Effects of Dietary Crude Protein and Protease Levels on Performance, Immunity Capacity, and AA Digestibility of Broilers

Abstract

Exogenous proteases are promising to stimulate the application of low-protein diets for broilers. A total of 540 1-day-old Arbor Acres male broilers were randomly assigned to 9 groups with 6 replicates of 10 birds. A 3 × 3 factorial, completely randomized arrangement was used to evaluate the effects of dietary crude protein (CP) and protease levels on growth and slaughter performance, immunity capacity, and apparent ileal digestibility (AID) of amino acids (AA). Dietary CP levels were 20.0%, 19.5%, or 19.0% during the starter phase, and 18.0%, 17.5%, or 17.0% during the finisher phase. Protease levels were 0, 250, or 500 mg/kg in diets throughout the trial. The trial lasted for 42 days. Weight gain and feed efficiency of broilers decreased as dietary CP lowered, but improved with protease supplementation. Dietary CP and protease levels had few effects and interactions on carcass characteristics, immune organ indexes, and immunoglobulin concentrations. The AID of most AA was improved by dietary CP decrease or protease supplementation. In conclusion, reducing dietary CP decreased the performance and immune capacity of broilers but increased the AID of AA. Almost independent of dietary CP level, dietary protease addition improved the performance of broilers, probably through the enhancement of AA digestibility, and had no effect on carcass traits.

1. Introduction

Protein raw materials are the most expensive ingredients of poultry feeds [1]. It is uneconomical and environmentally unfriendly when the crude protein (CP) level of broiler diets exceeds optimal levels. The digestion and absorption rate of nutrients will be reduced, excess protein in the lower gastrointestinal tract can also be used as food by opportunistic pathogens, leading to an increased risk of disease, and the excess protein in the body will be broken down and used for energy or excreted as uric acid [2]. Protein in poultry diets is mainly provided by soybean meal, but the animal feed industry in China is facing a shortage of soybean meal resources. Low-protein diets are one strategy to narrow the gap between yields and the consumption of soybean meal and also minimize the potential environmental pollution of nitrogen excretion. However, lowering the CP content in diets may compromise growth performance [3] and immune functions [4] because the content of essential or nonessential amino acids (AA) will be reduced when the dietary protein level is reduced [5]. A low CP diet may also lead to the accumulation of abdominal fat in broilers [6,7]. Unconventional protein feedstuffs, including cottonseed, rapeseed, corn gluten, and dried distillers grains with soluble, are potential replacements for soybean meal [8]. However, anti-nutritional factors in these miscellaneous meals can affect the contact between endogenous enzymes and substrates and reduce nutrition. Three classes of enzymes, phytases, carbohydrases, and proteases, can reduce the effect of antinutritional factors and improve feed utilization [9].

Protease, a protein-digesting enzyme, can break down protein-bound starch stored in feed ingredients and allow the energy in the protein-bound starch to be used by poultry [10]. Proteases can also effectively degrade protein anti-nutritional factors present in feed ingredients such as soybean meal [11]. Exogenous protease supplementation can improve CP and energy digestibility in the low-CP diets used for broiler chickens or ducks [12,13,14]. The effects of low-CP diets on growth performance are not consistent yet. Supplementation of protease to low-CP diets has no effects on feed intake (FI), body weight gain (BWG), or feed conversion ratio (FCR) [7]. However, supplementation of diets with exogenous proteases increased body weight (BW) and BWG [13]. The differences may result from the contents of low-CP and protease in diets. Based on the above, we propose a scientific hypothesis that proteases could probably improve the digestibility of broiler feed, thereby promoting the wide application of low-protein diets in broilers. Therefore, the current study aimed to determine the effects of dietary CP levels and protease concentrations on the broiler raising industry from the perspectives of growth performance, carcass traits, body health (serum parameters, immune indicators), and nutrient utilization (apparent ileal CP and AA digestibility) in broilers.

2. Materials and Methods

2.1. Broilers, Diets and Design of Experiment

Five hundred and forty Arbor Acres male chicks (one day old) were randomly allocated to nine dietary treatments consisting of six replicates. Each replicate with 10 birds was raised in one wire floor cage (120 × 100 × 48 cm) in a three-level battery in a room with automatic environmental control at the Nankou Animal Experiment Base (Beijing, China) in the spring. A two-phase feeding program (days 1–21 and 22–42) was used in the experiment on the basis of the actual production experience. The chicken room was kept under continuous light for the first three days and 23 h light for the rest time. The temperature was maintained at 32 °C for the first three days and then lowered by 2 °C at weekly intervals until it reached 24 °C. Mechanical longitudinal ventilation was used to keep the ammonia concentration in the room below 20 ppm. The humidity was maintained at 60~70% for the first two weeks and then at 50–60%. Birds had free access to feed and water and got vaccinations according to the established schedule. Details of the protein levels and protease concentrations in each treatment group are shown in

Table 1. Experimental design and dietary treatments.

Treatments	Protein Levels % 1		Protease, mg/kg
	Starter Phase	Finisher Phase
T1	20	18	0
T2	20	18	250
T3	20	18	500
T4	19.5	17.5	0
T5	19.5	17.5	250
T6	19.5	17.5	500
T7	19	17	0
T8	19	17	250
T9	19	17	500

¹ Starter phase: 1–21 days old; finisher phase: 22–42 days old.

. Pichia pastoris protease used in the current study was supplied by Challenge Corporation (Beijing, China). Pichia pastoris protease is an enzyme mainly composed of alkaline protease with an activity of 20,000 U. All diets were provided (cold pelleting, diameter 0.2 cm), with crumbles in the starter and pellets in the finisher. The experimental diets were prepared in a 3 × 3 factorial, completely randomized arrangement. There were both three levels for dietary CP content (20%, 19.5%, and 19% in the starter phase; 18%, 17.5%, and 17% in the finisher phase) and protease supplementation (0 mg/kg, 250 mg/kg, or 500 mg/kg throughout the trial). The protease levels were based on pre-experimental results. The ingredient and nutrient compositions of diets are listed in

Table 2. Ingredient composition and nutrient content of basal diets (dry matter basis).

Items	Starter Phase (Day 1~21)			Finisher Phase (Day 22~42)
	CP20%	CP19.5%	CP19%	CP18%	CP17.5%	CP17%
Ingredient, %
Corn	60.00	61.25	62.50	62.98	64.15	65.33
Soybean meal	24.49	23.68	22.87	15.23	14.56	13.90
Cottonseed meal	3.00	3.00	3.00	5.00	5.00	5.00
DDGS	3.00	3.00	3.00	5.00	5.00	5.00
Corn gluten meal	1.86	1.51	1.16	2.78	2.33	1.89
Soybean oil	2.61	2.53	2.46	4.35	4.30	4.26
NaCl	0.35	0.35	0.35	0.35	0.35	0.35
CaHPO4	1.71	1.71	1.71	1.35	1.35	1.36
Limestone	1.50	1.50	1.51	1.49	1.49	1.50
L-Lys, 79%	0.63	0.62	0.60	0.69	0.67	0.65
DL-Met, 99%	0.18	0.18	0.17	0.17	0.16	0.16
L-Thr, 98.5%	0.17	0.16	0.16	0.17	0.17	0.17
Choline chloride, 50%	0.20	0.20	0.20	0.20	0.20	0.20
Premix 1	0.30	0.30	0.30	0.30	0.30	0.30
Total	100	100	100	100	100	100
Nutrient levels 2, %
Metabolizable energy, MJ/kg	3000	3000	3000	3150	3150	3150
Crude protein	20.00	19.50	19.00	18.00	17.50	17.00
Lys	1.36	1.33	1.29	1.22	1.19	1.15
Met	0.51	0.49	0.48	0.46	0.45	0.44
Met + Cys	0.83	0.81	0.80	0.76	0.74	0.72
Thr	0.91	0.89	0.87	0.81	0.78	0.76
Trp	0.25	0.24	0.24	0.20	0.19	0.19
Arg	1.23	1.20	1.17	1.06	1.03	1.00
Leu	1.68	1.63	1.57	1.59	1.52	1.46
Ile	0.71	0.69	0.67	0.61	0.59	0.57
Phe	0.99	0.96	0.94	0.90	0.87	0.84
Phe + Tyr	1.69	1.64	1.59	1.53	1.48	1.43
His	0.47	0.46	0.45	0.41	0.40	0.39
Val	0.86	0.83	0.81	0.76	0.74	0.72
Calcium	1.00	1.00	1.00	0.90	0.90	0.90
Available Phosphorus	0.45	0.45	0.45	0.42	0.42	0.42

¹ In one kilogram of diets, for broilers with 1 to 21 days of age, vitamin A 8000 IU, vitamin D3 1000 IU, vitamin E 20 mg, vitamin K3 0.5 mg, vitamin B1 2.0 mg, vitamin B2 8 mg, vitamin B6 3.5 mg, vitamin B12 0.01 mg, pantothenic acid 10 mg, nicotinic acid 35 mg, folic acid 0.55 mg, biotin 0.18 mg, copper sulfate 8 mg, ferrous sulfate 80 mg, zinc sulfate 80 mg, manganese sulfate 80 mg, potassium iodide 0.7 mg; for broilers with 22 to 42 days of age, vitamin A 6 000 IU, vitamin D3 750 IU, vitamin E 10 mg, vitamin K3 0.5 mg, vitamin B1 2.0 mg, vitamin B2 5 mg, vitamin B6 3.0 mg, vitamin B12 0.01 mg, pantothenic acid 10 mg, nicotinic acid 30 mg, folic acid 0.55 mg, biotin 0.15 mg, copper sulfate 6.4 mg, ferrous sulfate 64 mg, zinc sulfate 64 mg, manganese sulfate 64 mg, potassium iodide 0.56 mg. ² Nutrient levels were calculated values. DDGS: distillers dried grains with soluble.

. Protease was premixed with soybean meal before being added to diets.

2.2. Growth and Slaughter Performance

After 8 h fasting, using replicate as a measuring unit, the BW of the birds was weighed on the mornings of days 21 and 42. Feed intake was recorded every week. Average daily gain (ADG), average daily feed intake (ADFI), death and culling rate (DCR), and ratio of feed to gain (FCR) were calculated.

At the end of the trial, one bird with the medium average BW of the replicate was chosen from each replicate, and blood was sampled from the heart after starving for 8 h. Then the selected birds were slaughtered to score the carcass, breast, leg yields, and abdominal fat after hot de-feathering and chilling at room temperature. Dressing was calculated as the weight of broilers after bloodletting and hair removal divided by the live weight. Half-eviscerated weight of broilers is the dressing weight minus the weight of the trachea, esophagus, crop, intestine, spleen, pancreas, gallbladder, reproductive organs, and the cuticular membrane and contents of the muscular stomach. Full-eviscerated weight is the weight of half-eviscerated weight minus the weight of the heart, liver, glandular stomach, muscle stomach, fat, and head and feet. Half- or full-eviscerated rates were calculated from the ratio of their weight to the live weight of broilers. Breast weight was recorded with both pectoralis major and minor without skin. Abdominal fat includes the tissues surrounding the gizzard and intestines, around the cloaca and bursa of Fabricius, and adjoining the abdominal muscle. Carcass yield was expressed as the percentage of live weight at 42 d. The ratios of breast yield, leg yield, and abdominal fat were calculated based on the chilled, full-eviscerated weight. The European performance index (EPI) was calculated using the formula.

$$\mathrm{EPI}=\mathrm{live}\mathrm{weight}, \mathrm{kg}\times (1-\mathrm{D}\mathrm{C}\mathrm{R})/(\mathrm{F}\mathrm{C}\mathrm{R}\times 42)\times \mathrm{10,000}$$

2.3. Immune Organ Indexes

After slaughter, the thymus, spleen, and bursa were isolated, and the blood on the surface of the organs was removed with filter paper. Fresh weight was weighed to calculate the immune organ index. The spleen, thymus, and bursa indexes were generated using the formula.

$$\mathrm{Immune}\mathrm{organ}\mathrm{index} (\%)=100\times \mathrm{immune}\mathrm{organ}\mathrm{weight} \left(\mathrm{g}\right)/\mathrm{B}\mathrm{W} \left(\mathrm{g}\right)$$

2.4. Serum Biochemical Indices

Blood samples were centrifuged (TDL-80-2B, Anke, Shanghai, China) for 10 min at 1500 rpm and 4 °C after standing for 30 min at room temperature to collect the serum, which was stored at −70 °C until analysis.

The contents of immunoglobulin A (IgA), immunoglobulin G (IgG) immunoglobulin M (IgM), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine (CREA), blood ammonia (BA), and uric acid (UA) in serum were measured by an automated system (7600 analyzer, Hitachi High Technologies Co., Tokyo, Japan) with commercial kits following manufacturer guidelines (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.5. Apparent Ileal Digestibility Assay

All broilers in the trial aged from 36 to 42 days were fed the corresponding experimental diets supplemented with 0.4% of titanium dioxide as an exogenous indicator to analyze the digestibility coefficients of crude protein and apparent ileal AA digestibility [15].

Three forty-two-day-old birds from each replicate were sacrificed. The contents of the ileum from Meckel’s diverticulum to about 50 mm before the ileocecal junction were gathered in a plastic dish. The contents were immediately frozen, stored at −20 °C, and then freeze-dried. The dried ileal digesta were ground to pass through a 0.45 mm particle size filter and stored in ziplock bags at −4 °C prior to analysis.

The total nitrogen levels of ileal contents were assessed by an automatic Kjeldahl analyzer (KDY-9830, Ketuo, Beijing, China). The AA of ileal digesta was determined by an automatic AA analyzer (Hitachi L-8800, Tokyo, Japan). Non-sulfur AA in samples was hydrolyzed using pretreatment with 6 M HCl for 24 h, and the hydrolysate was adjusted to pH 2.20, centrifuged, and filtered. Methionine and cysteine were transformed into methionine sulfone and cysteic acid with cold performic acid oxidation overnight and hydrolyzing with 7.5 N HCl at 110 °C for 24 h, and then analyzed using the AA analyzer (Hitachi L-8800). The TiO₂ concentration in diets and ileal digesta were analyzed in conformity with the previous report [2]. The AID for nitrogen and AA was calculated using the following formula.

$$\mathrm{AID}\mathrm{of}\mathrm{nitrogen}=\left[1-\left(Ni\times Td\right)/\left(Nd\times Ti\right)\right]\times 100$$

where Ni = nitrogen concentration in ileal digesta (%), Nd = nitrogen concentration in diet (%), Td = titanium dioxide concentration in diet (%), and Ti = titanium dioxide concentration in ileal digesta (%).

$$\mathrm{AID} \mathrm{of} \mathrm{AA}=\left[1-\left(Ai\times Td\right)/\left(Ad\times Ti\right)\right]\times 100$$

where Ai = AA concentration in ileal digesta (%), Ad = AA concentration in diet (%), Td = titanium dioxide concentration in diet (%), and Ti = titanium dioxide concentration in ileal digesta (%).

2.6. Statistical Analysis

The main effects of CP levels, protease concentrations, and their interaction were analyzed by a 3 × 3 factorial using the GLM procedure of SAS (v 9.1, SAS Inst. Inc., Cary, NC, USA). The data within one main factor were subjected to one-way ANOVA to determine the differences between three levels, taking the replicate as the experimental unit (n = 6). All data were tested for normality using the univariate procedure. p ≤ 0.05 was the criterion for significance.

3. Results

3.1. Growth and Slaughter Performance

The effects of dietary CP levels and protease concentrations on ADG, ADFI, F/G, and DCR of broilers are shown in

Table 3. Effects of dietary crude protein and protease levels on growth performance of broilers.

	Crude Protein Level			Protease, mg/kg			SEM	p-Value
	CP1	CP2	CP3	0	250	500		CP	PT	CP × PT
Day 1~21
ADG, g	44.95	43.60	44.15	42.59 y	43.97 xy	46.14 x	2.27	0.06	<0.01	0.09
ADFI, g	55.02 b	54.27 b	56.24 a	53.98 y	54.96 xy	56.59 x	1.15	0.05	<0.01	0.64
FCR, g/g	1.22 b	1.24 ab	1.27 a	1.27 x	1.25 xy	1.23 y	0.01	0.01	0.04	0.31
DCR, %	0.83	0.83	0.00	0.83	0.83	0.00	0.02	0.34	0.45	0.59
Day 22~42
ADG, g	85.57 a	80.49 b	80.07 b	80.14 y	81.46 xy	84.53 x	1.87	0.05	0.02	0.70
ADFI, g	150.89	146.36	143.55	146.34	144.66	149.80	2.71	0.23	0.45	0.98
FCR, g/g	1.76 b	1.82 a	1.79 ab	1.83 x	1.78 y	1.77 y	0.02	0.05	0.01	0.18
DCR, %	1.66	0.83	0.83	1.66	0.83	0.83	0.03	0.17	0.23	0.19
Day 1~42
ADG, g	65.36 a	62.13 b	62.31 b	61.37 y	62.80 xy	65.63 x	1.42	0.02	0.57	0.44
ADFI, g	103.81	101.04	101.59	101.38	101.21	103.85	1.39	0.37	0.70	0.96
FCR, g/g	1.59 b	1.63 a	1.63 a	1.65 x	1.61 y	1.58 y	0.01	0.01	0.27	0.06
DCR, %	2.49	1.66	0.83	2.49	1.66	0.83	0.02	0.13	0.24	0.08
EPI	408.14 a	382.47 b	385.81 b	368.86 z	390.03 y	418.33 x	10.33	<0.01	<0.01	0.05

CP1, CP2, and CP3 represent the groups fed diets containing 20%, 19.5%, and 19% crude protein, respectively, within 1 to 21 days, and 18%, 17.5%, and 17% crude protein, respectively, over 22 to 42 days. CP: crude protein level, PT: protease, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio, DCR: death and culling rate, EPI: European performance index, SEM: standard error of means, ^{a, b, x–z}: means without a common superscripted letter within a line are significantly different.

. In the starter phase, reducing dietary CP levels increased ADFI but had a negative effect on the F/G (p ≤ 0.05). Dietary protease supplementation increased ADG, ADFI, and decreased F/G of broilers (p ≤ 0.05). In the finisher and whole phase, lowering dietary CP contents decreased the ADG and increased F/G, while adding protease to diets increased the ADG and decreased F/G of broilers (p ≤ 0.05). The EPI of broilers was decreased by the reduction in dietary CP levels but increased by the dietary supplementation with protease (p ≤ 0.05). There were no interactions between dietary CP and protease on ADG, ADFI, F/G, and DCR of broilers except for EPI (p ≤ 0.05). In

Table 4. Effects of dietary crude protein and protease levels on carcass traits of broilers.

Item, %	Crude Protein Level			Protease, mg/kg			SEM	p-Value
	CP1	CP2	CP3	0	250	500		CP	PT	CP × PT
Dressing	92.85	91.55	90.95	91.38	91.74	92.22	1.73	0.55	0.97	0.58
Half eviscerated	86.40	85.88	84.44	84.84	85.89	85.98	1.81	0.84	0.07	0.87
Full eviscerated	73.99	73.72	73.14	73.32	73.12	74.41	1.83	0.06	0.86	0.41
Breast muscle	29.33	29.54	28.42	29.53	29.21	28.55	0.27	0.45	0.44	0.41
Leg muscle	22.02	21.97	21.92	21.84	22.49	21.51	0.22	0.99	0.46	0.48
Abdominal fat	2.30 b	2.49 ab	2.64 a	2.48	2.54	2.41	0.11	0.04	0.35	0.11

CP1, CP2, and CP3 represent the groups fed diets containing 20%, 19.5%, and 19% crude protein, respectively, within 1 to 21 days, and 18%, 17.5%, and 17% crude protein, respectively, over 22 to 42 days. CP: crude protein level, PT: protease, SEM: standard error of means. ^a,b: means without a common superscripted letter within a line are significantly different.

, dietary CP levels and protease concentrations had no effects or interactions on the carcass traits of broilers, including dressing and the rates of half-eviscerated, full-eviscerated, breast muscle, and leg muscle. The abdominal fat of broilers was increased by the low-protein diets (p ≤ 0.05), but not influenced by the supplementation of protease.

3.2. Blood Biochemistry and Immune Capacity

The effects of dietary CP levels and protease supplementation on serum biochemicals are shown in

Table 5. Effects of dietary crude protein and protease levels on serum biochemical parameters of broilers.

Item, %	Crude Protein Level			Protease, mg/kg			SEM	p-Value
	CP1	CP2	CP3	0	250	500		CP	PT	CP × PT
ALT, U/L	1.77	1.79	1.76	1.85	1.69	1.78	0.09	0.98	0.53	0.96
AST, U/L	172.35	181.62	161.09	185.48	147.29	182.30	17.56	0.69	0.21	0.09
ALP, U/L	6810.6 a	1830.2 b	2461.7 b	4573.1	2615.7	3913.6	1208.0	0.03	0.75	0.75
CREA, μmol/L	12.56 a	10.50 b	9.87 b	9.86	11.37	11.71	0.85	0.04	0.28	0.41
BA, μmol/L	43.19	44.64	45.12	39.65 z	45.21 y	48.11 x	1.83	0.82	0.05	0.45
UA, μmol/L	295.54 a	259.17 ab	222.69 b	325.77 x	245.39 y	206.23 z	16.67	0.04	<0.01	0.81

CP1, CP2, and CP3 represent the groups fed diets containing 20%, 19.5%, and 19% crude protein, respectively, within 1 to 21 days, and 18%, 17.5%, and 17% crude protein, respectively, over 22 to 42 days. CP: crude protein level, PT: protease, ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, CREA: creatinine, BA: blood ammonia, UA: uric acid, SEM: standard error of means, ^{a, b, x–z}: means without a common superscripted letter within a line are significantly different.

, including ALT, AST, ALP, CREA, BA, and UA. The ALP activity and concentrations of CREA and UA were reduced by the dietary CP content (p ≤ 0.05). Dietary protease supplementation increased the concentration of BA and decreased that of UA (p ≤ 0.05). No interactions were observed between dietary CP and protease on all of those biochemical indexes.

As shown in

Table 6. Effects of dietary crude protein and protease levels on immune organ indices and serum immunoglobulins of broilers.

Item, %	Crude Protein Level			Protease, mg/kg			SEM		p-Value
	CP1	CP2	CP3	0	250	500		CP	PT	CP × PT
Thymus	0.27	0.24	0.27	0.29	0.27	0.23	0.03	0.66	0.35	0.11
Spleen	0.11	0.09	0.11	0.10	0.10	0.11	0.01	0.12	0.72	0.42
Bursa of Fabricius	0.06	0.06	0.07	0.06	0.07	0.06	0.01	0.67	0.85	0.69
IgA, g/L	2.46 a	2.21 b	2.06 c	2.28	2.33	2.12	0.10	0.03	0.33	0.47
IgM, g/L	1.59	1.51	1.72	1.63	1.52	1.66	0.08	0.19	0.44	0.11
IgG, g/L	4.58	4.54	4.44	4.56	4.52	4.48	0.18	0.32	0.19	0.18

CP1, CP2, and CP3 represent the groups fed diets containing 20%, 19.5%, and 19% crude protein, respectively, within 1 to 21 days, and 18%, 17.5%, and 17% crude protein, respectively, over 22 to 42 days. CP: crude protein level, PT: protease, IgA: immunoglobulin A, IgG: immunoglobulin G, IgM: immunoglobulin M, SEM: standard error of means, ^a–c: means without a common superscripted letter within a line are significantly different.

, dietary CP level and protease concentration had no effect and no interactions on the weight indexes of the thymus, spleen, and bursa and the serum concentrations of IgM and IgG. The IgA concentration in serum was decreased by reducing the dietary CP level (p ≤ 0.05) but was not affected by protease supplementation in diets.

3.3. Apparent Ileal AA Digestibility

The effects of dietary CP level and protease supplementation on apparent ileal CP and AA digestibility coefficients are presented in

Table 7. Effects of dietary crude protein and protease levels on crude protein and apparent ileal essential AA digestibility coefficients of broilers.

Item, %	Crude Protein Level			Protease, mg/kg			SEM	p-Value
	CP1	CP2	CP3	0	250	500		CP	PT	CP × PT
Crude protein	68.94 b	73.57 a	75.43 a	71.04 y	74.86 x	72.04 xy	1.33	0.04	0.05	0.45
Indispensable AA
Arg	78.98 b	81.36 ab	83.59 a	80.33	83.47	80.13	1.79	0.02	0.08	0.76
Cys	58.41 b	68.35 a	65.00 a	61.00 y	65.04 x	65.72 x	1.95	<0.01	0.04	0.02
His	68.79 b	74.57 a	75.98 a	71.49 y	74.65 x	73.20 xy	2.04	<0.01	0.05	0.39
Ile	68.74 c	72.03 b	74.52 a	70.28 y	74.68 x	70.34 y	1.48	0.04	<0.01	0.62
Leu	73.06 b	77.01 a	78.29 a	74.84 y	78.95 x	74.57 y	1.36	0.03	<0.01	0.82
Lys	77.23 c	79.88 b	83.54 a	79.86	81.31	79.49	1.91	0.03	0.63	0.83
Met	81.26 b	84.51 a	84.92 a	83.69	85.07	81.94	1.32	0.03	0.33	0.87
Phe	75.22 b	76.94 ab	78.70 a	74.83 y	80.65 x	75.38 y	1.52	0.05	<0.01	0.74
Thr	62.68 c	69.83 b	72.79 a	67.07	69.70	68.53	0.94	<0.01	0.07	0.15
Tyr	73.90	73.41	74.38	72.72 y	77.19 x	71.79 y	2.36	0.96	0.04	0.75
Val	63.64 b	70.74 a	73.25 a	66.99 y	71.65 x	68.99 xy	1.77	<0.01	0.03	0.36
Dispensable AA
Ala	69.90 b	75.81 a	77.35 a	73.22	76.37	73.47	1.89	0.01	0.09	0.74
Asp	63.60 b	70.98 a	73.53 a	66.96 y	70.77 x	70.38 x	1.51	<0.01	<0.01	0.11
Glu	74.81 b	79.88 a	81.01 a	77.04 y	80.58 x	78.08 xy	1.48	0.02	0.05	0.59
Gly	59.25 c	66.81 b	69.48 a	62.68 y	66.86 x	66.00 x	2.01	<0.01	<0.01	0.09
Pro	68.58 b	75.16 a	75.95 a	71.77 y	75.53 x	72.40 xy	1.72	0.01	0.03	0.71
Ser	64.60 c	71.31 b	73.06 a	67.42 y	71.74 x	69.82 xy	1.12	<0.01	0.02	0.32

CP1, CP2, and CP3 represent the groups fed diets containing 20%, 19.5%, and 19% crude protein, respectively, within 1 to 21 days, and 18%, 17.5%, and 17% crude protein, respectively, over 22 to 42 days. CP: crude protein level, PT: protease, AA; Lys, lysine; Met, methionine; Thr, threonine; Val, valine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; Arg, arginine; His, histidine; Gly, glycine; Asp, aspartic acid; Ser, serine; Pro, proline; Ala, alanine; Cys, cysteine; Glu, glutamic acid; Tyr, tyrosine; SEM: standard error of means, ^{a–c, x–y}: means without a common superscripted letter within a line are significantly different.

. The apparent ileal digestibility coefficients of CP, arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, alanine, aspartic acid, glutamic acid, glycine, proline, and serine increased, along with the reduction in dietary CP content (p ≤ 0.05). Compared with the treatments without protease supplementation, dietary supplementation with 250 mg/kg of protease increased the apparent ileal digestibility coefficients of CP, cysteine, histidine, isoleucine, leucine, phenylalanine, tyrosine, valine, glutamic acid, glycine, proline, and serine, while adding 500 mg/kg of protease only increased those of cysteine, aspartic acid, and glycine (p ≤ 0.05). No significant interactions between dietary CP and protease levels were observed in apparent ileal CP and AA digestibility coefficients other than Cys.

4. Discussion

A large reduction in dietary protein content over 2 percentage points would lead to the poor growth performance of broilers [16,17,18]. Similar results were noted in the present study, where the weight gain and feed efficiency of broilers were depressed along with the reduction in dietary protein levels throughout the whole experimental period. However, feed intake was increased with a dietary protein reduction in the starter phase. One possible explanation is that birds tend to increase their feed intake to compensate for diets that are marginally deficient in CP.

It was widely demonstrated that the growth performance of broilers could be improved by the addition of exogenous proteases to their diets, owing to an increase in the digestibility of CP [13,19,20]. In the current study, the growth performance of birds fed diets supplemented with protease was significantly increased, including ADG and feed efficiency. The digestive system of broilers is immature, and pancreatic protease secretion is limited in the starter phase [21]. Exogenous proteases are beneficial because they help compensate for the deficiency of trypsin and increase the degradation of intestinal proteins [22]. Another possible factor is the indigestible ingredients of cottonseed meal, rapeseed meal, corn gluten meal, and dried distiller’s grains [23], whose digestibility was improved by the addition of exogenous enzymes in diets. It is supported by previous reports that dietary protease supplementation increases nitrogen utilization [14,24]. In addition, there are inconsistent reports that proteases added to a low-protein diet had a small positive effect on growth performance and even a negative effect in the finisher phase on the ADG, ADFI, and F/G of broilers [25]. In the present study, we found that broilers fed diets containing 500 mg/kg protease had a lower apparent ileal digestibility of CP and AA than those containing 250 mg/kg. The underlying reason is that it is possible that the addition of exogenous proteases may inhibit endogenous secretion and activity [26]. These results indicated that a high protease concentration in the diet was not conducive to the growth performance of broilers. Broilers fed the low protein diet had a higher abdominal fat rate [12,27], which was confirmed again in this study. One possibility was that the net energy of the diet was underestimated due to the protein level reduction in the diet [28], which led to increased abdominal fat deposition of broilers. Protease in diets did not affect the rate of fat deposition, which was consistent with the previous results [12].

ALP activity is an important serum biochemical indicator of liver disease, and elevated serum ALP activity probably reflects physiologic or pathologic changes in the liver [29]. The severity of acute kidney injury is classified according to the serum CREA level, which is widely interpreted as a measure of renal function [30]. In the current study, the reduction in dietary CP contents decreased ALP activity and CREA concentration. It suggested that low CP diets are beneficial to the function of the liver and kidney of broilers. There was a tendency for the CREA level to decrease with decreasing protein levels, although this decrease was not significant. CREA is converted from creatine and phosphocreatine, and its absolute conversion rate is usually proportional to body weight [31]. This statement coincides with the ADG of the broilers in this study. Creatine synthesis is a major component of arginine and glycine metabolism [32]. Therefore, the levels of arginine and glycine in the diet have an important effect on the synthesis of CREA. In this study, the digestibility of apparent ileal arginine and glycine increased with the decrease in dietary protein content, which indicates that arginine and glycine are insufficient in the diet. The degradation of AA, deamination of purines and pyrimidines, and absorption from the digestive tract are the main sources of BA. Concentrations of ammonia, together with UA and urea, in serum, were used as indicators of the AA utilization rate of diets for broilers [33]. The serum BA level of broilers fed low-CP diets was significantly greater than those fed normal diets [6]. It could be explained by the fact that low-protein diets reduce nitrogen metabolism pressure in animals. Protease increased the total tract retention of nitrogen and decreased the excreta ammonia of growing broilers [34]. In this study, serum BA was improved by the supplementation of protease but not affected by dietary CP contents. One possibility is that the addition of protease accelerates protein hydrolysis and leads to a high absorption rate in contrast to the absorption of intact protein. Serum UA produced by purine metabolism is a by product of protein catabolism and turnover in the body, balanced by production and the net reabsorption or secretion of the kidney and intestine [35]. A decrease in serum UA could be associated with lower dietary nutrition or less protein degradation [5,15]. In the current study, with the reduction in CP or protease contents in diets, serum UA content decreased, which is similar to the results reported previously [12]. The immune function was observed to be suppressed in cats and pigeons given a low-protein diet [36,37], which was also confirmed in broilers in the present study. It shows that low protein consumption is not conducive to immunity of animals. Enzyme-treated soy protein supplementation in a low-protein diet enhanced the immune function of immune organs in on-growing grass carp [38], whereas the immune response of broilers in the current study did not reveal any significant effect of protease enzyme supplementation, which is consistent with the previous report [39]. It could be deduced that the modulation of immune function by proteases may be related to crude protein levels in feed.

Broilers fed on a low-CP diet significantly increased the apparent ileal digestibility of CP and all AA except for Lys [40]. Significant increases in ileal digestibility were confined to Arg, Ile, Leu, Thr, Val, Cys, and Pro when the dietary protein level was reduced by 4.5 percentage units [3]. The secretion of digestive enzymes in the digestive tract will be lowered in response to the low protein intake [41]. In this study, as dietary protein levels decreased by one percentage unit, the apparent ileal AA digestibility improved except for Tyr. This may be because the ileal endogenous losses of AA of broilers fed low-protein diets were reduced. The addition of exogenous proteases to feed could increase the ileal digestibility of nutrients such as CP, energy, and AA in broilers, thereby effectively improving the growth performance of broilers [10,42,43,44,45]. Consistent with previous findings, in the current study, adding 250 mg/kg protease to diets increased the apparent ileal digestibility coefficients of broilers for cysteine, histidine, isoleucine, leucine, phenylalanine, tyrosine, valine, aspartic acid, glutamic acid, glycine, proline, and serine. Nevertheless, when the added amount was as high as 500 mg/kg in diets, the positive effects of protease on AA digestibility were greatly reduced, which may be owed to the fact that a higher amount of protease in diets significantly decreases trypsin activity and mRNA expression in broilers [46]. In contrast, some reports indicated that the addition of protease had no effect on growth performance or nutrient utilization [14], most likely due to the high digestibility of protein feedstuffs in the basal diet [47]. Therefore, it indicated that appropriate dietary protease increases digestion of CP and consequently promotes the growth performance of broilers. The underlying reason is probably that the addition of proteases plays a role in the pre-digestion of crude protein in feed.

5. Conclusions

Reducing dietary CP contents decreases the growth performance and immune capacity of broilers but significantly increases abdominal fat deposition and apparent ileal digestibility of protein and AA. Protease supplementation in diets increased the growth performance of broilers, probably through the enhancement of AA digestibility, but had no effect on carcass traits or body health. Both reduction in dietary CP levels and protease supplementation were beneficial for reducing fecal nitrogen emission. There were almost no interactions between dietary CP content and protease addition on broiler production, except for EPI.