Effects of rutin supplementation on intestinal morphology, antioxidant capacity, immunity, and gut microbiota of laying hens fed a diet containing stored soybean meal

Figures & data

Table 1. Composition and nutrient level of basal diet (as fed basis unless otherwise stated).

Item	Content
Ingredient (%)
Corn	64
Soybean meal	24
Limestone	9
Premix^a	3
Calculated nutrient levels
Apparent metabolizable energy (MJ/kg)	11.04
Crude protein (%)	16.16
Lysine (%)	0.80
Methionine + cystine (%)	0.62
Methionine (%)	0.34
Calcium (%)	3.6
Total phosphorus (%)	0.56
Available phosphorus (%)	0.37

^aSupplied per kg of diet: Vitamin A, 10,000 IU; vitamin D3, 3000 IU; vitamin E, 30 IU; vitamin K3, 1 mg; vitamin B1, 1 mg; vitamin B2, 6 mg; vitamin B6, 3 mg; pantothenic acid, 10 mg; vitamin B12, 0.02 mg; nicotinamide, 40 mg; folic acid, 0.3 mg; biotin, 0.1 mg; choline chloride, 500 mg; Fe (ferrous sulfate), 80 mg; Cu (copper sulfate), 8 mg; Mn (manganese sulfate), 100 mg; Zn (zinc oxide), 65 mg; I (calcium iodate), 0.8 mg; Se (sodium selenite), 0.3 mg; calcium, 3.6 g; phosphorus, 2.4 g; methionine, 1 g; sodium chloride, 3 g.

Table 2. Sequences for real-time PCR primers.

Genes^a	Gene Bank ID	Primer Sequence (5'-3')		Product length (bp)
GPX1	NM_001277853.1	F	CGCTACAGCCGCCACTT	287
		R	TTCGGAGAATCCCACAACG
SOD1	NM_205064.1	F	TTGTCTGATGGAGATCATGGCTTC	98
		R	TGCTTGCCTTCAGGATTAAAGTGAG
SOD2	NM_204211.1	F	CAGATAGCAGCCTGTGCAAATCA	86
		R	GCATGTTCCCATACATCGATTCC
Nrf2	NM 205117.1	F	GATGTCACCCTGCCCTTAG	216
		R	CTGCCACCATGTTATTCC
Keap1	XM_010728179.1	F	TTCCACGCCTGCATCAACT	203
		R	GGTTTGTGCAGGGTGAGG
NQO1	NM_001277619.1	F	GTTCAATGCCGTGCTCTCAC	146
		R	CCGCTTCAATCTTCTTCTGC
NF-κB	NM_205134.1	F	AGGACTTAAAATGGCAGGAGAG	141
		R	GCTGTTCGTAGTGGTAAGTCTG
IL-1β	NM_204524.1	F	TGCCTGCAGAAGAAGCCTCG	204
		R	GACGGGCTCAAAAACCTCCT
TNF-α	NM204267	F	TGTGTATGTGCAGCAACCCGTAGT	229
		R	GGCATTGCAATTTGGACAGAAGT
IFN-γ	NM_205149.1	F	ATGTAGCTGACGGTGGACCT	196
		R	TTCACGCCATCAGGAAGGTT
IL-4	NM_001007079.1	F	GTGCCCACGCTGTGCTTAC	82
		R	AGGAAACCTCTCCCTGGATGTC
β-actin	NM_205518	F	TGCTGTGTTCCCATCTATCG	150
		R	TTGGTGACAATACCGTGTTCA

^aGPX1: glutathione peroxidase 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; Nrf2: nuclear factor E2-related factor 2; Keap1: Kelch-like ECH-associated protein 1; NQO1: NADPH:quinone oxidoreductase 1; NF-κB: nuclear factor-κB, IL-1β: interleukin-1β; TNF-α: tumour necrosis factor-α; IFN-γ: interferon-γ; IL-4: interleukin-4.

Figure 1. Effects of rutin on the intestinal morphology of laying hens fed a diet containing stored soybean meal. (A) representative images of the jejunum of laying hens, stained with haematoxylin-eosin staining (HE); (B) representative images of the ileum of laying hens, stained with haematoxylin-eosin staining (HE); CON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

Table 3. Effects of rutin on the intestinal morphology of laying hens fed a diet containing stored soybean meal.

Items^a	Groups^b				SEM^c	P-value^d
	CON	RT	R	RT + R		SBM	R	SBM × R
Jejunum
VH (μm)	990.94	720.31	1021.12	1014.20	47.06	0.122	0.073	0.141
CD (μm)	169.67	138.32	178.39	193.52	7.92	0.589	0.040	0.128
VH/CD	6.22	5.38	6.11	5.53	0.25	0.166	0.975	0.801
Ileum
VH (μm)	816.99	648.34	806.03	830.54	32.72	0.262	0.185	0.136
CD (μm)	179.47	140.59	166.79	150.94	8.25	0.107	0.944	0.489
VH/CD	4.88	4.85	5.24	5.96	0.20	0.394	0.072	0.347

^aVH: villus height; CD: crypt depth; VH/CD: villus height/crypt depth.

^bCON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

^cSEM: standard error of mean.

^dSBM: soybean meal.

Table 4. Effects of rutin on antioxidant indices in the intestinal mucosa of laying hens fed a diet containing stored soybean meal.

Items^c	Groups^d				SEM^e	P-value^f
	CON	RT	R	RT + R		SBM	R	SBM × R
Jejunum
MDA (nmol/mg prot.)	1.01	1.00	1.20	1.17	0.06	0.841	0.131	0.947
T-SOD (U/mg prot.)	175.61^b	194.11^ab	222.80^a	188.67^ab	5.86	0.461	0.056	0.018
T-AOC (U/mg prot.)	0.94^b	0.84^b	1.31^b	2.68^a	0.16	0.002	<0.001	<0.001
GSH-Px (U/mg prot.)	17.64	22.54	14.97	15.88	1.88	0.450	0.229	0.603
Ileum
MDA (nmol/mg prot.)	0.79	1.05	0.79	0.34	0.15	0.748	0.237	0.240
T-SOD (U/mg prot.)	215.39	231.74	217.60	215.90	6.03	0.560	0.588	0.474
T-AOC (U/mg prot.)	0.70	0.72	0.76	0.70	0.06	0.889	0.880	0.760
GSH-Px (U/mg prot.)	60.64^b	36.28^b	104.5^a	67.1^ab	6.67	0.007	0.001	0.543

^a,bMeans within a row with different superscripts are different at p < 0.05.

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

^dCON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

^eSEM: standard error of mean.

^fSBM: soybean meal.

Table 5. Effects of rutin on immunoglobulin and inflammatory cytokine in the intestinal mucosa of laying hens fed a diet containing stored soybean meal.

Items^c	Groups^d				SEM^e	P-value^f
	CON	RT	R	RT + R		SBM	R	SBM × R
Jejunum
sIgA (μg/mg prot.)	2.22	2.42	2.33	2.10	0.06	0.916	0.371	0.087
IgG (μg/mg prot.)	95.60	102.51	100.48	94.60	1.85	0.889	0.685	0.094
IgM (μg/mg prot.)	11.54	12.88	11.87	10.74	0.35	0.880	0.187	0.075
IL-1β (pg/mg prot.)	5.04	5.02	5.72	4.95	0.15	0.180	0.303	0.198
TNF-α (pg/mg prot.)	17.34^ab	19.07^a	17.51^ab	15.85^b	0.44	0.966	0.071	0.046
IFN-γ (pg/mg prot.)	22.11^ab	25.47^a	20.44^b	18.07^b	0.71	0.655	<0.001	0.014
IL-4 (pg/mg prot.)	8.48	8.36	8.86	8.10	0.18	0.233	0.874	0.391
Ileum
sIgA (μg/mg prot.)	1.53	1.37	1.42	1.35	0.04	0.123	0.374	0.529
IgG (μg/mg prot.)	81.52	80.42	74.77	73.37	1.68	0.707	0.045	0.965
IgM (μg/mg prot.)	7.12	7.54	7.02	7.46	0.34	0.549	0.901	0.988
IL-1β (pg/mg prot.)	5.12^ab	5.37^a	4.17^b	4.17^b	0.17	0.667	0.001	0.662
TNF-α (pg/mg prot.)	18.17^a	19.46^a	14.80^b	14.90^b	0.52	0.380	<0.001	0.451
IFN-γ (pg/mg prot.)	17.60^a	18.97^a	11.67^b	12.54^b	0.70	0.213	<0.001	0.780
IL-4 (pg/mg prot.)	9.01^a	9.33^a	6.24^b	5.89^b	0.35	0.982	<0.001	0.443

^a,bMeans within a row with different superscripts are different at p < 0.05.

^csIgA: secretory immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; IL-1β: interleukin-1β; TNF-α: tumour necrosis factor-α; IFN-γ: interferon-γ; IL-4: interleukin-4.

^dCON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

^eSEM: standard error of mean.

^fSBM: soybean meal; R: rutin.

Table 6. Effects of rutin on mRNA levels of antioxidant and immune function related genes in the intestinal mucosa of laying hens fed a diet containing stored soybean meal.

Items^d	Groups^e				SEM^f	P-value^g
	CON	RT	R	RT + R		SBM	R	SBM × R
Jejunum
GPX1	1.00	0.86	1.28	1.03	0.11	0.369	0.296	0.819
SOD1	1.00^ab	1.05^ab	1.23^a	0.85^b	0.05	0.050	0.879	0.011
SOD2	1.00^b	0.96^b	2.12^a	1.01^b	0.12	0.003	0.002	0.005
Nrf2	1.00	1.10	1.18	1.22	0.03	0.279	0.025	0.692
Keap1	1.00	1.23	0.89	1.06	0.08	0.220	0.371	0.846
NQO1	1.00^b	1.03^ab	1.23^a	1.19^ab	0.03	0.966	0.003	0.567
NF-κB	1.00^a	0.88^ab	0.41^c	0.52^bc	0.06	0.938	<0.001	0.253
IL-1β	1.00^a	1.01^a	0.60^b	0.95^a	0.07	0.050	0.002	0.057
TNF-α	1.00	0.85	1.12	0.73	0.08	0.103	0.978	0.454
IFN-γ	1.00^a	1.08^a	0.47^b	0.38^b	0.07	0.971	<0.001	0.441
IL-4	1.00^ab	1.18^a	0.59^c	0.74^bc	0.06	0.054	<0.001	0.628
Ileum
GPX1	1.00^ab	0.78^b	1.86^a	1.30^ab	0.13	0.095	0.005	0.452
SOD1	1.00	0.98	0.93	0.92	0.05	0.897	0.534	0.951
SOD2	1.00	1.17	1.41	0.86	0.15	0.537	0.871	0.247
Nrf2	1.00	1.12	1.20	1.29	0.04	0.197	0.033	0.872
Keap1	1.00	0.73	1.12	0.92	0.09	0.223	0.423	0.855
NQO1	1.00^b	0.88^b	1.44^a	1.22^ab	0.05	0.013	<0.001	0.419
NF-κB	1.00^a	0.82^ab	0.46^b	0.55^b	0.06	0.665	0.001	0.232
IL-1β	1.00	0.97	0.64	0.85	0.06	0.418	0.038	0.288
TNF-α	1.00^ab	1.15^a	0.96^ab	0.84^b	0.04	0.824	0.023	0.067
IFN-γ	1.00	1.10	0.85	0.89	0.05	0.441	0.049	0.694
IL-4	1.00^a	1.11^a	0.81^ab	0.62^b	0.05	0.630	<0.001	0.067

^a,b,cMeans within a row with different superscripts are different at p < 0.05.

^dGPX1: glutathione peroxidase 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; Nrf2: nuclear factor E2-related factor 2; Keap1: Kelch-like ECH-associated protein 1; NQO1: NADPH: quinone oxidoreductase 1; NF-κB: nuclear factor-κB, IL-1β: interleukin-1β; TNF-α: tumour necrosis factor-α; IFN-γ: interferon-γ; IL-4: interleukin-4.

^eeCON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

^fSEM: standard error of mean.

^gSBM: soybean meal.

Figure 2. Alpha diversity. (A) Chao1; (B) ACE; (C) Shannon; (D) Simpson; values on each bar with no common letter differ significantly (p < 0.05); CON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

Figure 3. Principal coordinates analysis (PCoA); CON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

Figure 4. Composition and distribution of caecal microbiota at phylum and family levels of laying hens. (A) overall microbiota compositions at the phylum level; (B) the relative abundances of microbiota at the phylum level; (C) overall microbiota compositions at the family level; (D) the relative abundances of microbiota at the family level; values on each bar with no common letter differ significantly (p < 0.05); CON: a basal diet with soybean meal stored in cold storage warehouse; RT: a basal diet with soybean meal stored in room temperature warehouse; SBM: soybean meal; R: rutin.

Data availability statement

The data that support the findings of this study are available from the corresponding author, Yanmin Zhou, upon reasonable request.