Potential use of cowpea protein hydrolysate as a dietary supplement in broiler chickens: effects on growth, intestinal morphology, muscle lipid profile, and immune status

Figures & data

Table 1. Amino acid composition (g amino acid/100 g protein) of cowpea protein isolate.

Amino acids	Concentration (g / 100 g)
Aspartic	10.71
Glutamic	17.85
Lysine	2.754
Arginine	8.466
Histidine	3.366
Threonine	3.978
Serine	3.876
Proline	1.3566
Glycine	2.652
Alanine	4.182
Valine	3.672
Methionine	0.816
Cystine	1.224
Isoleucine	5.712
Leucine	8.67
Tyrosine	4.284
Phenylalanine	5.814
Tryptophane	1.122

Table 2. The proximate chemical composition of the experimental diets as fed basis (%).

		Starter Period (4–10 d)					Grower Period (11–23 d)					Finisher Period (24–35 d)
Ingredients	Unit	CPH0	CPH2	CPH4	CPH6	CPH8	CPH0	CPH2	CPH4	CPH6	CPH8	CPH0	CPH2	CPH4	CPH6	CPH8
yellow corn	%	55.86	55.87	55.88	55.9	55.92	59.29	59.29	59.31	59.33	59.34	62.27	62.27	62.29	62.3	62.32
Soybean meal, 48% CP	%	33.67	33.67	33.67	33.67	33.67	28.08	28.08	28.08	28.08	28.08	23.62	23.62	23.62	23.62	23.62
Corn gluten, 60%CP	%	3.725	3.525	3.325	3.125	2.925	5.3	5.1	4.9	4.7	4.5	6	5.8	5.6	5.4	5.2
CPH	%	–	0.2	0.4	0.6	0.8	0	0.2	0.4	0.6	0.8	0	0.2	0.4	0.6	0.8
Soybean oil	%	2.2	2.2	2.2	2.2	2.2	3	3	3	3	3	4	4	4	4	4
Calcium carbonate	%	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.1	1.1	1.1	1.1	1.1
Calcium dibasic phosphate	%	1.5	1.5	1.5	1.5	1.5	1.4	1.4	1.4	1.4	1.4	1.3	1.3	1.3	1.3	1.3
Common salt	%	0.15	0. 15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15
Premix^1	%	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
DL- Methionine, 98%	%	0.4	0.4	0.4	0.4	0.4	0.3	0.3	0.3	0.3	0.3	0.33	0.33	0.33	0.33	0.33
Lysine, Hcl, 78% CP	%	0.47	0.46	0.45	0.43	0.41	0.45	0.45	0.43	0.41	0.4	0.4	0.4	0.38	0.37	0.35
Choline	%	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07	0.07
Threonine	%	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Phytase	%	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005
NACO3	%	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Antimycotoxin	%	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Chemical analysis
Moisture	%	11.32	12.1	11.4	11.6	11.1	12.04	11.8	11.34	12.2	11.5	11.20	11.46	12.03	12.10	11.7
ME	(kcal/kg)	3000	2999	2998	2997	2996	3100	3099	3099	3098	3097	3202	3201	3200	3200	3199
CP	%	23.03	23.04	23.05	23.05	23.05	21.53	21.55	21.55	21.55	21.56	20.04	20.06	20.06	20.06	20.06
Lysine	%	1.47	1.48	1.48	1.48	1.47	1.31	1.33	1.32	1.32	1.32	1.16	1.17	1.16	1.17	1.16
Methionine	%	0.72	0.72	0.72	0.72	0.72	0.61	0.61	0.61	0.61	0.61	0.62	0.63	0.63	0.63	0.63
Threonine	%	0.83	0.83	0.83	0.84	0.48	0.77	0.77	0.77	0.78	0.78	0.71	0.71	0.72	0.72	0.73
Calcium	%	0.94	0.94	0.94	0.94	0.94	0.90	0.90	0.90	0.90	0.90	0.83	0.83	0.83	0.83	0.83
Available Phosphorus	%	0.48	0.48	0.48	0.48	0.48	0.45	0.45	0.45	0.45	0.45	0.42	0.42	0.42	0.42	0.41

^* Premix per kg of diet: vitamin A, 1500 IU; vitamin D3, 200 IU; vitamin E, 10 mg; vitamin K3, 0.5 mg; thiamine, 1.8 mg; riboflavin, 3.6 mg; pantothenic acid, 10 mg; folic acid, 0.55 mg; pyridoxine3.5 mg; niacin, 35 mg; cobalamin, 0.01 mg; biotin, 0.15 mg; Fe, 80 mg; Cu, 8 mg; Mn, 60 mg; Zn, 40 mg; I, 0.35 mg; Se, 0.15 mg. Cp: Crude protein, ME: metabolisable energy. CPH: Cowpea protein hydrolysate. CPH0, CPH2, CPH4, CPH6, and CPH8: basal diets supplemented with 0, 0.2 or 0.4 or 0.6 or 0.8% Cowpea protein hydrolysate, respectively. Composition of CPH: CP: 70%, ash 2.5%, fat 0.5%, Crude fibre 1.5%, Nitrogen free extract 15%, lysine 6.54%, methionine 1.31%. NACO3: Sodium carbonate.

Figure 1. Degree of hydrolysis (%) of cowpea protein isolate treated with pepsin (Enzyme/ substrate ratio 1:300) for 5 h.

Table 3. Effect of dietary Cowpea protein hydrolysate (CPH) on the growth performance of broiler chickens.

	CPH g‎ kg^-1 diet						P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
Starter (4-10 d)
Int. Wt.	86.80	87.16	86.54	86.34	86.75	0.20	0.81	0.771	0.754
BW, g	315	324	323	311	322	3.030	0.670	0.934	0.802
BWG, g	228	237	236	225	235	3.075	0.675	0.901	0.778
FI, g	245	255	242	236	248	2.649	0.189	0.226	0.330
FCR	1.07	1.07	1.02	1.04	1.05	0.012	0.596	0.280	0.327
Grower (11–23 d)
BW, g	1161	1113	1066	1138	1135	23.88	0.825	0.825	0.700
BWG, g	846	789	743	826	813	24.17	0.769	0.883	0.349
FI, g	1102	950	1016	1084	1066	22.63	0.203	0.675	0.142
FCR	1.30	1.21	1.40	1.32	1.31	0.029	0.390	0.533	0.653
Finisher (24-35 d)
BW, g	2251	2270	2323	2244	2214	46.20	0.974	0.793	0.616
BWG, g	1090	1158	1257	1106	1079	40.24	0.662	0.808	0.248
FI, g	1815	1701	1812	1760	1791	36.95	0.898	0.975	0.772
FCR	1.71	1.48	1.45	1.60	1.66	0.044	0.272	0.911	0.042
Overall performance (4-35 d)
BWG, g	2164	2183	2237	2157	2127	46.23	0.974	0.795	0.615
FI, g	3167	2905	3070	3079	3104	49.87	0.616	0.899	0.359
FCR	1.46	1.33	1.37	1.42	1.45	0.022	0.258	0.628	0.084

BW: body weight; BWG: body weight gain; FI: feed intake; FCR: feed conversion ratio.

Table 4. Effect of dietary Cowpea protein hydrolysate (CPH) on the carcase traits (%) relative to live weight.

	CPH g kg^-1 diet					P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
Carcase dressing	63.52	66.48	63.57	65.50	62.21	0.540	0.054	0.251	0.053
Intestine	4.86	4.86	5.95	4.39	5.59	0.283	0.466	0.636	0.914
Liver	1.76	1.55	1.90	1.71	1.92	0.058	0.247	0.242	0.513
Spleen	0.058	0.070	0.079	0.081	0.104	0.008	0.511	0.103	0.818
Bursa of Fabricius	0.124	0.075	0.131	0.139	0.149	0.011	0.278	0.158	0.441
Gizzard	2.42	2.25	2.44	2.27	2.69	0.093	0.631	0.430	0.343

Figure 2. Photomicrograph of the small intestine (duodenum (I), jejunum (II), and ileum (III)) stained with haematoxylin and eosin. A: Cowpea protein hydrolysate (CPH)0, B: CPH2, C: CPH4, D: CPH6, and E: CPH8.

Table 5. Effect of dietary Cowpea protein hydrolysate (CPH) on the morphometric measures (μm) of the small intestine of broiler chickens.

	CPH g kg^-1 diet					P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
Duodenum
VH	708^b	728^b	986^a	1134.1^a	984^a	45.64	0.000	<0.001	0.004
VW	150.6^a	113.0^ab	139.0^ab	138.0^ab	85.77^b	7.873	0.029	0.027	0.257
CD	137^b	146^b	174^b	253^a	162^b	11.59	0.000	<0.001	0.002
VH:CD	5.20^bc	5.00^bc	5.67^ab	4.47^c	6.10^a	0.160	0.000	0.035	0.015
GCC	124^b	149^ab	159^a	166^a	152^ab	4.426	0.006	0.004	0.005
Jejunum
VH	971^c	1052^c	1311^ab	1471^a	1137^bc	51.31	0.000	<0.001	<0.001
VW	103	119	115	114	111	2.915	0.580	0.650	0.200
CD	190^c	230^bc	275^b	342^a	212^c	14.90	0.000	0.001	<0.001
VH:CD	5.133^ab	4.57^ab	4.80^ab	4.30^b	5.40^a	0.134	0.042	0.719	0.012
GCC	195^c	198^c	263^b	318^a	231^bc	12.76	0.000	<0.001	<0.001
Ileum
VH	461^b	461^b	490^b	604^a	447^b	16.83	0.001	0.076	0.008
VW	227	227	95.23	112	132	28.05	0.425	0.149	0.437
CD	113^ab	113^ab	121^ab	134^a	108^b	2.978	0.019	0.433	0.021
VH:CD	4.10	4.10	4.067	4.533	4.133-	0.064	0.072	0.190	0.493
GCC	91.80^b	91.80^b	101^ab	109^a	94.27^b	2.054	0.009	0.041	0.021

^a,b,c Means with different superscripts within the same row differ significantly (p < 0.05). VH: villus height, VW: villus width, CD: crypt depth, GCC: goblet cell count

Table 6. Effect of dietary Cowpea protein hydrolysate (CPH) on the fatty acid composition of the breast muscle in broiler chickens.

	CPH g kg^-1 diet					P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
18:2n-6	0.797^b	0.863^ab	0.857^ab	0.900^a	0.873^ab	0.011	0.046	0.013	0.099
18:3n-3	0.026^b	0.053^ab	0.056^ab	0.066^a	0.080^a	0.005	0.006	<0.001	0.490
20:4n-6	1.163^b	1.263^ab	1.293^a	1.263^ab	1.306^a	0.016	0.011	0.003	0.075
20:5n-3	0.0167^b	0.0367^ab	0.040^ab	0.046^a	0.056^a	0.004	0.006	<0.001	0.375
22:6n-3	0.013^b	0.030^ab	0.33^a	0.036^a	0.040^a	0.004	0.004	<0.001	0.080
ω-3 (%)	0.073^b	0.157^ab	0.170^ab	0.196^a	0.230^a	0.016	0.004	<0.001	0.295
ω-6 (%)	1.96^b	2.13^a	2.15^a	2.16^a	2.18^a	0.023	0.000	<0.001	0.005
ω-3: ω-6	0.038^b	0.074^ab	0.079^ab	0.091^a	0.105^a	0.007	0.009	<0.001	0.347

^a,b Means with different superscripts within the same row differ significantly (p < 0.05).

Table 7. Effect of dietary Cowpea protein hydrolysate (CPH) on blood biochemical parameters of broiler chickens.

	CPH g kg^-1 diet					P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
T3 (ng/mL)	4.37	4.90	5.17	5.11	5.35	0.128	0.110	0.017	0.327
T4 (ng/mL)	18.69^b	23.20^a	24.76^a	25.16^a	24.65^a	0.675	0.000	<0.001	<0.001
Glucose (mg/dL)	336	341	339	343	341	1.064	0.289	0.089	0.402
GH (ng/mL)	2.80^c	3.433^c	4.967^b	5.467^ab	6.033^a	0.338	0.000	<0.001	0.189
Leptin (ng/mL)	2.19	2.28	2.043	2.15	2.15	0.058	0.835	0.667	0.761
TP (g/dL)	3.39^c	4.46^b	5.19^a	5.29^a	5.19^a	0.197	0.000	<0.001	<0.001

^a,b Means with different superscripts within the same row differ significantly (p < 0.05). T3: Triiodothyronine, T4: thyroxine, GH: growth hormone, TP: total protein, SEM: Standard error of means.

Table 8. Effect of dietary Cowpea protein hydrolysate (CPH) on immune indices of broiler chickens.

	CPH g‎ kg^-1 diet					P-value
	0	2	4	6	8	SEM	ANOVA	Linear	Quadratic
INF-γ	6.27^b	11.83^a	13.83^a	12.00^a	13.80^a	0.821	0.001	<0.001	0.009
Albumin (g/dL)	1.21^c	1.47^c	1.97^b	2.29^a	2.01^ab	0.107	0.000	<0.001	<0.001
Globulin (g/dL)	2.17^b	2.99^a	3.22^a	2.99^a	3.18^a	0.106	0.000	<0.001	<0.001
Lysozyme (μg/mL)	133^c	177^b	188^ab	190^ab	193^a	6.026	0.000	<0.001	<0.001
IL10 (pg/mL)	1.167^c	2.97^b	3.63^ab	4.20^ab	4.43^a	0.335	0.000	<0.001	0.021
C3 (mg/dL)	1.08^b	1.21^ab	1.19^ab	1.21^ab	1.29^a	0.021	0.007	0.001	0.524

^a,b Means with different superscripts within the same row differ significantly (p < 0.05). INF-γ: interferon-gamma, IL10: interleukin 10, C3: complement 3, SEM: Standard error of means.

Figure 3. Photomicrograph of splenic tissues immunostained with IgG antibody.A: Cowpea protein hydrolysate (CPH)0, B: CPH2, C: CPH4, D: CPH6, and E: CPH8. Bar = 50 µm. (F) Percent of the immunolabelling index of IgG assessed by determination of positive immunostaining area.

Data availability statement

The datasets generated and/or analysed during the current study are not publicly available due to institution roles and permission but are available from the corresponding author upon reasonable request.

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