Histomorphometric traits, microbiota, nutrient digestibility, growth performance, carcass traits and meat quality parameters of chickens fed diets supplemented with different levels of Bacillus protease

Figures & data

Table 1. Ingredient (%) and chemical composition (g/kg DM unless otherwise stated) of experimental diets for broiler chicks at the starter phase (0–3 weeks).

Ingredient	PROT0	PROT10	PROT15	PROT20	PROT25
Yellow maize	62.98	62.98	62.98	62.98	62.98
Soybean meal	27.38	27.38	27.38	27.38	27.38
Sunflower meal	4.00	4.00	4.00	4.00	4.00
Fish meal	2.50	2.50	2.50	2.50	2.50
Canola oil	0.11	0.11	0.11	0.11	0.11
Limestone	1.32	1.32	1.32	1.32	1.32
MonoCaP	0.44	0.44	0.44	0.44	0.44
Salt	0.28	0.28	0.28	0.28	0.28
Methionine	0.23	0.23	0.23	0.23	0.23
Threonine	0.05	0.05	0.05	0.05	0.05
Lysine	0.32	0.32	0.32	0.32	0.32
Choline Cl	0.10	0.10	0.10	0.10	0.10
^1VMP	0.20	0.20	0.20	0.20	0.20
^2Maxiban	0.05	0.05	0.05	0.05	0.05
^3Surmax	0.04	0.04	0.04	0.04	0.04
Protease	0.00	0.10	0.15	0.20	0.25
Total	100.00	100.00	100.00	100.00	100.00
Chemical composition
Moisture	11.11	11.19	11.00	11.18	11.11
ME (kcal/kg)	2988.96	2988.86	2988.06	2988.99	2988.69
Crude protein	23.83	23.90	23.79	23.85	23.88
Crude fat	4.01	4.03	4.00	4.01	4.02
NDF	15.32	15.27	15.39	15.28	15.30
ADF	3.99	3.95	3.97	4.00	3.98
Calcium	0.93	0.94	0.92	0.93	0.94
Phosphorus	0.66	0.68	0.65	0.69	0.67

Notes: MonoCaP = Monocalcium Phosphate. Choline Cl = Choline Cloride. VMP = Vitamine mineral premix. ME = Metabolizable energy. NDF = Neutral detergent fibre. ADF = Acid detergent fibre. PROT0 (only basal diet; BD), PROT10 (BD + 1 g protease), PROT15 (BD + 1.5 g protease), PROT20 (BD + 2 g protease) and PROT25 (BD + 2.5 g protease). ^1a2.5 kg of vitamin premix contained: 2700 mg retinal, 400 mg calcidiol, 18 g tocopheryl acetate, 2000mg menadione, 1800 mg thiamine, 6600 mg riboflavin, 10 g niacin, 30 g calcium pantothenate, 3 g pyridoxine, 1 g folic acid, 15 mg cobalamin, 250 g choline chloride, 100 mg biotin. ^b2.5 kg of trace mineral premix contained: 100 g Mn, 50 g Fe, 100 g Zn, 10 g Cu, 1 g I, 200 mg Se. ²1000 g of Maxiban contained: 80 g/kg narasin, 80 g/kg nicarbazin. ³1000 g of Surmax contained: 100 g/kg avilamycin.

Table 2. Ingredient (%) and chemical composition (g/kg DM unless otherwise stated) of experimental diets for broilers at the finisher phase (4–6 weeks).

Ingredient	PROT0	PROT10	PROT15	PROT20	PROT25
Yellow maize	72.36	72.36	72.36	72.36	72.36
Soybean meal	24.55	24.55	24.55	24.55	24.55
Canola oil	0.24	0.24	0.24	0.24	0.24
Limestone	1.25	1.25	1.25	1.25	1.25
MonoCaP	0.17	0.17	0.17	0.17	0.17
Salt	0.39	0.39	0.39	0.39	0.39
Methionine	0.21	0.21	0.21	0.21	0.21
Tryptophan	0.05	0.05	0.05	0.05	0.05
Threonine	0.05	0.05	0.05	0.05	0.05
Lysine	0.34	0.34	0.34	0.34	0.34
Choline Cl	0.10	0.10	0.10	0.10	0.10
^1VMP	0.20	0.20	0.20	0.20	0.20
^2Maxiban	0.05	0.05	0.05	0.05	0.05
^3Surmax	0.04	0.04	0.04	0.04	0.04
Protease	0.00	0.10	0.15	0.20	0.25
Total	100.00	100.00	100.00	100.00	100.00
Chemical composition
Moisture	11.01	11.03	11.00	11.02	11.00
ME (kcal/kg)	3110.94	3111.76	3111.00	3111.85	3111.93
Crude protein	19.75	19.91	19.70	19.83	19.85
Crude fat	4.13	4.12	4.12	4.13	4.12
NDF	18.23	18.32	18.28	18.21	18.22
ADF	4.85	4.97	4.82	4.95	4.80
Calcium	0.94	0.92	0.94	0.93	0.93
Phosphorus	0.61	0.62	0.61	0.61	0.61

Notes: MonoCaP = Monocalcium Phosphate. Choline Cl = Choline Cloride. VMP = Vitamine mineral premix. ME = Metabolizable energy. NDF = Neutral detergent fibre. ADF = Acid detergent fibre. PROT0 (only basal diet; BD), PROT10 (BD + 1 g protease), PROT15 (BD + 1.5 g protease), PROT20 (BD + 2 g protease) and PROT25 (BD + 2.5 g protease). ^1a2.5 kg of vitamin premix contained: 2700 mg retinal, 400 mg calcidiol, 18 g tocopheryl acetate, 2000mg menadione, 1800 mg thiamine, 6600 mg riboflavin, 10 g niacin, 30 g calcium pantothenate, 3 g pyridoxine, 1 g folic acid, 15 mg cobalamin, 250 g choline chloride, 100 mg biotin. ^b2.5 kg of trace mineral premix contained: 100 g Mn, 50 g Fe, 100 g Zn, 10 g Cu, 1 g I, 200 mg Se. ²1000 g of Maxiban contained: 80 g/kg narasin, 80 g/kg nicarbazin. ³1000 g of Surmax contained: 100 g/kg avilamycin.

Figure 1. Histomicrograph of the Jejunum of broilers fed PROT0, PROT10, PROT15, PROT20 and PROT25 which shows the villi height, crypt depth, thickness of the epithelium, and muscularis. Two birds from each cell/replicate were used for the analysis.

Figure 2. Histomicrograph of the ileum of broilers fed PROT0, PROT10, PROT15, PROT20 and PROT25 which shows the villi height, crypt depth, thickness of the epithelium, and muscularis. Two birds from each cell/replicate were used for the analysis.

Table 3. The effect of dietary Bacillus protease supplementation on the gut histomorphometric traits of broiler birds.

	Diets
	PROT0	PROT10	PROT15	PROT20	PROT25	SEM	p-value
Duodenum
Villus height (µm)	1301.75^e	1407.15^d	1512.81^c	1610.74^b	1650.35^a	19.87	0.05
Crypt depth (µm)	280.46^a	251.99^b	231.79^bc	183.14^c	148.91^d	13.55	0.04
VH/CD ratio	4.64^d	5.58^c	6.53^c	8.81^b	11.08^a	3.03	0.00
TE (µm)	53.44^b	73.32^ab	72.59^ab	80.18^a	78.29^a	5.92	0.02
TM (µm)	196.44^b	212.48^ab	215.54^ab	217.43^ab	238.86^a	12.45	0.04
Jejunum
Villus height (µm)	1197.63^de	1295.81^d	1418.72^c	1445.51^b	1481.62^a	18.99	0.05
Crypt depth (µm)	225.66^a	185.78^b	167.84^bc	166.71^bc	124.94^c	13.01	0.03
VH/CD ratio	5.31^c	6.97^c	8.45^b	8.67^b	11.86^a	4.07	0.01
TE (µm)	42.92^c	48.69^b	50.91^b	55.88^ab	61.66^a	5.81	0.05
TM (µm)	147.13	146.55	151.32	150.91	154.84	11.00	0.10
Ileum
Villus height (µm)	1016.95^e	1127.47^d	1227.87^c	1339.08^b	1374.86^a	17.98	0.04
Crypt depth (µm)	202.54^a	160.38^bc	161.92^bc	175.35^b	146.54^c	11.99	0.05
VH/CD ratio	5.79^c	7.03^b	7.54^b	6.52^b	9.38^a	4.05	0.00
TE (µm)	48.91	47.79	45.57	50.99	51.46	6.42	0.08
TM (µm)	187.71^c	207.44^b	211.93^b	230.53^a	245.86^a	11.11	0.03

Notes: ^a,b,cRow means with different superscripts differ significantly. SEM = Standard error of the mean. PROT0 = Basal Diet: BD (without PROT; protease). PROT10 = BD + 1 g PROT. PROT15 = BD + 1.5 g PROT. PROT20 = BD + 2 g PROT. PROT25 = BD + 2.5 g PROT. VH/CD ratio = Villi height: Crypt depth ratio. TE = Thickness of epithelium. TM = Thickness of muscularis. Two birds from each cell/replicate were used for the analysis.

Table 4. The effect of Bacillus protease inclusion on feed intake, body weight gain, and feed conversion ratio of broiler chicken.

Treatment	PROT0	PROT10	PROT15	PROT20	PROT25	P-value	SEM
Items
BWG (g)
1–21 d	950.40^b	951.20^b	953.80^b	966.20^b	1075.00^a	0.04	19.05
22–42 d	1579.40^c	1580.60^c	1623.80^b	1725.00^ab	1950.00^a	0.03	19.08
1–42 d	2566.80^c	2579.80^c	2609.60^bc	2712.20^b	3060.00^a	0.05	20.33
FI (g/bird)
1–21 d	1276.00^a	1221.00^a	1209.50^a	1265.00^a	1097.80^b	0.04	19.10
22–42 d	3014.00^a	2827.80^ab	2731.30^b	2768.90^ab	2396.50^c	0.02	21.64
1–42 d	4280.10^a	4039.40^ab	3930.80^b	4023.10^ab	3484.40^c	0.03	23.04
FCR
1–21 d	1.34^a	1.30^a	1.31^a	1.30^a	1.02^b	0.01	0.00
22–42 d	1.91^a	1.79^ab	1.68^b	1.61^b	1.23^c	0.02	0.01
1–42 d	1.67^a	1.57^a	1.51^a	1.48^ab	1.14^b	0.01	0.01

Notes: ^a,b,cRow means with common superscripts do not differ significantly. SEM = Standard error of the mean. BWG = Body weight gain. FI = Feed intake. FCR = Feed conversion ratio. PROT0 (only basal diet; BD), PROT10 (BD + 1 g protease), PROT15 (BD + 1.5 g protease), PROT20 (BD + 2 g protease) and PROT25 (BD + 2.5 g protease). Three hundred birds were used for the analysis.

Table 5. The effect of dietary protease supplementation on protein intake and protein efficiency ratio of broiler birds.

Treatments	PROT0	PROT10	PROT15	PROT20	PROT25	p-value	SEM
Items
DPI (g)	20.54^a	19.38^ab	19.86^ab	19.30^ab	17.71^b	0.04	0.84
DPER (g)	0.07^b	0.07^b	0.08^b	0.07^b	0.10^a	0.01	0.00
DMEI (kcal/bird)	312.61^a	295.12^a	287.33^ab	293.56^a	254.28^c	0.05	3.85
DEER(kcal/100 kcal)	0.49^b	0.50^b	0.50^b	0.50^b	0.68^a	0.02	0.07
Starter phase
PI (g)	301.31^a	288.21^a	285.49^ab	298.50^a	258.91^b	0.05	2.87
PER (g)	3.14^b	3.27^b	3.33^b	3.21^b	4.11^a	0.03	0.01
MEI (kcal/bird)	3784.07^a	3614.84^a	3582.90^ab	3743.67^a	3248.17^c	0.05	13.78
EER(gain/100 kcal)	24.93^bc	26.04^b	26.56^b	25.54^b	32.79^a	0.03	0.81
Finisher phase
PI (g)	606.83^a	569.31^ab	559.70^ab	557.29^ab	482.08^b	0.05	3.10
PER (g)	2.82^b	2.84^b	2.87^b	2.83^b	4.03^a	0.01	0.01
MEI (kcal/bird)	9345.44^a	8780.26^a	8585.01^ab	8585.70^ab	7431.56^b	0.05	12.89
EER(gain/100 kcal)	18.36^b	18.38^b	18.51^b	18.27^b	26.10^a	0.02	0.93
Overall performance
PI (g)	908.14^a	857.52^ab	855.19^ab	855.79^ab	740.99^b	0.04	4.19
PER (g)	2.93^b	2.98^b	3.02^b	2.96^b	4.06^a	0.02	0.01
MEI (kcal/bird)	13129.50^a	12395.10^ab	12267.90^ab	12329.40^ab	10679.70^b	0.05	39.67
EER(gain/100 kcal)	20.59^bc	20.97^b	21.19^b	20.84^b	28.56^a	0.03	0.90

Notes: ^a,b,cRow means with different superscripts differ significantly. SEM = Standard error of the mean. DPC = daily protein intake. DPER = daily protein efficiency ratio. DMEI = daily metabolizable energy intake. DEER = daily energy efficiency ratio. PC = protein intake. PER = protein efficiency ratio. MEI = metabolizable energy intake. EER = energy efficiency ratio. PROT0 (only basal diet; BD), PROT10 (BD + 1 g protease), PROT15 (BD + 1.5 g protease), PROT20 (BD + 2 g protease) and PROT25 (BD + 2.5 g protease). Three hundred birds were used for the analysis.

Table 6. Effects of dietary protease supplementation on apparent digestibility (%) of dry matter, crude protein, crude fat, and crude fibre in broiler finisher diets.

	Diets
	PROT0	PROT10	PROT15	PROT20	PROT25	SEM	p-value
Parameters
Crude fibre	11.99^b	12.29^b	16.56^ab	17.29^a	18.55^a	0.07	0.04
Crude protein	78.42^c	79.55^c	84.04^b	87.99^a	89.97^a	0.18	0.02
Crude Fat	76.85	77.79	78.04	78.56	77.26	0.16	0.08
Dry Matter	75.00^b	74.11^b	77.01^ab	81.90^a	82.83^a	0.19	0.03

Notes: ^a,b,cRow means with different superscripts differ significantly. SEM = Standard error of the mean. PROT0 (only basal diet; BD), PROT10 (BD + 1 g protease), PROT15 (BD + 1.5 g protease), PROT20 (BD + 2 g protease) and PROT25 (BD + 2.5 g protease). Two birds from each cell/replicate were used for the analysis.

Figure 3. The effect of dietary Bacillus protease on Lactobacillus, Bifidobacteria and E. coli counts (log₁₀ cfu/g) in the ileum section of the intestine. Two birds from each cell/replicate were used for the analysis.

Figure 4. The effect of dietary Bacillus protease on Lactobacillus, Bifidobacteria, and E. coli counts (log₁₀ cfu/g) in the caecum section of the intestine. Two birds from each cell/replicate were used for the analysis.

Table 7. The effect of dietary Bacillus protease supplementation on carcass characteristics of broiler birds.

Treatment	PROT0	PROT10	PROT15	PROT20	PROT25	SEM	p-value
Slaughter weight (g)	2556.00^c	2569.20^c	2599.80^c	2802.80^b	3050.00^a	20.01	0.04
Carcass weight (g)	2006.80^cd	2014.05^cd	2050.09^c	2220.90^b	2508.30^a	18.97	0.02
Neck weight (g)	85.02	80.00	80.02	80.77	83.33	2.32	0.11
Wing weight (g)	83.05	77.07	80.01	78.33	82.65	2.06	0.09
Drumstick weight (g)	81.00^bc	85.67^b	88.27^b	89.11^b	108.47^a	2.57	0.01
Thigh weight (g)	80.33^bc	81.93^bc	88.33^b	89.67^b	110.47^a	2.98	0.04
Breast weight (g)	379.66^c	390.48^b	400.20^b	515.00^a	535.60^a	5.76	0.02
Shank weight (g)	38.20	34.80	35.00	35.27	39.66	0.98	0.17
Carcass yield (%)	77.26^b	78.19^b	79.78^b	79.79^b	84.24^a	1.97	0.01
Neck yield (%)	3.83	3.98	3.97	3.94	3.88	0.07	0.07
Wing yield (%)	3.74	3.84	3.89	3.72	3.78	0.06	0.10
Drumstick yield (%)	3.83^b	3.93^b	3.89^b	3.96^b	4.32^a	0.08	0.02
Thigh yield (%)	3.97^c	4.03^c	4.04^c	4.18^b	4.40^a	0.09	0.02
Breast yield (%)	18.30^b	17.44^b	17.93^b	20.24^a	21.35^a	0.76	0.05
Shank yield (%)	1.72	1.73	1.74	1.72	1.70	0.02	0.15

Notes: ^a,b,cRow means with different superscripts differ significantly. SEM = Standard error of the mean. PROT0 = Basal Diet: BD (without PROT; protease). PROT10 = BD + 1 g PROT. PROT15 = BD + 1.5 g PROT. PROT20 = BD + 2 g PROT. PROT25 = BD + 2.5 g PROT. Five birds from each cell/replicate were used for the analysis.

Table 8. The effect of dietary protease supplementation on meat quality attributes of broiler birds.

Treatment	PROT0	PROT10	PROT15	PROT20	PROT25	SEM	p-value
Colour
a*(redness)	1.32	1.08	1.44	1.14	1.62	0.00	0.11
b*(yellowness)	15.92	15.64	14.98	16.81	14.15	0.13	0.31
L*(lightness)	49.87	46.96	46.97	49.41	49.27	0.67	0.20
Cooking loss (%)	17.53^a	16.96^a	14.83^b	13.98^b	13.26^bc	0.13	0.03
Shear force (N)	8.32	9.37	9.60	9.81	9.87	0.07	0.09
Drip loss (%)	8.12	7.89	6.74	5.63	5.10	0.02	0.07
WHC (%)	18.17^b	19.10^ab	19.81^ab	20.29^ab	21.92^a	0.25	0.02
pH0	6.04	6.05	6.16	6.24	6.30	0.02	0.23
pHu	5.64	5.72	5.58	5.73	5.67	0.02	0.15
Temp1 (°C)	34.75	34.54	34.55	35.19	34.67	0.46	0.22
Temp2 (°C)	11.24	11.56	11.64	11.79	11.69	0.11	0.10

Notes: ^a,b,c Row means with different superscripts differ significantly. SEM = Standard error of the mean. WHC = water holding capacity. PROT0 = Basal Diet: BD (without PROT; protease). PROT10 = BD + 1 g PROT. PROT15 = BD + 1.5 g PROT. PROT20 = BD + 2 g PROT. PROT25 = BD + 2.5 g PROT. Five birds from each cell/replicate were used for the analysis.