Effects of protease supplementation of low protein and/or energy diets on growth performance and blood parameters in broiler chickens under heat stress condition

Figures & data

Figure 1. The experimental design and experimental diets. RPE: recommended-CP and recommended-ME diet; RPLE: recommended-CP and low-ME diet; LPRE: low-CP and recommended-ME diet; LPE: low-CP and low-ME diet; Diets (except RPE) compounded was subsequently divided into two, one serving as the control and the other supplemented with the enzyme to serve as the test diet; ¹Heat stress was started from d 22–42 in chamber B.

Table 1. Ingredient composition (as fed basis) of the starter and finisher diets.

Ingredient, %	Starter (d 1–21)					Finisher (d 22–42)
	Control	RPLE	LPRE	LPE		Control	RPLE	LPRE	LPE
Corn	55.25	56.52	63.66	64.93		55.67	56.90	63.40	64.69
Soybean meal	31.20	30.95	23.62	23.39		23.47	23.26	16.50	16.23
Canola meal	4.00	4.00	4.00	4.00		8.00	8.00	8.00	8.00
Palm kernel meal	1.50	1.50	1.50	1.50		3.00	3.00	3.00	3.00
Palm olein	4.34	3.31	3.05	2.02		6.91	5.89	5.72	4.70
Limestone	1.29	1.29	1.34	1.33		1.06	1.08	1.10	1.10
Sodium chloride	0.34	0.34	0.34	0.34		0.33	0.33	0.33	0.33
MDCP	1.54	1.54	1.57	1.57		1.22	1.21	1.25	1.25
L-lysine HCl	0.17	0.17	0.35	0.35		0.06	0.06	0.23	0.23
DL-methionine	0.15	0.15	0.18	0.18		0.11	0.10	0.14	0.14
L-threonine	0.07	0.08	0.18	0.18		0.02	0.02	0.13	0.13
L-tryptophan	0.00	0.00	0.05	0.05		0.00	0.00	0.04	0.04
L-valine	0.00	0.00	0.01	0.01		0.00	0.00	0.01	0.01
Vitamin premix	0.03	0.03	0.03	0.03		0.03	0.03	0.03	0.03
Mineral premix	0.10	0.10	0.10	0.10		0.10	0.10	0.10	0.10
Antioxidant	0.02	0.02	0.02	0.02		0.02	0.02	0.02	0.02

Control: recommended-CP and recommended-ME diet; RPLE: recommended-CP and low-ME diet; LPRE: low-CP and recommended-ME diet; LPE: low-CP and low-ME diet; Diets (except RPE) compounded was subsequently divided into two, one serving as the control and the other supplemented with the enzyme to serve as the test diet; ME: Metabolisable energy; Antioxidant: Butyrated hydroxytoluene.

Vitamin premix per kg of diet: vitamin A (retinyl acetate), 8000 U; vitamin D₃ (cholecalciferol), 1000 U; vitamin E (DL-α-tocopherol), 30 U; vitamin K₃ (menadione dimethylpyrimidinol), 2.5 mg; vitamin B₁ (thiamine mononitrate), 2 mg; vitamin B₂ (riboflavin), 5 mg; vitamin B₆ (pyridoxine), 2 mg; vitamin B₁₂, 0.01 mg; niacin, 30 mg; d-biotin, 0.25 mg; vitamin C (ascorbic acid), 50 mg; d-calcium pantothenate, 8 mg, folic acid, 5 mg.

Mn (manganese oxide), 70 mg; Zn (zinc sulphate monohydrate), 70 mg; Fe (ferrous sulphate monohydrate), 35 mg; Cu (cupric sulphate pentahydrate), 8 mg; I (calcium iodate), 1 mg; Mo (sodium molybdate), 1 mg; Se (sodium selenite), 0.25 mg; Co (cobalt carbonate), 0.2 mg. MDCP: monodicalcium phosphate.

Table 2. Nutrient composition of the starter and finisher diets.

Item	Starter (d 1–21)					Finisher (d 22–42)
	Control	RPLE	LPRE	LPE		Control	RPLE	LPRE	LPE
Nutrient (calculated, % unless stated otherwise)
ME, Kcal/kg	3035	2985	3035	2985		3180	3130	3180	3130
Crude protein	21.00	21.00	18.50	18.50		19.00	19.00	16.70	16.70
Lysine	1.31	1.31	1.31	1.31		1.07	1.07	1.07	1.07
Methionine	0.48	0.48	0.48	0.48		0.42	0.42	0.42	0.42
Threonine	0.88	0.88	0.88	0.88		0.76	0.76	0.76	0.76
Tryptophan	0.27	0.27	0.27	0.27		0.24	0.24	0.24	0.24
Valine	1.02	1.02	1.02	1.02		0.94	0.94	0.94	0.94
Arginine	1.47	1.47	1,24	1.24		1.32	1.32	1.10	1.10
Leucine	1.79	1.80	1.61	1.61		1.63	1.63	1.46	1.46
Isoleucine	0.91	0.91	0.77	0.77		0.81	0.81	0.68	0.68
Histidine	1.30	1.29	1.05	1.04		1.07	1.06	0.83	0.83
Glycine + Serine	1.91	1.91	1.63	1.63		1.72	1.72	1.47	1.47
Analysed composition, %
Crude protein	21.38	21.22	17.64	17.82		18.27	18.58	16.67	17.03
Lysine	1.31	1.29	1.38	1.08		0.99	1.01	0.98	0.98
Methionine	0.50	0.47	0.50	0.44		0.46	0.41	0.40	0.44
Threonine	0.95	0.96	1.01	0.84		0.75	0.79	0.78	0.83

Control: recommended-CP and recommended-ME diet; RPLE: recommended-CP and low-ME diet; LPRE: low-CP and recommended-ME diet; LPE: low-CP and low-ME diet; Diets (except RPE) compounded was subsequently divided into two, one serving as the control and the other supplemented with the enzyme to serve as the test diet.

Table 3. Effect of diet on growth performance and mortality rate in broiler chickens from day 1 to 21.

Item	Feed intake, g/bird	Weight gain, g/bird	FCR, feed/gain	Mortality rate, %
Diet
Control	1303	932	1.400^b	2.000
RPLE	1308	909	1.440^ab	2.000
RPLEP	1306	937	1.390^b	2.000
LPRE	1293	892	1.450^ab	2.000
LPREP	1273	912	1.400^b	2.000
LPE	1285	895	1.440^ab	2.000
LPEP	1320	893	1.480^a	4.000
SEM	55.700	42.200	0.060	6.670
p value	.572	.092	.010	>.050

Data represent mean values of 5 cages per treatment.

^a,bMeans within a column without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease; SEM: standard error of the mean; FCR: feed conversion ratio.

Table 4. Effects of diet and temperature on growth performance and mortality rate in broiler chickens from day 22 to 42 and 1 to 42.

Item	Feed intake, g/bird			Weight gain, g/bird		FCR, feed/gain		Mortality rate, %
	d 22–42		d 1–42	d 22–42	d 1–42	d 22–42	d 1–42	d 22–42
Diet
Control	3129		4433	1551	2483^a	2.040^ab	1.790^b	4.000
RPLE	3066		4375	1518	2427^ab	2.020^b	1.800^b	6.000
RPLEP	3160		4466	1558	2495^a	2.030^b	1.790^b	2.000
LPRE	2964		4257	1429	2321^ab	2.070^ab	1.830^ab	4.000
LPREP	3072		4345	1439	2350^ab	2.140^ab	1.850^ab	2.000
LPE	2982		4267	1361	2256^b	2.190^a	1.890^a	2.000
LPEP	2861		4181	1327	2220^b	2.160^ab	1.880^a	2.000
SEM	364		389	231	245	0.130	0.060	7.510
Temperature
Normal	3191^a		4497^a	1562^a	2476^a	2.040^b	1.820^b	0.570
Heat stress	2876^b		4167^b	1347^b	2253^b	2.130^a	1.850^a	5.710
SEM	327		348	212	230	0.140	0.070	6.910
Analysis of variance (p value)
Diet		.446	.563	.084	.043	.003	<.001	>.050
Temperature		<.001	<.001	<.001	<.001	.001	.014	>.050
Diet × temperature		.670	.750	.592	.794	.078	.452	>.050

Data represent mean values of 5 cages per treatment.

^a,b,cMeans within a column without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease; SEM: standard error of the mean; FCR: feed conversion ratio.

Table 5. Effects of diet and temperature on serum metabolites in broiler chickens at 42 days of age.

Item	GLU, mmol/L	TG, mmol/L	TP, g/L	CK, U/L
Diet
Control	14.740	0.460^b	26.660	4115^bc
RPLE	14.930	0.500^b	27.110	3714^c
RPLEP	15.400	0.400^b	28.980	5211^a
LPRE	15.400	0.690^a	27.490	5344^a
LPREP	14.850	0.730^a	24.660	5244^a
LPE	15.720	0.720^a	27.950	4955^ab
LPEP	15.930	0.700^a	27.690	5177^a
SEM	1.640	0.250	3.960	1383
Temperature
Normal	14.450^b	0.460^b	25.760^b	4498^b
Heat stress	16.040^a	0.740^a	28.740^a	5179^a
SEM	1.450	0.240	3.800	1442
Analysis of variance (p value)
Diet	.173	<.001	.068	<.001
Temperature	<.001	<.001	<.001	.001
Diet × temperature	.696	.105	.355	.002

Data represent mean values of 10 birds per treatment.

^a,b,cMeans within a column without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease; SEM: standard error of the mean; GLU: glucose; TG: triglycerides; TP: total protein; CK: creatine kinase.

Table 6. Creatine kinase where the interaction between diet and temperature were significant.

Diet		Creatine kinase, U/L
		Normal		Heat stress		SEM	p value
Control		3502^b		4881		1656	.098
RPLE		2430^c,y		4869^x		1320	.001
RPLEP		4724^a,y		5753^x		984	.036
LPRE		5018^a		5670		1026	.172
LPREP		5299^a		5183		1297	.849
LPE		4803^a		5107		1437	.788
LPEP		5612^a		4618		1031	.076
SEM		1387		1131
p value		<.001		.314

^a–cMeans within a column without common superscripts differ at p < .05.

^x,yMeans within a row without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease.

Table 7. Effects of diet and temperature on the percentage of breast yield and abdominal fat in broiler chickens at 43 days of age.

Item	Breast yield, %	Abdominal fat, %
Diet
Control	34.990	2.840^b
RPLE	34.770	2.930^b
RPLEP	35.690	2.400^c
LPRE	34.670	3.560^a
LPREP	34.430	3.550^a
LPE	34.880	3.580^a
LPEP	33.670	3.190^ab
SEM	2.210	0.670
Temperature
Normal	35.300^a	3.210
Heat stress	34.160^b	3.090
SEM	2.170	0.780
Analysis of variance (p value)
Diet	.136	<.001
Temperature	.002	.328
Diet × temperature	.213	.768

Data represent mean values of 10 birds per treatment.

^a,bMeans within a column without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease; SEM: standard error of the mean. Breast meat and abdominal fat, as percentage of live weight.

Table 8. Effects of diet and temperature on serum ceruloplasmin, ovotransferrin, α1-acid glycoprotein, corticosterone and brain heat shock protein70 in broiler chickens at 42 days of age.

Item	CPN, mg/mL	OVT, mg/mL	AGP, mg/mL	CORT, ng/mL	HSP 70, arbitrary unit
Diet
RPE	0.790	0.260	2.360	1.930	0.560^c
RPLE	0.980	0.300	2.780	1.760	0.570^bc
RPLEP	0.790	0.290	2.660	1.610	0.570^bc
LPRE	0.930	0.260	1.780	2.260	0.610^ab
LPREP	0.730	0.270	1.700	2.330	0.600^abc
LPE	1.020	0.290	2.570	2.050	0.630^a
LPEP	0.940	0.280	1.970	2.280	0.610^ab
SEM	0.760	0.080	1.560	1.610	0.110
Temperature
Normal	0.610^b	0.240^b	1.670^b	1.050^b	0.510^b
Heat stress	1.160^a	0.320^a	2.860^a	3.010^a	0.670^a
SEM	0.700	0.070	1.470	1.260	0.080
Source of variation
Diet	0.424	0.814	0.083	0.465	0.006
Temperature	<0.001	<0.001	<0.001	<0.001	<0.001
Diet × temperature	0.100	0.792	0.254	0.884	0.002

Data represent mean values of 10 birds per treatment.

^a,b,cMeans within a column without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease; SEM: standard error of the mean; CPN: ceruloplasmin; OVT: ovotransferrin; AGP: α1-acid glycoprotein; CORT: corticosterone; HSP: heat shock protein.

Table 9. Heat shock protein 70 where the interaction between diet and temperature were significant.

Item		Heat shock protein 70				SEM
		Normal		Heat stress			p value
Diet
Control		0.523^y		0.588^b,x		0.040	.006
RPLE		0.530^y		0.603^b,x		0.070	.037
RPLEP		0.465^y		0.672^a,x		0.080	<.001
LPRE		0.517^y		0.705^a,x		0.060	<.001
LPREP		0.530^y		0.674^a,x		0.060	<.001
LPE		0.536^y		0.722^a,x		0.090	<.001
LPEP		0.511^y		0.702^a,x		0.070	<.001
SEM		0.070		0.060
p value		.355		<.001

^a,bMeans within a column without common superscripts differ at p < .05.

^x,yMeans within a row without common superscripts differ at p < .05.

Control: recommended-CP and recommended-ME; RPLE: recommended-CP and low-ME; RPLEP: recommended-CP and low-ME with protease; LPRE: low-CP and recommended-ME; LPREP: low-CP and recommended-ME with protease; LPE: low-CP and low-ME; LPEP: low-CP and low-ME with protease.