data is expressed as the standard error of the means (SEM). The alpha level used for the determination of significance was 0.05 and trends were discussed at P < 0.10. RESULTS Growth Performance During days 8 to 21 and 1 to 35, broilers fed the diets supplemented with protease had better BWG, FCR and/or FI (P < 0.05) than did those fed the diets without protease supplementation (Table 2). There was a trend (P < 0.10) for the addition of EO to improve BWG (days 1 to 7), FI (days 8 to 21), and FCR (days 1 to 35). Table 2. Effects of the protease and EO on body weight gain, feed intake and feed conversion ratio in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3body weight gain. 4feed intake. 5feed conversion ratio. View Large Table 2. Effects of the protease and EO on body weight gain, feed intake and feed conversion ratio in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO 1–7 days  BWG3, g 149 137 141 138 3.91 0.367 0.063 0.273  FI4, g 165 154 156 162 4.76 0.952 0.628 0.187  FCR5 1.11 1.13 1.11 1.18 0.04 0.479 0.284 0.512 8–21 day  BWG, g 662 662 794 790 9.34 0.018 0.853 0.818  FI, g 815 826 848 925 24.22 0.022 0.088 0.194  FCR 1.23 1.25 1.07 1.18 0.04 0.045 0.103 0.2238 22–35 days  BWG, g 1113 1151 1156 1125 41.11 0.844 0.925 0.405  FI, g 2092 2276 2141 2156 93.13 0.147 1.134 0.843  FCR 1.88 1.98 1.85 1.92 0.05 0.461 0.147 0.868 1–35 days  BWG, g 1925 1952 2091 2053 43.88 0.018 0.893 0.464  FI, g 2682 2677 2652 2655 103.51 0.703 0.295 0.375  FCR 1.75 1.69 1.68 1.65 0.04 0.034 0.067 0.893 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3body weight gain. 4feed intake. 5feed conversion ratio. View Large Blood Cell Profiles Concentrations of white blood cell, red blood cell, and lymphocytes were not influenced by the addition of protease and/or EO (Table 3). Table 3. Effects of the protease and EO on red blood cell, white blood cell and lymphocyte in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3red blood cell. 4white blood cell. View Large Table 3. Effects of the protease and EO on red blood cell, white blood cell and lymphocyte in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO RBC3, 106/μL 2.18 2.23 2.22 2.14 0.06 0.952 0.835 0.492 WBC4, 103/μL 429 381 422 374 27.72 0.325 0.638 0.421 Lymphocyte, % 78.0 74.0 76.5 65.2 5.25 0.988 0.624 0.192 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3red blood cell. 4white blood cell. View Large Apparent Total Tract Retention The ATTR of DM, nitrogen and energy was greater in broiler chicks fed the diet with the protease than in those fed diets without protease (P ≤ 0.05; Table 4). The ATTR of energy for broilers fed diets with EO was higher than for broilers fed diets without EO (P < 0.05). In addition, there was an interaction among protease and EO addition for the ATTR of nitrogen (P < 0.05). Table 4. Effects of the protease and EO on total tract retention of dry matter, nitrogen, or energy in broilers.1 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,bMeans in the same row with different superscripts differ (P < 0.05). View Large Table 4. Effects of the protease and EO on total tract retention of dry matter, nitrogen, or energy in broilers.1 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 Items, % P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Dry matter 70.85 70.98 71.63 72.16 0.23 0.042 0.174 0.403 Nitrogen 68.62b 69.49a,b 70.15a,b 73.63a 0.62 0.050 0.202 0.011 Energy 72.54 76.51 75.27 75.83 0.33 0.023 0.017 0.134 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,bMeans in the same row with different superscripts differ (P < 0.05). View Large Ileal Microbiota There were no differences in bacterial counts for Lactobacillus and E. coli for birds fed diets with protease, but Lactobacillus counts increased and E. coli counts decreased with the addition of EO to the diets (P < 0.05; Table 5). Table 5. Effects of the protease and EO on ileal Lactobacillus and E. coli microbiota in broilers.1 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. View Large Table 5. Effects of the protease and EO on ileal Lactobacillus and E. coli microbiota in broilers.1 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 Items, log10 cfu/g P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Lactobacillus 7.15 7.95 6.90 7.19 0.10 0.172 0.034 0.664 E. coli 6.57 5.84 5.72 5.11 0.15 0.488 0.015 0.275 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. View Large Excreta Noxious Gas Emissions Protease or EO supplementation did reduce (P < 0.05) the release of ammonia gas from excreta (Table 6). In addition, there was an interaction (P < 0.05) between protease and EO for excreta noxious gas emission. Hydrogen sulfide and total mercaptans were not influenced by the addition of the protease or EO. Table 6. Effects of the protease and EO on ammonia, hydrogen sulfide and methyl mercaptan gas emission in broilers.1 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,b,cMeans in the same row with different superscripts differ (P < 0.05). View Large Table 6. Effects of the protease and EO on ammonia, hydrogen sulfide and methyl mercaptan gas emission in broilers.1 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 Items, ppm P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO Ammonia 88.50a 78.40b 84.50a,b 71.70c 6.18 0.025 0.031 0.043 Hydrogen sulfide 3.60 3.80 3.30 3.30 1.23 0.766 0.372 0.528 Methyl mercaptan 1.50 1.80 1.50 1.20 1.11 0.851 0.675 0.496 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. a,b,cMeans in the same row with different superscripts differ (P < 0.05). View Large Meat Quality and Relative Organ Weight There were no differences in breast meat pH, color (L*, a*, and b*), drip loss and WHC between birds fed the diets with protease or EO and those fed diets with none added. Moreover, no differences were observed for the relative weight of breast muscle, abdominal fat, liver, bursa of Fabricius, spleen, and gizzard of between birds fed the treatments and controls (Table 7). Table 7. Effects of the protease and EO on chemical characteristics of breast meat and relative organ weight in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3water holding capacity. View Large Table 7. Effects of the protease and EO on chemical characteristics of breast meat and relative organ weight in broilers.1 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 Items P − P + SEM2 P-value EO − EO + EO − EO + P EO P × EO pH value 5.54 5.92 5.88 5.84 0.22 0.562 0.475 0.358 Breast muscle color  Lightness 51.00 54.27 52.57 53.34 1.05 0.764 0.175 0.255  Redness 15.71 14.71 15.62 14.58 0.76 0.882 0.198 0.972  Yellowness 13.65 13.75 14.39 14.85 0.83 0.285 0.734 0.833 Drip loss, %  d 1 1.52 2.43 2.56 3.74 1.25 0.354 0.443 0.944  d 3 2.86 4.43 3.84 5.65 1.34 0.430 0.243 0.938  d 5 4.02 7.18 5.45 7.65 1.99 0.641 0.218 0.814  d 7 5.74 9.08 8.34 10.59 1.67 0.253 0.131 0.752 WHC3,% 65.52 64.33 64.26 65.39 1.81 0.952 0.986 0.537 Relative organ weight, %  Breast muscle 16.56 17.28 14.81 17.25 0.84 0.315 0.157 0.303  Liver 2.44 2.27 2.26 2.14 0.20 0.491 0.492 0.893  Bursa of Fabricius 0.16 0.16 0.15 0.15 0.10 0.447 0.841 0.848  Abdominal fat 2.42 2.44 2.43 2.47 0.16 0.818 0.783 0.918  Spleen 0.08 0.10 0.09 0.08 0.01 0.258 0.165 0.103  Gizzard 1.20 1.21 1.23 1.24 0.08 0.673 0.841 0.984 1P = protease, EO = essential oils, and + or— = supplemented with or without 0.02% protease and 0.03% EO, respectively. 2standard error of the mean. 3water holding capacity. View Large DISCUSSION This experiment was designed to determine the effects of dietary combinations of a protease and EO on growth, nutrient utilization, blood cell profiles, ileal microbiota, and meat quality in broiler chicks. Results revealed that broilers fed diets supplemented with the protease had improved BWG and FI from day 7 to day 21 and overall (P < 0.05). Proteases play a critical role in several physiological processes, such as degradation of food proteins, protein turnover, cell division, blood coagulation, transport of polypeptide hormones, and activation of zymogens (Rao et al., 1998). In particular, proteases have been suggested to optimize feed protein utilization in poultry (Caine et al., 1998). Fru-Nji et al. (2011) reported that proteases used as feed additives could supplement the effects of endogenous pepsin and pancreatic enzymes by augmenting the hydrolysis and solubilization of protein in vitro and in vivo. These effects were also confirmed in other broiler studies, where improved protein and/or fat retention and greater growth performance were observed (Angel et al., 2011; Freitas et al., 2011). In the present study, EO supplementation showed a trend for increased BWG (day 1 to 7; P = 0.063) and FI (day 8 to 21; P = 0.088), and improved FCR (day 1 to 35; P = 0.067). Essential oils have been shown to exert antibacterial, antifungal, and antiviral activities in vitro (Windisch et al., 2008) and numerous studies have documented the benefits of EO on growth performance in poultry (Giannenas et al., 2003; Jang et al., 2007; Franz et al., 2010). Other effects of EO have been suggested to include enhanced voluntary feed intake and, thus, greater body weight gain (Zeng et al., 2015). Phytogenic compounds, which include EO, stimulate bile secretion (Lee et al., 2004; Platel and Srinivasan, 2004). Dietary EO blend including thymol, eugenol, and piperine have been shown to stimulate secretion of pancreatic digestive enzymes (e.g., α-amylase, maltase, and trypsin) in broilers (Jang et al., 2007). These results suggest that dietary EO could affect nutrient retention via quite different mechanisms from the direct effects of proteases. The present study indicates that supplementation with a combination of a protease and EO may synergistically improve nitrogen retention in broilers (protease x EO interaction, P = 0.011). We found no studies regarding the synergistic effects of protease and EO on nutrient retention, but the findings of our current study imply that improvement in growth performance of broilers fed protease and EO may result from higher nutrient retention. Aengwanich et al. (2009) suggest that changes in blood cell profiles can be used to determine the physiological, pathological, and nutritional status of an animal. White blood cells including lymphocytes can increase in response to infection or stress because they are one of the first lines of defense in the body (Wang et al., 2003). As far as we know, there are currently no reports of blood cell profiles including RBC, WBC and lymphocyte due to protease supplementation in broiler chicks, and whether proteases contributed to increase in blood cells is unknown. In contrast, the blood cell profiles of broilers fed EO derived from plants are inconsistent among researchers (Bardzardi et al., 2014; Torki et al., 2015; Attia et al., 2017). The results of the present study indicated that supplementation of protease and EO did not affect blood cell profiles (RBC, WBC and lymphocyte) in broilers. Intestinal microbiota of livestock and poultry plays an important role in supporting health and growth performance. The microbial community in a chicken's ileum is mainly dominated by Lactobacillus spp. (Bjerrum et al., 2006). Du et al. (2015) observed strong antibacterial activity of EO (thymol and carvacrol), in an in vitro minimum inhibitory concentration assay, against pathogenic E. coli, C. perfringens, and Salmonella strains. Other in vivo studies demonstrated inhibiting effects against pathogens such as C. perfringens, E. coli, or Eimeria species (Bozkurt et al., 2013). In the present study, Lactobacillus populations were increased and E. coli populations were decreased in ileal digesta following the addition of EO to broiler diets. Essential oils are a rich source of a variety of phytochemicals and bioactive components that could cause such changes in gut microflora and those changes may be responsible for the improved nutrient retention observed in our experiment. In the present study, emission of ammonia gas from excreta of broilers fed diets with EO was lower than that from birds fed diets without EO. In addition, these results suggest a synergistic effect of EO with a protease (protease × EO interaction, P = 0.043). The interaction between the protease and EO indicated that the reduction of ammonia gas emission from birds fed diets with EO was even more effective with the addition of the protease. There are no previous reports on the influence of EO on excreta ammonia gas emission in broilers. However, Cho et al. (2006) suggested that EO (from fenugreek, clove, and cinnamon) could reduce environmental pollutants, such as fecal ammonia and hydrogen sulfide, by improving feed efficiency and nutrient digestibility in pigs. Ammonia gas is mainly generated because of inefficient utilization of feed protein and the resulting excretion of nitrogen as uric acid. Considering that excreta mal odor and ammonia gas are related to the intestinal microbial ecosystem and nutrient utilization, the reduction in ammonia gas in excreta of broilers fed with protease or EO is easily understood. Meat pH is generally highly associated with meat color, WHC, and drip loss of chicken meat. Higher meat pH causes darker meat, whereas lower meat pH causes lighter meat (Livingston and Brown, 1981). Water holding capacity is the ability of meat to retain its water during processing, storage, and cooking. It is affected by pH and influences the drip loss and eating quality (Kim et al., 2014). Meat pH, color, drip loss, and the WHC of breast muscle were unaffected by protease or EO treatments in the present study. Therefore, the lack of change in meat quality characteristics can be explained by similar pH values among the treatments. Previous reports on the effects of enzyme and phytogenic feed additives on meat quality and organ weight in broilers have been inconsistent. Some authors reported advantages of administration of enzymes or EO (Omojola and Adesehinwa, 2007; Symeon et al., 2009), whereas others did not observe improvement when a protease or an EO was used (Tabook et al., 2006; Zakaria et al., 2010; Hassanein, 2011; Dalólio et al., 2015). Further study with more focus on meat quality associated with these additives is recommended. CONCLUSIONS Our results indicated that supplementation of protease and EO in diets for broiler chicks improved growth performance, increased ileal Lactobaicllus populations, and reduced ileal E. coli populations. Although blood profiles and breast meat quality were not affected, these additives reduced the concentrations of excreta ammonia emission and increased nitrogen retention. Additionally, supplementation with a combination of a protease and EO synergistically improved nitrogen retention and deceased excreta ammonia emission. 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Poultry ScienceOxford University Press

Published: Jul 11, 2018

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