Dietary effects of enzymolytic soybean meal inclusion on antioxidant capacity, intestinal morphology and caecal microbiota of Rex rabbits

Figures & data

Table 1. Nutrient composition and biochemical characteristics of SBM and ESBM.

Items	SBM	ESBM
Nutrient composition, %
Dry matter	89.47	90.90
Ash	6.20	8.04
Gross energy	19.51	19.33
Crude fibre	5.15	4.94
Crude protein	46.33	45.49
Ether extract	1.10	0.51
Calcium	0.61	0.46
Total phosphorus	0.57	0.62
Biochemical characteristics
Small peptide, %	0.68	14.94
Glycinin, mg/g	104.77	2.06
β-conglycinin, mg/g	47.12	3.35

SBM: soybean meal; ESBM: enzymolytic soybean meal.

Table 2. Composition and nutrient levels of the experimental diets (air-dry basis).

Items	Control	T1	T2	T3
Ingredients, %
Corn	8.00	8.00	8.00	8.00
Wheat bran	33.00	33.00	33.00	33.00
Soybean meal	11.00	10.50	10.00	9.50
Enzymolytic soybean meal	0.00	0.50	1.00	1.50
Wheat middlings	4.00	4.00	4.00	4.00
Peanut seedling	15.00	15.00	15.00	15.00
Peanut shell	12.50	12.50	12.50	12.50
DL-methionine	0.15	0.15	0.15	0.15
Stone powder	1.05	1.05	1.05	1.05
L-lysine sulphate	0.30	0.30	0.30	0.30
Salt	0.50	0.50	0.50	0.50
Calcium hydrogen phosphate	0.50	0.50	0.50	0.50
Rice bran	5.00	5.00	5.00	5.00
Wormwood leaf powder	8.00	8.00	8.00	8.00
Premix^a	1.00	1.00	1.00	1.00
Nutrient levels^b
Digestible energy, MJ/kg	10.92	10.40	10.72	10.58
Dry matter, %	88.83	88.58	89.00	88.86
Crude protein, %	15.49	15.53	15.86	16.52
Ash, %	11.96	12.30	12.18	12.62
Ether extract, %	3.16	2.87	3.21	3.31
Crude fibre, %	14.75	14.53	14.18	14.17
Calcium, %	1.50	1.60	1.68	1.71
Total phosphorus, %	0.70	0.70	0.70	0.73

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

^aPremix provides the following (per kg of the premix): Vitamin A 10,000 IU, Vitamin B₁ 2 mg, Vitamin B₂ 6 mg, Vitamin B₃ 50 mg, Vitamin B₄ 1,000 mg, Vitamin B₅ 50 mg, Vitamin B₆ 2 mg, Vitamin B₇ 0.2 mg, Vitamin B₁₂ 0.02 mg, Vitamin D 900 IU, Vitamin E 50 mg, Vitamin K 2 mg, Cu (as copper sulphate) 20 mg, Fe (as ferric sulphate) 70 mg, Mn (as manganese sulphate) 10 mg, Zn (as zinc sulphate) 70 mg, Se (as sodium sulphate) 0.25 mg, Co (as cobalt sulphate) 0.15 mg, I (as iodine sulphate) 0.2 mg.

^bDigestible energy was a calculated value, while the others were measured values.

Table 3. Effect of enzymolytic soybean meal on the antioxidant capacity of Rex rabbits (n = 6).

Items	Control	T1	T2	T3	SEM	p-Value
Liver
SOD, U/mL	161.44^b	159.30^b	172.90^b	208.88^a	5.61	<0.001
MDA, nmol/mL	9.31^a	8.45^b	9.05^a	9.34^a	0.11	0.013
GSH-Px, U/mL	278.17^b	274.08^b	276.43^b	295.96^a	3.20	0.045
T-AOC, ng/mL	22.36^bc	21.47^c	22.87^ab	23.90^a	0.28	0.012
Jejunum
SOD, U/mL	168.12	173.06	178.57	190.38	6.45	0.683
MDA, nmol/mL	9.49^ab	8.85^b	9.41^ab	9.91^a	0.14	0.035
GSH-Px, U/mL	321.89	315.06	326.81	338.07	5.33	0.511
T-AOC, ng/mL	18.98	19.25	22.76	22.28	0.86	0.280

SOD: superoxide dismutase; MDA: malondialdehyde; GSH-Px: glutathione peroxidase; T-AOC: total antioxidant capacity; SEM: standard error of mean.

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

^a,b,cMeans in a row without the same superscript are different significantly (p < 0.05).

Table 4. Effect of enzymolytic soybean meal on the intestinal morphology of Rex rabbits (n = 6).

Items	Control	T1	T2	T3	SEM	p-Value
Jejunum
Villus height, μm	497.88	496.37	576.68	636.61	46.81	0.727
Crypt depth, μm	83.07	67.81	74.69	72.31	7.14	0.924
Villus height : crypt depth	6.82	7.60	7.85	9.10	0.59	0.653
Ileum
Villus height, μm	484.93	428.63	409.93	358.81	39.99	0.792
Crypt depth, μm	72.60	79.43	60.26	64.79	4.94	0.594
Villus height:crypt depth	6.92	6.51	6.60	5.28	0.74	0.902

SEM: standard error of mean.

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Table 5. Effect of enzymolytic soybean meal on the intestinal pH of Rex rabbits (n = 6).

Items	Control	T1	T2	T3	SEM	p-Value
Jejunum pH	7.10	6.99	6.92	7.09	0.03	0.184
Ileum pH	7.34	7.10	7.02	7.34	0.06	0.140
Caecum pH	6.52	6.67	6.58	6.58	0.02	0.131

SEM: standard error of mean.

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Figure 1. Rarefaction curves of samples. The Illumina NovaSeq PE250 sequencing platform was used to detect the V3-V4 region of 16S rDNA in the caecum content samples of Rex rabbits. The sequences were selected randomly, the number of species they represented was counted and the dilution curves were plotted by the number of selected sequences and corresponding species. T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Figure 2. Venn diagram of feature. According to the feature abundance table, the number of features in each group was calculated, and the number of common and unique features in each group was visualised by Venn diagram, if the feature existed in only one group, it was unique to that group. T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Table 6. Alpha diversity indices of caecal microbiota (n = 4).

Items	Control	T1	T2	T3	SEM	p-Value
Observed_otus	1583.50	1196.25	1136.50	1178.25	65.40	0.166
Chao1	1603.15	1210.18	1148.62	1192.78	66.99	0.183
Goods_coverage	0.9981	0.9989	0.9992	0.9989	0.00	0.277
Shannon	8.45	8.43	8.15	8.22	0.07	0.447
Simpson	0.9884	0.9919	0.9892	0.9893	0.00	0.349

Note. Chao1 and Observed_otus indices mainly reflect the richness of the species in the sample; Goods_coverage reflects the low abundance feature coverage of the sample; Simpson and Shannon reflect the richness and evenness of the species together.

SEM: standard error of mean.

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Figure 3. Principal co-ordinates analysis diagram of caecal microflora composition. Principal Coordinate Analysis (PCoA) is based on a sample distance matrix for ranking. The PCoA analysis allows the difference among the groups to be observed. The different colours in the results represent the different groupings and the closer the distance, the more similar the structure of the microbial composition and the less difference. T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Figure 4. Relative abundance of caecal microbiota at phylum level. T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Table 7. Distribution of caecal microbiota at phylum level (%) (n = 4).

Items	Control	T1	T2	T3	SEM	p-Value
Firmicutes	67.49	74.94	68.92	73.56	1.29	0.169
Bacteroidetes	20.12	14.56	19.71	16.28	1.15	0.340
Verrucomicrobia	4.47	2.05	4.03	1.94	0.77	0.628
Proteobacteria	1.99	2.83	2.67	2.84	0.22	0.209
Actinobacteria	1.97	2.37	2.59	3.31	0.33	0.729
Tenericutes	0.29	0.58	0.52	0.60	0.10	0.675
Patescibacteria	0.22	0.68	0.36	0.43	0.07	0.264
Epsilonbacteraeota	0.43	0.29	0.33	0.34	0.06	0.568
Synergistetes	0.51	0.37	0.29	0.14	0.09	0.750

SEM: standard error of mean.

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Figure 5. Relative abundance of caecal microbiota at genus level. T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Table 8. Distribution of caecal microbiota at genus level (%) (n = 4).

Items	Control	T1	T2	T3	SEM	p-Value
Clostridiales_vadinBB60_group_unclassified	10.55	15.51	10.62	12.29	1.01	0.349
Ruminococcaceae_NK4A214_group	10.05	9.67	10.80	11.42	0.49	0.335
Muribaculaceae_unclassified	9.78	3.67	7.27	6.58	1.43	0.587
Christensenellaceae_R-7_group	5.44	5.48	6.32	9.35	0.63	0.330
Firmicutes_unclassified	4.53	3.91	6.10	4.88	0.60	0.767
Ruminococcaceae_V9D2013_group	5.20	5.03	3.82	4.96	0.51	0.658
Ruminococcus_1	4.18	3.05	3.32	2.94	0.37	0.831
Ruminiclostridium_6	2.19	3.33	3.82	3.11	0.32	0.169
Akkermansia	4.47	2.05	4.03	1.94	0.77	0.628
Lachnospiraceae_unclassified	2.07	3.50	2.26	2.06	0.30	0.336
Alistipes	1.86	1.62	2.41	3.42	0.55	0.758
Ruminococcaceae_UCG-010	1.76	1.98	1.68	2.34	0.20	0.464
Ruminococcaceae_UCG-014	1.96	1.67	2.09	1.97	0.18	0.905
Ruminococcaceae_UCG-013	1.56^c	1.69^bc	2.41^a	2.18^ab	0.12	0.006
Coriobacteriaceae_UCG-002	1.47	1.71	1.92	2.46	0.25	0.639
dgA-11_gut_group	1.92	2.73	2.27	1.93	0.45	0.836
Ruminococcaceae_unclassified	2.71^a	1.08^b	1.17^b	1.11^b	0.21	0.034
Bacteroides	1.14	2.00	2.68	1.27	0.36	0.322
Barnesiellaceae_unclassified	0.89	2.04	1.81	1.45	0.28	0.481
Clostridium	1.16	1.30	1.01	0.99	0.10	0.399
Ruminococcaceae_UCG-005	1.07	1.37	0.60	0.82	0.12	0.057
Rikenellaceae_RC9_gut_group	2.85	0.00	0.00	0.00	0.71	0.371
Parabacteroides	0.63	0.67	1.53	0.59	0.20	0.449
Betaproteobacteria_unclassified	0.40	0.56	0.57	1.11	0.10	0.160
Bacteroidetes_unclassified	0.44	1.24	0.57	0.36	0.20	0.592

SEM: standard error of mean.

^a,b,cMeans in a row without the same superscript are different significantly (p < 0.05).

T1 means 0.5% enzymolytic soybean meal addition group, T2 means 1.0% enzymolytic soybean meal addition group, and T3 means 1.5% enzymolytic soybean meal addition group.

Data availability statement

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