Effects of different levels of rutin on growth performance, immunity, intestinal barrier and antioxidant capacity of broilers

Figures & data

Table 1. Ingredient and nutrient levels of the basal diets (fed basis).

Item	Starter period (1–21d)	Grower period (22–42d)
Ingredient (%)
Corn	57.02	61.36
Soybean meal	31.30	28.30
Corn gluten meal	3.70	1.70
Soy oil	3.00	4.00
Dicalcium phosphate	2.00	1.60
Limestone	1.20	1.40
L-lysine	0.33	0.31
DL-methionine	0.15	0.13
Sodium chloride	0.30	0.30
Premix^a	1.00	1.00
Total	100.00	100.00
Nutrient levels^b
ME (MJ/kg)^c	12.57	12.91
Crude protein (%)	21.42	19.23
Lysine (%)	1.20	1.10
Methionine (%)	0.50	0.44
Calcium (%)	1.00	0.93
Available phosphorus (%)	0.46	0.30

^aPremix provided per kilogram of diet: VA 10,000 IU, VD₃ 3,000 IU, VE 30 IU, VK₃ 1.3 mg, VB₁₂ 0.013 mg, biotin 0.04 mg, selenium 0.3 mg, folic acid 1 mg, iodine 1.1 mg, thiamine 2.2 mg, pyridoxine 4 mg, riboflavin 8 mg, copper 8 mg, calcium pantothenate 10 mg, niacin 40 mg, zinc 65 mg, iron 80 mg, manganese 110 mg, choline chloride 600 mg.

^bNutrient levels were calculated values.

^cME: metabolisable energy.

Table 2. Sequences used for real-time PCR primers.

Gene name^a	Accession No	Primer sequence (5′ to 3′)	Product size (bp)
Nrf2	NM_205117.1	F: CGCTTTCTTCAGGGGTAGCA	170
		R: AGTTCGGTGCAGAAGAGGTG
NQO1	NM_001277621.1	F: GGCAATGGCAGCAGCAG	138
		R: AAGCACTCGGGGTTCTTGAG
SOD	NM_205064.1	F: CCGGCTTGTCTGATGGAGAT	124
		R: TGCATCTTTTGGTCCACCGT
GSH-Px	NM_001277853.1	F: GACCAACCCGCAGTACATCA	205
		R: GAGGTGCGGGCTTTCCTTTA
NF-κB	NM_001001472.2	F: AACAACCTCAGCCAGACTCC	231
		R: ATGGCCGTTGTAGTTGGTGG
TNF-α	NM_204267.1	F: AGCAGGGCTGACACGGAT	216
		R: ATGAAGGTGGTGCAGATGGG
MyD88	NM_001030962.1	F: ATCCGGACACTAGAGGGAGG	115
		R: GGCAGAGCTCAGTGTCCATT
IL-2	XM_015276098.2	F: TTCTATGGGGGAGTGGGACA	85
		R: CACTCACTTTTGGCGCTCAC
INF-γ	NM_205149.1	F: CACTGACAAGTCAAAGCCGC	87
		R: ACCTTCTTCACGCCATCAGG
MUC2	JX284122.1	F: AGCGAGATGTTGGCGATGAT	166
		R: AAGTTGCCACACAGACCACA
OCLN	NM_205128.1	F: CCGTAACCCCGAGTTGGAT	214
		R: ATTGAGGCGGTCGTTGATG
CLDN2	NM_204202.1	F: CCCGTCCCGTTGTTGTTTTG	126
		R: CCCCTTCAACCTTCCCGAAA
ZO-1	XM_413773.4	F: TGTAGCCACAGCAAGAGGTG	159
		R: CTGGAATGGCTCCTTGTGGT
BAX	FJ977571.1	F: AGCTGGCGAACACCATCAAA	166
		R: AGCACGATAATTCTGCCCCA
Bcl-2	NM_205339.2	F: CACAGGTGCCTACTGTCGTT	226
		R: CACACTGGGATTCTTCCGCT
Ki67	NM_205505	F: AGGTCCGTTCCCTCGTT	270
		R: CATTGTCGTCTGGGTCATC
HO-1	NM_205344.1	F: ACGAGTTCAAGCTGGTCACG	244
		R: GGATGCTTCTTGCCAACGAC
Caspase3	NM_204725.1	F: TGGTGGAGGTGGAGGAGC	205
		R: CCTGAGCGTGGTCCATCTTT

^aBAX: B-cell lymphoma 2 associated X; Bcl-2: B-cell lymphoma 2; CLDN2: claudin-2; GSH-Px: glutathione peroxidase; HO-1: haem oxygenase-1; IL-2: interleukin-2; IFN-γ: interferon-γ; MUC2: mucin 2; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor kappa-B; NQO1: NAD(P)H quinone oxidoreductase 1; Nrf2: nuclear factor erythroid-2-related factor 2; OCLN: occludin; SOD: superoxide dismutase; TNF-α: tumour necrosis factor-α; ZO-1:zonula occludens-1.

Table 3. Effects of dietary rutin on growth performance of broilers.

Item^A	Dietary treatments^B				SEM^C	p Value^D
	0 (control)	250	500	1,000		P	L	Q
Body weight, g
1 d	42.76	43.17	42.90	43.17	0.157	0.753	0.510	0.820
21 d	733.93^b	812.53^a	836.66^a	834.66^a	8.785	<0.001	<0.001	<0.001
42 d	2127.50^c	2236.56^b	2416.88^a	2388.02^a	33.922	0.003	0.001	0.228
Starter period (1–21d)
ADFI, g/d	49.14^b	49.14^b	52.10^a	53.20^a	0.494	0.001	<0.001	0.493
ADG, g/d	32.91^b	36.64^b	37.80^a	37.69^a	0.418	<0.001	<0.001	<0.001
FCR	1.49^a	1.34^b	1.38^b	1.41^b	0.016	0.002	0.087	0.001
Grower period (22–42d)
ADFI, g/d	132.12^b	132.14^b	137.76^ab	139.81^a	1.227	0.042	0.007	0.651
ADG, g/d	66.36^bc	67.81a^bc	75.25^a	73.97^ab	1.322	0.030	0.008	0.572
FCR	2.00	1.97	1.85	1.90	0.035	0.442	0.190	0.552
Whole trial period (1–42d)
ADFI, g/d	90.63^b	90.64^b	94.93^a	96.50^a	0.753	0.003	<0.001	0.535
ADG, g/d	49.64^c	52.22^bc	56.52^a	55.83^ab	0.807	0.003	0.001	0.228
FCR	1.83^a	1.74^ab	1.69^b	1.73^ab	0.018	0.033	0.024	0.052

^AADFI: average daily feed intake; ADG: average daily gain; FCR: feed conversion ratio.

^B0 (control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a–cMeans in the same row with no common superscript differ significantly (p < 0.05).

Table 4. Effects of dietary rutin on serum GH/IGF-1 concentration of broilers (day 42).

Items^A	Dietary treatments^B				SEM^C	p value^D
	0 (Control)	250	500	1,000		P	L	Q
GH (μg/L)	16.01^b	17.08^ab	18.63^a	18.80^a	0.373	0.014	0.002	0.482
IGF-1 (ng/mL)	18.24	18.00	18.63	19.46	0.245	0.165	0.269	0.182

^AGH: growth hormone; IGF-1: insulin-like growth factor-1.

^B0 (control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a,bMeans in the same row with no common superscript differ significantly (p < 0.05).

Table 5. Effects of dietary rutin on jejunal tissue morphology of broilers (day 42).

Items^A	Dietary treatments^B				SEM^C	p Value^D
	0 (Control)	250	500	1,000		P	L	Q
VH (µm)	1117.59^b	1089.12^b	1314.36^a	1073.14^b	24.955	<0.001	0.519	0.003
VW (µm)	171.77	181.26	190.27	174.67	2.975	0.119	0.482	0.035
CD (µm)	178.81	171.74	184.58	171.09	2.535	0.190	0.639	0.515
VH:CD (µm/µm)	6.26^b	6.36^b	7.16^a	6.31^b	0.132	0.040	0.379	0.056
VA (mm^2)	0.30^b	0.31^b	0.39^a	0.29^b	0.011	<0.001	0.338	0.001

^AVH: villous height; VW: villus width; CD: crypt depth; VH:CD: villus height to crypt depth ratio; VA: villus area.

^B0 (control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a,bMeans in the same row with no common superscript differ significantly (p < 0.05).

Figure 1. Effects of dietary rutin on the tight junctions (TJs) of the jejunal mucosa of 42-day-old broilers (representative images of jejunum using transmission electron microscopy). Note: red arrows, the TJs; A, control group, basal diet supplemented 0 mg rutin/kg; B, 500 mg rutin/kg group, basal diet supplemented 500 mg rutin/kg; Scale bar, 1.0 μm.

Figure 2. Effects of dietary rutin on the mRNA expressions of proliferation and apoptosis related genes in jejunum mucosa. Note: a, b a, ba,b a, ba,b Means within the same gene of the histogram with different superscript differ significantly (P < 0.05). BAX, B-cell lymphoma 2 associated X; Bcl-2, B-cell lymphoma 2. 0, basal diet (control group); 250, 500 and 1,000, basal diet further supplemented with 250, 500 and 1,000 mg rutin/kg, respectively. Q and L are the quadratic and linear responses, respectively, to the levels of dietary supplementation with rutin.

Table 6. Effects of dietary rutin on immune organ indexes and serum immunoglobulin and inflammatory factors of broilers (day 42).

Items^A	Dietary treatments^B				SEM^C	p Value^D
	0 (Control)	250	500	1,000		P	L	Q
Thymus(g/kg)	1.30	1.36	1.35	1.32	0.015	0.449	0.808	0.128
Spleen (g/kg)	2.13	1.98	2.15	2.30	0.077	0.565	0.338	0.363
Bursa (g/kg)	1.43	1.54	1.41	1.51	0.028	0.359	0.694	0.928
IgA (ug/mL)	342.39^b	384.16^ab	393.78^a	415.49^a	7.914	0.004	0.001	0.441
IgM (ug/mL)	8.00	8.32	8.24	8.70	0.156	0.477	0.164	0.818
IgG (ug/mL)	395.48	415.55	405.68	414.52	3.106	0.070	0.076	0.335
TNF-α (ng/L)	39.67^a	35.43^b	35.24^b	35.72^b	0.579	0.010	0.011	0.023

^AIgA: immunoglobulin A; IgM: immunoglobulin M; IgG: immunoglobulin G; TNF-α: tumour necrosis factor-α.

^B0 (Control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a,bMeans in the same row with no common superscript differ significantly (p < 0.05).

Figure 3. Effects of dietary rutin on the mRNA expressions of immune-related genes in jejunal mucosa of 42-day-old broilers. Note: a, bMeans within the same gene of the histogram with different superscript differ significantly (P < 0.05). NF-κB, nuclear factor kappa-B; MyD88, myeloid differentiation factor 88; IL-2, interferon-2; IFN-γ, interferon -γ; TNF-α, tumor necrosis factor-α. 0, basal diet (control group); 250, 500 and 1,000, basal diet further supplemented with 250, 500 and 1,000 mg rutin/kg, respectively. Q and L are the quadratic and linear responses, respectively, to the levels of dietary supplementation with rutin.

Table 7. Effects of dietary rutin on serum intestinal barrier indicators of broilers (day 42).

Items^A	Dietary treatments^B				SEM^C	p Value^D
	0(control)	250	500	1,000		P	L	Q
D-LA (ug/mL)	10.68^a	10.40^ab	7.63^c	9.43^b	0.269	0.010	<0.001	0.002
DAO (U/L)	33.27^a	27.52^ab	25.37^b	25.61^b	1.005	<0.001	0.003	0.088
LPS (EU/mL)	7.18^a	6.08^ab	4.87^b	5.62^b	0.230	0.001	0.001	0.014

^AD-LA: D-lactic acid; DAO: diamine oxidase; LPS: lipopolysaccharide.

^B0 (control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a,bMeans in the same row with no common superscript differ significantly (p < 0.05).

Figure 4. Effects of dietary rutin on the mRNA expressions of intestinal barrier-related genes in jejunal mucosa of 42-day-old broilers. Note: a, bmeans within the same gene of the histogram with different superscript differ significantly (P < 0.05). MUC2, mucin2; OCLN, occludin; CLDN2, claudin-2; ZO-1, zonula occludens-1; 0, basal diet (control group); 250, 500 and 1,000, basal diet further supplemented with 250, 500 and 1,000 mg rutin/kg, respectively. Q and L are the quadratic and linear responses, respectively, to the levels of dietary supplementation with rutin.

Table 8. Effects of dietary rutin on antioxidant capacity of jejunal mucosa of broilers (day 42).

Items^A	Dietary treatments^B				SEM^C	p Value^D
	0 (control)	250	500	1,000		P	L	Q
MDA (nmol/mg prot)	1.30^a	1.26^a	0.94^b	1.18^ab	0.042	0.011	0.036	0.053
T-SOD (U/mg prot)	214.62^b	227.21^ab	249.73^a	239.73^ab	4.525	0.005	0.012	0.175
CAT (U/mg prot)	20.70	20.77	20.68	19.82	0.352	0.581	0.407	0.520
GSH-Px (U/mg prot)	33.70	33.08	32.58	34.73	0.770	0.460	0.720	0.394
T-AOC (U/mg prot)	1.83^b	1.82^b	2.35^a	2.32^a	0.074	0.012	0.001	0.925

^AMDA: malondialdehyde; T-SOD: total superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; T-AOC: total antioxidant capacity.

^B0 (Control), 250, 500 and 1,000, basal diet further supplemented with 0 (control group), 250, 500 and 1,000 mg rutin/kg, respectively.

^CSEM: pooled standard error of means.

^DL: linear; Q: quadratic. Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of dietary rutin (n = 8 birds).

^a,bMeans in the same row with no common superscript differ significantly (p < 0.05).

Figure 5. Effects of dietary rutin on the mRNA expressions of antioxidant-related genes in jejunal mucosa of 42-day-old broilers. Note: a, b means within the same gene of the histogram with different superscript differ significantly (P < 0.05). Nrf2, nuclear factor erythroid-2-related factor 2; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase. 0, basal diet (control group); 250, 500 and 1,000, basal diet further supplemented with 250, 500 and 1,000 mg rutin/kg, respectively. Q and L are the quadratic and linear responses, respectively, to the levels of dietary supplementation with rutin.