Effects of enzyme + bacteria treatment on growth performance, rumen bacterial diversity, KEGG pathways, and the CAZy spectrum of Tan sheep

Figures & data

Table 1. Composition and nutrient levels of diets (DM basis)%

Raw material	Diet composition
	Control group	Trial group
Alfalfa	56	56
Buckwheat straw	14	14
Corn	15	15
Soybean meal	10	10
Wheat bran	1	1
Jute cake	2	2
NaCl	1	1
Premix^1)	1	1
Total	100	100
Nutrient levels^2)2
ME MJ/kg	9.39	9.83
CP	11.81	12.82
Ca	0.92	1.32
P	0.24	0.48

¹⁾Each kg of premix contains: va 200,000–400,000 iu, ve 500,000–2,000,000 iu, vd330000-80,000 iu, copper 400–600 mg, manganese 800–1600 mg, zinc 1200–2400 mg, iodine 6–80 mg, cobalt 5–40 mg, selenium 10–25 mg, calcium 20–300 g, phosphorus 20–150 g, sodium chloride ≥ 15%, water ≤ 10%.

²⁾ME was a calculated value,while the others are measured values.

Table 2. Effect of compound bacteria treatment of buckwheat straw and alfalfa on growth performance of Tan sheep

Items	Control group	Trial group
Initial body weight (kg)	33.34 ± 1.85	32.12 ± 1.65
Final body weight (kg)	38.48 ± 2.58	39.76 ± 1.36
Total gain (kg)	5.15 ± 1.23^A	7.63 ± 1.43^B
ADG (g/d)	90.12 ± 0.02^A	130.25 ± 0.03^B
DMI (g/d)	665.09 ± 0.02	634.32 ± 0.02
F/G	7.38 ± 3.50^A	4.87 ± 1.89^B

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow

Table 3. Number of OTUs and species in each group

Items	Control group	Trial group
Coverage	0.986 ± 0.00^B	0.988 ± 0.00^A
observed species	1722.00 ± 86.42	1567.33 ± 146.00
OTUs	1722.00 ± 86.42	1657.33 ± 146.00

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Table 4. Alpha diversity detected by 16srDNA

Items	Control group	Trial group
Chao	2787.80 ± 138.34^A	2457.44 ± 188.74^B
ACE	2898.43 ± 94.54^A	2543.55 ± 202.80^B
Shannon	5.90 ± 0.48	5.81 ± 0.45
Simpson	0.93 ± 0.02	0.93 ± 0.02

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Table 5. Abundance of dominant bacteria in the whole sample(%)

Phylum	Control group	Trial group
Proteobacteria	34.02 ± 2.07	31.72 ± 6.00
Firmicutes	35.87 ± 4.01^b	53.19 ± 0.07^a
Bacteroidetes	26.59 ± 5.21^A	12.99 ± 5.18^B

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Figure 1. Principal coordinate analysis of OTUs of rumen bacteria

Note: QM-1 is the control group and QM-2 is the experimental group.

Figure 2. Difference between species via Welch’s t test

Note: QM-3 is the control group and QM1-3 is the experimental group. The same as blow.

Figure 3. Differences among species via Welch’s t test

Note: QM-3 is the control group and QM1-3 is the experimental group. The same as blow.

Table 6. Abundance of dominant bacteria in the whole sample(%)

Genus	Control group	Trial group
Comamonas	15.44 ± 8.38	20.95 ± 7.72
Prevotella_1	19.65 ± 3.81^A	7.73 ± 3.77^B
Acinetobacter	14.37 ± 7.74	9.24 ± 3.05
Lysinibacillus	12.39 ± 1.71	7.24 ± 3.75
Kurthia	12.63 ± 3.64^B	26.04 ± 9.26^A

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Table 7. Ratio of high abundance families of reads to KEGG (level B)

Pathway	Control group	Trial group
Purine metabolism	16302.34 ± 218.68^B	17140.08 ± 53.88^A
Pyrimidine metabolism	14464.11 ± 188.29^B	15473.46 ± 88.33^A
Two-component system	13005.87 ± 159.65^B	13411.08 ± 67.78^A
ABC transporters	11432.27 ± 477.79^B	13429.50 ± 277.15^A
Ribosome	11868.33 ± 171.02^B	12570.48 ± 25.35^A
Quorum sensing	9730.45 ± 304.23^B	10476.79 ± 240.40^A
Starch and sucrose metabolism	10132.83 ± 63.73	10134.92 ± 81.93
Amino sugar and nucleotide sugar metabolism	9924.84 ± 87.16	9913.34 ± 42.73
Homologous recombination	8281.83 ± 73.53	8530.82 ± 34.55
Aminoacyl-tRNA biosynthesis	7869.20 ± 191.02^B	8739.45 ± 61.82^A
Glycolysis/gluconeogenesis	7650.30 ± 121.05	7737.06 ± 51.01
Alanine,aspartate and glutamate metabolism	7336.03 ± 76.75	7210.78 ± 86.93
Mismatch repair	6809.54 ± 102.13	6950.78 ± 3.33
Galactose metabolism	6839.14 ± 57.18^A	6457.29 ± 51.34^B
DNA replication	6239.30 ± 22.03	6233.71 ± 26.58
Cysteine and methionine metabolism	6484.43 ± 104.75^B	6702.27 ± 38.39^A
Peptidoglycan biosynthesis	6101.20 ± 107.23^B	6479.57 ± 36.86^A
Pyrimidine metabolism	5676.18 ± 134.75^B	6095.63 ± 53.77^A
Glycine,serine, and threonine metabolism	5619.56 ± 136.77^B	5765.88 ± 60.25^A
Fructose and mannose metabolism	5471.47 ± 26.79^A	4947.29 ± 8.74^B
Oxidative phosphorylation	5110.35 ± 67.54^B	5187.79 ± 19.71^A
Phenylalanine,tyrosine and tryptophan biosynthesis	4863.86 ± 31.32^A	4688.70 ± 72.82^B
Pentose phosphate pathway	4587.27 ± 119.61^B	5060.22 ± 3.96^A
Glyoxylate and dicarboxylate metabolism	4900.22 ± 72.16^B	5011.26 ± 34.89^A
Methane metabolism	4112.53 ± 92.70^B	4611.81 ± 48.42^A

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Figure 4. KEGG annotation(level A)

Figure 5. CAZy annotation(level A)

Figure 6. Analysis of CAZy abundance of Deseq2 between the control group and the experimental group

Table 8. Ratios of high abundance families of reads to CAZy (level B)

LevelA	Lever B	Control group	Trial group
GHs	GH13	234522.33 ± 38501.27	298726.33 ± 44494.17
	GH43	206457.00 ± 33966.33	205827.67 ± 30207.29
	GH35	213555.67 ± 37592.17	160965.67 ± 26533.15
	GH3	158770.33 ± 22828.70	200406.00 ± 29957.74
	GH2	168750.67 ± 26648.80	185595.00 ± 27565.72
GTs	GT2	2161276.00 ± 2500701.36	1056991.33 ± 160265.16
	GT4	1050591.75 ± 1249907.22	548267.67 ± 80354.93
	GT51	256062.00 ± 303796.92	132761.33 ± 19784.53
	GT0	167363.00 ± 194928.96	83135.33 ± 12818.98
	GT68	150532.00 ± 177063.56	76488.67 ± 10768.53
CBMs	CBM50	258514.33 ± 42836.47	318397.67 ± 47961.44
	CBM13	99698.00 ± 16243.93	117958.00 ± 17240.03
	CBM32	81680.33 ± 13184.67	107784.00 ± 17255.26
	CBM6	75331.33 ± 11870.31	92017.00 ± 14040.68
	CBM2	59042.33 ± 9750.56	72814.33 ± 11243.32

The same letter or no letter in shoulder mark of peer data shows no significant difference (P > 0.05), different letters show significant difference (P < 0.05), and different capital letters show significant difference (P < 0.01).The same as blow.

Table 9. Differences in CAZy abundance of DEseq2 between groups

Change	Control group vs Trial group
Upregulation	CBM83,CBM60,PL15,GH52,PL24,GH152,CBM53,GT95,CBM8,GT97,GH124,GT72. (P < 0.05)
Downregulation	AA1,GH126,AA12. (P < 0.05)