Effects of fermented Chinese herb residues on growth performance, nutrient apparent digestibility, serum biochemical indices and faecal microbial flora in beef cattle

Abstract

This study aimed to evaluate the effects of fermented Chinese herb residues on the growth performance, nutrient apparent digestibility, serum biochemical indices and microflora of Simmental beef cattle during the fattening period. Twenty-four healthy Simmental beef cattle with an average body weight of 358.54 ± 65.63 kg were randomly divided into four treatment groups, with 6 replicates. The dietary treatments were as follows: I: basal diet; II, III and IV: 10% corn husk in the diet was replaced by Chinese herb residue, enzyme-fermented residue, and enzyme bacteria co-fermented residue, respectively. The experimental period was 49 days. There was no significant difference in the growth performance of beef cattle (p > 0.05). The ether extract (EE) apparent digestibility of groups III and IV was significantly higher than that of group I (p < 0.05); the apparent digestibility values of nitrogen free extract (NFE) and total phosphorus (P) in group IV were significantly lower than those in the other groups (p < 0.05). The maleic dialdehyde (MDA) contents in groups I and II were significantly higher than those in groups III and IV (p < 0.05). The ACE and Chao1 indices of groups I and III were significantly higher than those of groups II and IV (p < 0.05). The Shannon indices of groups I and III were significantly higher than that of group II (p < 0.05). The abundance of Firmicutes in group III was significantly lower than that in group II (p < 0.05). In conclusion, fermented Chinese herb residues could replace 10% corn husk and be used in beef cattle breeding.

HIGHLIGHTS

1. Dietary replacement of 10% corn husk with Chineseherb residue can improve nutrient digestibility of fattening cattle.

2. Concentrate containing 10% fermented Chinese herb residues as a corn husk replacement can be used for feedlot cattle without adverse effects.

Keywords:

• Fermented Chinese herb residue
• Simmental beef cattle
• growth performance
• nutrient apparent digestibility
• serum biochemistry
• faecal microbes

Subject classification codes:

• Ruminant nutrition and feeding

Introduction

Chinese herb residue, the residue after effective extraction of Chinese medicinal materials, is rich in crude fat, crude protein, polysaccharides, alkaloids and other nutrients and active ingredients and has broad prospects for the development of feed resources (Liu et al. 2022). According to relevant statistics, the annual output of solid waste in the form of Chinese herb residue is approximately 30 million tons (Liu et al. 2023). The comprehensive utilisation of Chinese herb residue is of great significance for protecting the environment, saving resources, and promoting sustainable economic development of the animal husbandry industry. Microbial fermentation can significantly reduce the contents of cellulose and lignin in Chinese herb residues, increase the contents of protein, amino acids and other nutrients, improve dietary palatability, and improve the efficiency of feed utilisation of Chinese herb residues (Wang 2019; Wei 2021).

Several studies have found that the addition of fermented red ginseng residue to animal diets can improve the spleen and testicular organ indices (Huang et al. 2017); the use of Astragalus residue in the diet can promote growth performance (He et al. 2014); the addition of fermented Trollius chinensis residue can increase immune function, antioxidant capacity and disease resistance (Zhang et al. 2019); and the addition of fermented Chinese medicine residues can expand economic benefits (Li et al. 2021). However, most of the research on the fermentation of pharmaceutical residues focuses on their application as effective additives, and few studies have focused on the application of Chinese herb residues as feed resources instead of raw feed materials in animal husbandry production.

Previously obtained data showed that Chinese herb residue contained 10.28% crude protein, 1.93% crude fat, 25.06% crude fibre, 53.14% neutral detergent fibre, 36.63% acid detergent fibre, 13.79% crude ash and 1.36 mg/g flavonoids (Liu et al. 2022), which is a nutritional composition close to that of corn husk used as feed material (Li et al. 2021). The residue also has the effect of detoxification. The main common diseases in cattle production are mycoplasma pneumonia and calf diarrhoea (Qin et al. 2019). Heat-clearing and detoxifying drugs may have a good growth-promoting effect. Therefore, this study was conducted to evaluate the effect of replacing corn husk with fermented residues on Simmental beef cattle during the fattening period, and the results can provide a theoretical reference for the development and utilisation of Chinese herb residues.

Materials and methods

The animal experiment procedure was approved by the Animal Ethics Committee of Jilin Agricultural Science and Technology University and carried out according to the animal experiment guidelines formulated by the National Institute of Animal Health (20231025).

Experimental materials

The residues used in the experiment were by-products of the extraction or decoction of traditional Chinese medicines, with radix ophiopogonis, radix Isatidis, and radix scrophulariae as raw materials, which were provided by Jilin Tonghua Modification Pharmaceutical Co. Ltd. (Tonghua, China). Group II comprised the residues without an additive. Group III comprised residues fermented by cellulase (the added amount was 0.50%, and the enzyme activity was 20000 U/g). Group IV consisted of mixed fermentation residues of Lactiplantibacillus plantarum and Bacillus subtilis. The amount of bacteria added was 3 mL, and the total number of viable bacteria was ≥ 5 × 10⁸ CFU/mL.

The initial water content of the fermented residues in groups II, III and IV was set at 55%. After evenly stirring the residues with corn husk, soybean meal, and corn flour, they were put into a sealed fermentation bag with a breathing valve. The samples in groups II and III were placed in a 37 °C incubator for continuous fermentation for 72 h, and the samples in group IV were placed in a 40 °C incubator for continuous fermentation for 72 h. After 72 h, the fermentation residues were mixed and used to replace corn husk to prepare a concentrate. The concentrate in the experimental basal diet was a full-priced concentrate supplement, which was processed at Jilin Dabei Agriculture and Animal Husbandry Technology Co., Ltd. (Changchun, China). Simmental beef cattle were selected from the second Taonan Ranch of Zhongnong Jimu Agricultural Development Co., Ltd (Taonan, China).

Experimental design and feeding management

Twenty-four healthy male Simmental beef cattle (average body weight 358.54 ± 65.63 kg) aged 12 months were randomly divided into four groups, with 6 replicates per group and 1 bull per replicate. All the cattle were reared with free access to feed and clean water in individual enclosures (5 m × 4 m), which were separated by an iron fence and contained an individual trough and drinking bowl. Each group of cattle was raised in a separate fenced area. The dietary treatments were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue. The dietary concentrate composition and nutrients are detailed in Table 1. The bioactive composition and content of the residues used in groups II, III and IV are shown in Table 2.

Table 1. Concentrate composition and nutrient levels of the basal diet (air-dried basis), %.

Items	Content
	I	II	III	IV
Ingredients
Corn	32	32	32	32
Sprayed corn husks	10	10	10	10
Corn husk	10	0	0	0
Fermented Chinese herb residues	0	10	10	10
Sesame seed meal	5	5	5	5
DDGS	35	35	35	35
Syrup	3	3	3	3
NaCl	1	1	1	1
Mineral meal	3	3	3	3
Premix ^1)	1	1	1	1
Total	100	100	100	100
Chemical composition^2)
Dry matter (DM)	97.82	97.77	97.68	97.53
Organic matter (OM)	79.37	79.09	77.43	79.04
Crude protein (CP)	20.79	22.44	21.71	22.11
Ether extract (EE)	4.94	5.25	5.30	5.06
Crude fibre (CF)	10.43	10.36	7.19	7.49
Neutral detergent fibre (NDF)	38.51	32.79	31.72	33.43
Acid detergent fibre (ADF)	11.04	10.47	10.29	10.62
Ca	1.42	1.07	1.18	1.27
P	0.95	1.10	1.07	0.85
Gross Energy (MJ·kg^−1)	18.09	17.93	17.91	17.98

Note: 1) The premix provided the following per kg of diet: vitamin A 190 000 IU, vitamin E 2000 IU, vitamin D₃ 70 000 IU, Cu 460 mg, Mn 3 300 mg, Se 10 mg, Co 10 mg, Fe 2 100 mg, Zn 3 400 mg, I 19 mg, Ca 130 g, P 30 g.

2) Nutrient levels were measured in values.

The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Table 2. Bioactive substance composition and content of the residues used in groups II, III and IV.

Items	Groups			P value
	II	III	IV
Flavonoids (mg/g)	2.91 ± 0.26^b	3.92 ± 0.28^a	2.45 ± 0.18^b	0.0008
Reducing sugars (mg/g)	46.85 ± 0.43^c	94.29 ± 3.08^a	79.42 ± 2.05^b	<.0001
Alkaloids (μg/g)	4.66 ± 2.09	1.52 ± 0.15	2.57 ± 0.92	0.0676
Lactic acid (μmol/g)	308.41 ± 10.17	303.69 ± 7.26	310.57 ± 5.72	0.5836

Note: Values in the same row with different superscript letters are significantly different (p < 0.05).

The groups were as follows: II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

The diet of the four experimental groups comprised a 50:50 mixture of concentrate and roughage according to the Chinese Beef Cattle Feeding Standards (NY/T 815-2004). The roughage consisted of 25% maize straw and 75% rice straw. During feeding, roughage was fed first, and then concentrate was fed. The concentrated feed was sprinkled on the chopped roughage, and the beef cattle were able to eat the feed components in the tank together. The experimental period was from Jul. 1st, 2022 to Aug. 19th, 2022 at the Taonan Second Ranch of the Zhongnong Jimu Group (Taonan, China). Before the trial began, the cattle were dewormed, and the barn was cleaned and disinfected. The experiment lasted for 49 d. During the experiment, the experimental cattle drank water freely and were fed quantitatively at 6:00 and 15:30.

Sample collection and preservation

Blood sample collection

On the last day of the experimental period, venous blood collection was performed using a vacuum blood collection coagulant tube before morning feeding at the second Taonan Ranch of Zhongnong Jimu Agricultural Development Co., Ltd. (Taonan, China). The samples were separated at 3500 r/min for 10 min and stored at −80 °C for testing.

Faecal sample collection

Nutrient apparent digestibility was determined by the total faecal collection method. For 3 consecutive days before the end of the experimental period, special guards were assigned 24 h a day to collect faecal samples after morning feeding. After discharge, the faeces were immediately collected in a self-sealing bag. The faecal samples were accurately collected, weighed and recorded every day.

Samples weighing 10% of the fresh faeces weight were collected. After removing the gravel and hair from each sample, 10% dilute sulphuric acid was sprayed to fix nitrogen; the sample was dried at 65 °C until constant weight and then crushed to determine the contents of nutrients on an air-dried basis.

On the last day of the experiment, fresh faeces of each animal were collected before morning feeding, divided into 2-mL freezing tubes, immediately placed in liquid nitrogen for quick freezing, and then stored at −80 °C for testing.

Sampling and analyses

Growth performance

The initial body weight (IBW) was measured at the beginning of the experiment, and the final body weight (FBW) was measured at the end of the experiment. These parameters were used to calculate the total weight gain (TWG), average daily gain (ADG) and feed-to-gain ratio (F/G). (1) $$\text{ADG}=\left(\text{FBW}-\text{IBW}\right)/\text{experimental days}$$(1) (2) $$\mathrm{F}/\mathrm{G}=\text{ average daily feed intake }\left(\text{ADFI}\right)/\text{ADG}$$(2)

Nutrient apparent digestibility

The determination methods of dry matter (DM) content, crude protein (CP) content, ether extract (EE) content, crude fibre (CF) content, neutral detergent fibre (NDF) content, acid detergent fibre (ADF) content, ash content, calcium (Ca) content and total phosphorus (P) content in feed samples and faecal samples were performed according to Feed Analysis and Feed Quality Detection Technology (Zhang 2016). (3) $$\text{Organic matter }\left(\text{OM}\right)\%=\text{ DM}\%-\text{Ash}\%$$(3) (4) $$\text{Hemicellulose }\left(\text{ADS}\right)\%=\text{NDF}\%-\text{ ADF}\%$$(4) (5) $$\text{Nitrogen}-\text{free extract }\left(\text{NFE}\right)\%=\text{ DM}\%-\text{ Ash}\%-\text{ CP}\%-\text{ EE}\%-\text{ CF}\%$$(5) (6) $$\text{Apparent digestibility of nutrients }\left(\%\right)=[(\text{nutrient intake}-\text{nutrient content in faeces})/\text{nutrient intake}]\times 100\%$$(6)

Detection of bioactive components

The reducing sugar and flavonoid contents were measured using commercial kits (Beijing Solarbio Technology Co. Ltd., Beijing, China). The alkaloid and lactic acid contents were determined using commercial kits (Jiangsu Keming Biotechnology Co. Ltd., Jiangsu, China).

Determination of serum samples

The triglyceride (TG), cholesterol (CHO), aspartate transaminase (AST), glucose (GLU), alkaline phosphatase (ALP), albumin (ALB), total protein (TP), urea (urea) and alanine aminotransferase (ALT) contents were all measured using commercial kits (Zhongsheng Beikong Biotechnology Co., Ltd., Beijing, China). The above parameters were evaluated by an automatic biochemical analyser (Selectra-E, Netherlands) in strict accordance with the reagent instructions. Globulin (GLOB) content was calculated as the TP content minus ALB content (Si et al. 2019).

The total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, glutathione (GSH) content and superoxide dismutase (SOD) activity were all measured using commercial kits (Nanjing Jiancheng Technology Co., Ltd., Beijing, China), and values were determined by a microplate spectrophotometer (Biotek Epoch2, USA) in strict accordance with the kit instructions.

Immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) were all measured using commercial kits (Jiangsu Feiya Biotechnology Co., Ltd., Beijing, China) and were determined by a microplate spectrophotometer (Biotek Epoch2, USA) in strict accordance with the kit instructions.

High-throughput sequencing of microbial 16S amplicons

The genomic DNA of beef cattle faeces was extracted using the sodium dodecyl sulphate (SDS) method (Habibi et al. 2022). The V3-V4 variable region was amplified by PCR (forward primer sequence: 5′-CCTACGGGNGGCWGCAG-3′; reverse primer sequence: 5′-GACTACHVGGGTATCTAATCC-3′). The recovered PCR products were quantified and mixed using the QuantiFluor TM-ST Blue Fluorescence Quantitative System (Promega). Then, the mixed PCR products were purified by the AxyPrep DNA gel extraction kit (AXYGEN). The library was constructed using the NEB Next®Ultra™ DNA Library Prep Kit (NEB, USA), qualified by a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system (Agilent), and then sequenced on the Illumina NovaSeq 6000 platform (Illumina, Inc). The determinations were performed at Shanghai Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China).

After filtering the valid data obtained by sequencing, UPARSE software (Version7.0.1001) was used for clustering. The default sequences were clustered into OTUs with 97% consistency, and then the selected OTU representative sequences were annotated. The database for annotation is SILVA database (138.1, 2020). The α and β diversity indices were analysed using QIIME (Version 1.8.0) software. R software was used to draw principal coordinate analysis (PCoA) maps, and analysis of similarities (ANOSIM) was used to test the difference between different groups.

Statistical analysis

Data were processed using Excel 2019 (Microsoft) and analysed by SAS version 9.4 (SAS Institute Inc., Cary, NC). One-way analysis of variance (ANOVA) was used to test growth performance, nutrient apparent digestibility, serum biochemical indices and intestinal microbial flora. Duncan’s method was used for multiple comparisons. The composition of the bacterial community was compared by ANOSIM with principal coordinate analysis based on UniFrac distance with the vegan package (2.5) in R software (3.6.3). Pearson correlations between parameters were performed with R software (3.6.3). In this study, all the data are presented as the mean ± standard deviation. P values <0.05 were considered indicative of a significant difference between treatments; P values > 0.05 indicated that the difference was not statistically significant.

Results

Growth performance

No significant difference was found in initial weight among the groups (p > 0.05). There were no significant differences in FBW, TWG, ADG or F/G among all groups (p > 0.05, Table 3). However, compared with group I, TWG and ADG in groups II, III and IV showed a gradually increasing trend. F/G showed a gradually decreasing trend. The TWG and ADG of group IV were the highest, which were 2.04% and 21.42% higher than those of group I, respectively, and the F/G of group IV was the lowest, which was 15.69% lower than that of group I.

Table 3. Effects of fermented Chinese herb residues on the growth performance of Simmental beef cattle during the fattening period.

Items	Groups				P value
	I	II	III	IV
IBW, kg	358.83 ± 71.08	358.67 ± 84.22	358.33 ± 53.48	358.33 ± 53.73	1.0000
FBW, kg	400.00 ± 73.53	401.00 ± 84.69	404.33 ± 44.57	408.17 ± 53.70	0.9964
TWG, kg	41.17 ± 8.08	42.33 ± 7.66	46.00 ± 12.54	46.83 ± 12.86	0.4955
ADG, kg/d	0.84 ± 0.16	0.87 ± 0.16	0.94 ± 0.26	1.02 ± 0.26	0.4917
ADFI, kg/d	44.28	44.28	44.28	44.28	–
DMI, kg/d	8.07 ± 1.28	8.14 ± 1.27	8.40 ± 0.63	8.72 ± 1.18	0.7475
F/G	9.05 ± 1.64	8.86 ± 2.07	8.53 ± 2.98	7.63 ± 1.76	0.6863

Note: Values in the same row with different superscript letters are significantly different (p < 0.05).

IBW, initial body weight; FBW, final body weight; TWG, total weight gain; ADG, average daily gain; ADFI, average daily feed intake; DMI, dry matter intake; F/G, feed to gain ratio.

The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Nutrient apparent digestibility

The apparent digestibility of EE in group I was significantly lower than that in groups III and IV (p < 0.05, Table 4), and there was no significant difference in the apparent digestibility of EE among groups I and II (p > 0.05). The apparent digestibility of hemicellulose (ADS) in groups III and IV was significantly lower than that in groups I and II (p < 0.05). The apparent digestibility of nitrogen-free extract (NFE) in group IV was significantly lower than that in the other groups (p < 0.05). The apparent digestibility of P in group IV was significantly lower than that in the other groups (p < 0.05). There were no significant differences in the apparent digestibility of DM, organic matter (OM), CP, CF, NDF, ADF or Ca among all groups (p > 0.05).

Table 4. Effects of fermented Chinese herb residues on nutrient apparent digestibility of Simmental beef cattle during the fattening period (%).

Items	Groups				P value
	I	II	III	IV
DM	77.75 ± 3.79	78.13 ± 4.17	77.96 ± 2.83	77.49 ± 3.11	0.9536
OM	80.54 ± 3.40	81.42 ± 3.74	80.63 ± 2.85	79.33 ± 3.02	0.2959
EE	83.26 ± 4.37^b	85.54 ± 3.66^ab	86.66 ± 3.28^a	87.05 ± 3.03^a	0.0152
CP	81.53 ± 7.98	79.96 ± 4.28	77.33 ± 3.06	78.24 ± 3.84	0.0793
CF	71.31 ± 4.20	71.18 ± 7.04	68.97 ± 7.05	69.83 ± 4.45	0.5775
NDF	74.39 ± 4.82	73.11 ± 5.39	72.46 ± 4.12	72.54 ± 4.64	0.6006
ADF	67.90 ± 5.72	67.08 ± 6.50	67.04 ± 5.27	66.44 ± 4.95	0.8921
ADS	81.30 ± 4.08^a	81.26 ± 4.17^a	77.75 ± 3.59^b	78.11 ± 4.88^b	0.0134
NFE	84.96 ± 3.89^a	85.80 ± 2.82^a	85.16 ± 2.71^a	82.42 ± 3.28^b	0.0123
Ca	68.39 ± 6.17	68.45 ± 7.93	65.42 ± 7.33	68.22 ± 6.42	0.5238
P	69.44 ± 5.61^a	69.64 ± 6.72^a	68.05 ± 5.58^a	53.66 ± 8.27^b	<.0001

Note: Values in the same row with different superscript letters are significantly different (p < 0.05).

DM, dry matter; OM, organic matter; EE, ether extract; CP, crude protein; CF, crude fibre; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADS, hemicellulose; NFE, nitrogen-free extract; Ca, calcium; P, total phosphoru.

The groups were as follows:I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Serum biochemical indices

There were no significant differences in the contents of GLU, CHO and TG in serum among all groups (p > 0.05, Table 5). No significant differences were found in the activities of serum AST, ALT and ALP among all groups (p > 0.05). No significant differences were found in serum TP, ALB, GLOB and urea contents among all groups (p > 0.05).

Table 5. Effects of fermented Chinese herb residues on serum biochemical indices of Simmental beef cattle during the fattening period.

Items	Groups				P value
	I	II	III	IV
GLU, mmol/L	4.75 ± 1.43	3.87 ± 0.35	4.36 ± 0.37	4.48 ± 0.77	0.3403
CHO, mmol/L	2.92 ± 0.53	3.32 ± 0.31	2.97 ± 0.33	3.07 ± 0.66	0.5132
TG, mmol/L	0.14 ± 0.03	0.16 ± 0.03	0.16 ± 0.02	0.18 ± 0.02	0.3153
AST, U/L	88.89 ± 7.77	77.35 ± 7.63	78.93 ± 12.93	74.60 ± 9.46	0.1283
ALT, U/L	32.12 ± 9.04	25.89 ± 2.91	31.20 ± 5.40	26.44 ± 3.87	0.1692
ALP, U/L	142.52 ± 50.60	100.52 ± 35.00	97.78 ± 36.30	112.49 ± 25.42	0.2345
TP, g/L	65.72 ± 5.72	64.39 ± 3.38	69.14 ± 3.13	63.02 ± 4.03	0.0936
ALB, g/L	34.57 ± 3.78	34.88 ± 2.04	34.89 ± 2.21	33.84 ± 1.41	0.8618
GLOB, g/L	31.16 ± 5.38	29.51 ± 3.16	34.25 ± 4.70	29.18 ± 3.82	0.1906
Urea, mmol/L	3.00 ± 0.94	4.19 ± 1.21	3.48 ± 0.40	3.97 ± 0.60	0.1246

Note: Values in same row with different superscript letters are significantly different (p < 0.05).

GLU, glucose; CHO, cholesterol; TG, triglyceride; AST, aspartate transaminase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; GLOB, globulin.

The groups were as follows:I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

The T-AOC and SOD activity in group II were significantly higher than those in the other groups (p < 0.05, Figure 1). The MDA contents in groups I and II were significantly higher than those in groups III and IV (p < 0.05). There were no significant differences in GSH content among all groups (p > 0.05).

Figure 1. Effects of fermented Chinese herb residues on serum antioxidant indices of Simmental beef cattle during the fattening period.T-AOC, total antioxidant capacity; SOD, superoxide dismutase；MDA, malondialdehyde; GSH, glutathione.The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

There were no significant differences in serum IgA, IgG and IgM contents among all groups (p > 0.05, Figure 2). However, compared with group I, the IgA content in the serum of groups III and IV was increased by 11.03% and 12.78%, respectively. The IgG content was increased by 2.44% and 8.46%, respectively. The IgM content was increased by 11.07% and 2.38%, respectively.

Figure 2. Effects of fermented Chinese herb residues on serum immune indices of Simmental beef cattle during the fattening period.IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Faecal microflora composition

Comparative analysis of the faecal microbial α diversity index

A total of 1,592,529 valid sequences were obtained by 16S rDNA sequencing based on the Illumina NovaSeq 6000 sequencing platform. By default, the effective sequences were clustered with 97% consistency, as shown in Figure 3. A total of 4,154 OTUs were generated, among which 1,825 were shared by all four groups. The number of unique OTUs in group I was 359, accounting for 43.93% of the total OTUs. There were 107 unique OTUs in group II. The number of OTUs in group III was 508. The number of unique OTUs in group IV was 112.

Figure 3. OTU Venn diagram of intestinal flora in 4 groups of beef cattle.The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

According to the results of the OTU cluster analysis, the α diversity analysis showed that the sequencing coverage of all samples in this experiment was above 0.993. The ACE index and Chao1 index of the four groups showed the same trend, and the ACE index and Chao1 index of groups I and III were significantly higher than those of groups II and IV (p < 0.05, Figure 4). The Shannon index of groups I and III was significantly higher than that of group II (p < 0.05) but was not significantly different from that of group IV (p > 0.05). The observed_species index and PD_whole_tree index of groups I and III were significantly higher than those of groups II and IV (p < 0.05). There was no significant difference in the Simpson index among all groups (p > 0.05).

Figure 4. Comparisons of the α diversity indices of beef gut microbiota among the four groups.The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Comparative analysis of the faecal microbial β diversity index

Analysis of the difference in β diversity based on the unweighted UniFrac and weighted UniFrac distances showed that there was no significant difference among the four groups (p > 0.05. Figure 5).

Figure 5. Differences in intestinal microbial β diversity between groups of Simmental beef cattle during the fattening period based on unweighted UniFrac (A) and weighted UniFrac (B).The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

The results of the principal axis analysis showed that there was no significant separation between the four groups based on the unweighted UniFrac and weighted UniFrac distances (p > 0.05, Figure 6).

Figure 6. Principal coordinates analysis (PCoA) of intestinal microbiota in Simmental beef cattle during the fattening period based on unweighted UniFrac (A) and weighted UniFrac (B).the groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Comparative analysis of faecal microflora at selected taxonomic levels

In this study, the top 10 intestinal microorganisms with the highest abundance at the phylum level in Simmental beef cattle were selected for analysis. The dominant bacteria in the four groups were Firmicutes, Bacteroidetes and Proteobacteria. The abundance of Firmicutes in group III was significantly lower than that in group II (p < 0.05, Table 6), but there was no significant difference among groups I, II and IV (p > 0.05). The abundance of Actinobacteria in group II was significantly lower than that in groups I and III (p < 0.05). The abundance of Verrucomicrobia in group I was significantly lower than that in the other groups (p < 0.05). The Cyanobacteria abundances in groups III and IV were significantly lower than those in group I (p < 0.05). Acidobacteria abundance in groups I and IV was significantly lower than that in groups II and III (p < 0.05).

Table 6. Intestinal microbiome composition at the phylum level among the four groups of Simmental beef cattle (%).

Items	Groups				P value
	I	II	III	IV
Firmicutes	51.09 ± 3.14^ab	53.55 ± 2.49^a	47.52 ± 5.35^b	49.29 ± 2.39^ab	0.0474
Bacteroidetes	37.19 ± 2.89	37.91 ± 2.47	39.59 ± 2.93	38.73 ± 3.54	0.5495
Proteobacteria	4.72 ± 2.61	4.14 ± 2.48	6.76 ± 4.47	8.03 ± 4.33	0.2358
Spirochaetes	1.47 ± 0.37	1.51 ± 0.78	0.99 ± 0.24	0.89 ± 0.31	0.0710
Tenericutes	0.99 ± 0.20	0.81 ± 0.31	1.42 ± 1.37	1.10 ± 0.47	0.5512
Actinobacteria	0.86 ± 0.64^a	0.28 ± 0.14^b	1.00 ± 0.57^a	0.48 ± 0.25^ab	0.0404
Verrucomicrobia	1.03 ± 0.73^a	0.44 ± 0.18^b	0.45 ± 0.25^b	0.23 ± 0.11^b	0.0155
Patescibacteria	0.32 ± 0.09	0.49 ± 0.15	0.49 ± 0.23	0.47 ± 0.17	0.2880
Cyanobacteria	0.50 ± 0.13^a	0.40 ± 0.13^ab	0.30 ± 0.17^b	0.29 ± 0.08^b	0.0382
Acidobacteria	0.60 ± 0.49^a	0.001 ± 0.001^b	0.42 ± 0.33^a	0.003 ± 0.003^b	0.0036
Others	1.24 ± 0.42^a	0.48 ± 0.11^b	1.07 ± 0.48^a	0.49 ± 0.21^b	0.0010

Note: Values in the same row with different superscript letters are significantly different (p < 0.05).

The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

In this study, the top 10 intestinal microorganisms with the highest abundance at the genus level in Simmental beef cattle were selected for analysis. The dominant bacterial genera in the four groups were the Rikenellaceae RC9 gut group, Ruminococcaceae UCG-005 and Ruminococcaceae UCG-010, and there was no significant difference in relative abundance among the four groups (p > 0.05, Table 7). The abundance of Bacteroides in group III was significantly lower than that in groups I and II (p < 0.05). The abundance of Prevotellaceae UCG-003 in group IV was significantly higher than that in groups I and II (p < 0.05).

Table 7. Intestinal microbiome composition at the genus level among the four groups of Simmental beef cattle %.

Items	Groups				P value
	I	II	III	IV
Rikenellaceae RC9 gut group	10.58 ± 2.11	11.59 ± 0.49	10.70 ± 1.78	9.52 ± 0.95	0.1534
Ruminococcaceae UCG-005	10.52 ± 2.77	12.49 ± 1.19	10.04 ± 1.88	10.98 ± 1.11	0.1558
Ruminococcaceae UCG-010	9.47 ± 1.78	12.05 ± 1.46	10.09 ± 1.76	11.07 ± 1.35	0.0548
uncultured bacterium	7.36 ± 1.23	7.38 ± 0.61	7.54 ± 0.95	8.29 ± 0.79	0.2837
Bacteroides	4.40 ± 0.52^a	4.20 ± 0.66^ab	3.35 ± 0.56^c	3.52 ± 0.70^bc	0.0195
Alistipes	4.81 ± 0.70	5.00 ± 0.84	4.56 ± 0.69	3.96 ± 0.62	0.0986
[Eubacterium] coprostanoligenes group	3.90 ± 0.34	3.91 ± 0.33	4.07 ± 0.77	3.73 ± 0.45	0.7138
Uncultured	3.07 ± 0.23^a	2.61 ± 0.25^b	3.17 ± 0.43^a	2.82 ± 0.29^ab	0.0219
Prevotellaceae UCG-003	2.08 ± 0.49^b	2.48 ± 0.84^b	2.84 ± 1.65^ab	3.97 ± 0.96^a	0.0360
Ruminococcaceae UCG-013	2.61 ± 0.42	2.89 ± 0.30	2.61 ± 0.57	2.64 ± 0.26	0.5903
Others	41.18 ± 8.17	35.41 ± 1.44	41.04 ± 8.66	39.50 ± 4.76	0.3948

Note: Values in the same row with different superscript letters are significantly different (p < 0.05).

The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

Prediction and analysis of intestinal microbial function

In this study, the top 10 Kyoto Encyclopaedia of Genes and Genomes (KEGG) functional categories of intestinal microorganisms with the highest relative abundance at the secondary level in Simmental beef cattle were selected, and a relevant histogram was generated (Figure 7A). The enriched microbial communities in the four groups showed membrane transport, carbohydrate metabolism, amino acid metabolism, replication and repair, and there were many functional genes in related metabolic pathways.

Figure 7. Bar chart of the relative abundance of intestinal microbial functional categories of Simmental beef cattle in each group. KEGG (A) and COG (B).The groups were as follows: I: basal diet; II: diet prepared by replacing 10% corn husk with Chinese medicinal residue; III: diet prepared by replacing 10% corn husk with enzyme-fermented Chinese medicinal residue; IV: diet prepared by replacing 10% corn husk with enzymatic bacteria co-fermented Chinese herb residue.

In this study, the top 10 Clusters of Orthologous Groups (COG) functional categories of intestinal microorganisms with the highest relative abundance at the secondary level in Simmental beef cattle were selected, and a relevant histogram was generated (Figure 7B). Among the four groups, the enriched microbial communities showed general function prediction only, translation, amino acid transport and transcription. There were many functional genes in carbohydrate-related metabolic pathways, such as metabolism, carbohydrate transcription and metabolism.

Discussion

Growth performance can reflect the growth status of animals and has an important impact on the economic benefits of animal husbandry. In recent years, Chinese herbs have been commonly used as substitutes for antibiotics to treat diseases and improve the growth performance of animals (Yin et al. 2021). Adding appropriate Chinese herbs or fermenting Chinese herbs can improve the growth performance of livestock and poultry and improve product quality (Shan 2019; Gao et al. 2022). Studies have shown that dietary supplementation with different proportions of Chinese herb residues can significantly improve the fattening effect of lambs (Lin et al. 2017). The addition of fermented Chinese herb residues to the beef cattle diet can increase the dry matter intake and daily gain of beef cattle more than Chinese herb residues and improve feed utilisation (Li et al. 2020). There were no significant differences in FBW, TWG, ADG or F/G among all groups, which was consistent with the results of Zhuang et al. (2021) and Li et al. (2022). However, compared with the control group, TWG and ADG in the three fermented Chinese residue groups gradually increased, and F/G gradually decreased. This may be because the nutrients and some of the active ingredients of traditional Chinese medicine remaining in the residue promote the growth and development of animals, improve intestinal health, and thus promote the healthy growth of animals. The experiment further proved that fermented residue could be used as a feed resource to replace conventional feed with similar nutritional value. This approach may solve problems related to raw feed material shortages and price fluctuations in the future.

Nutrient apparent digestibility reflects the digestibility and utilisation of the diet. Chinese herb residues added to the diet can help promote the digestion and absorption of nutrients by animals (Ju et al. 2022) because the nutritious material contained in Chinese herb residues can improve and balance the diet composition, thus improving feed conversion efficiency (Li et al. 2021). Studies reported in the literature showed that the digestibility of crude fat, CF, CP, DM and OM was significantly higher than that of the control group after adding Chinese herb residues or fermented residues to the diets of animals (Zhu et al. 2018; Li et al. 2022). The results of this study are not consistent with those reported above. The gradual increase in crude fat digestibility may be due to the improvement in rumen fermentation after the addition of fermented Chinese herb residues, which enhanced rumen microbial activity, increased the contact area between fat and lipase, improved the activity of pancreatic lipase, and promoted the digestion and absorption of fat. The apparent digestibility of NFE and TP in group IV was the lowest, and there was no significant difference in the other three groups, indicating that dietary supplementation with fermented Chinese herb residues does not affect the digestion and absorption of mineral elements in beef cattle. However, further studies are needed to better clarify the possible reasons for the obtained results.

Serum biochemical indices can reflect the growth, metabolism and physiological health of animals to some extent (Hosoda et al. 2005). Changes in serum GLU, CHO, and TG contents can reflect the metabolic status of carbohydrates and fat in the body to some extent (Schade et al. 2020). Serum TP, ALB and GLOB can reflect the dietary nutrient levels and protein digestion and metabolism of the body (Yin et al. 2022). Serum urea is an important indicator of renal function and protein metabolism levels (Chi 2021). ALP, AST and ALT can directly reflect the status of liver function in the body (Hernandez et al. 2022), and they play an important role in amino acid metabolism, metabolic transformation, digestion and absorption of protein, fat and sugar (Li et al. 2017). In this experiment, there were no significant effects on serum biochemical indices of beef cattle among the four groups, indicating that adding fermented herb residues had no adverse effects on the liver, kidney and other organ functions and had no negative effects on blood glucose, blood lipid and protein metabolism of beef cattle during the fattening period.

Animals can continuously secrete a large amount of antioxidant substances to remove excess free radicals in the body to maintain the dynamic balance of the body, maintain health and prevent oxidative damage (Guan et al. 2023). SOD directly reflects the ability of the body to remove free radicals (Holdom et al. 2000). MDA can reflect the degree of lipid peroxidation and the severity of free radical attack on cells (Long et al. 2021). GSH can prevent oxidative damage to tissues and protect the structure and functional integrity of cell membranes (Enns and Cowan 2017). Studies have shown that dietary supplementation with fermented Chinese herb residues can significantly improve the activity of serum SOD and T-AOC in laying hens (Shi et al. 2020). It can improve the SOD activity and T-AOC of fattening sheep (Li et al. 2021) and can significantly reduce the MDA content of piglets (An et al. 2021). The contents of GSH and MDA in cattle can be significantly reduced (Wang et al. 2021). The results of this experiment were consistent with those of the abovementioned studies. Supplementation with fermented Chinese herb residues significantly increased the activity of SOD and T-AOC in serum, significantly decreased the content of MDA in serum, and increased the content of GSH. Supplementation with fermented Chinese herb residues can improve the antioxidant capacity of beef cattle, which may be related to residual flavonoids and other bioactive substances in Chinese herb residues. Appropriate flavonoids can be used as reducing agents and hydrogen donors in body redox to neutralise oxygen free radicals and thus improve antioxidant capacity (Kumar and Mandal 2017).

Immunoglobulins (IgA, IgM, IgG) are important non-specific immune factors in the body (Zhao et al. 2022). Immunoglobulins are proteins produced by lymphocytes of the animal immune system and can be converted into antibodies after a series of transformations (Megha and Mohanan 2021; Bu et al. 2023). Studies have shown that adding Chinese herb residues can promote the production of IgA and IgG in beef cattle serum and improve the immune capacity of beef cattle (Shan et al. 2018; Li et al. 2020). It can significantly increase the contents of IgG and IgM in the serum of broilers (Li 2021; Zhang et al. 2021). The contents of IgA, IgG, and IgM in the serum of weaned piglets were significantly increased (Xie et al. 2022). In this experiment, the contents of IgA, IgG, and IgG in the serum of beef cattle in the experimental groups were higher than those in the control group, indicating that the addition of fermented residue could improve the immune ability and resistance to adverse external environments of beef cattle to some extent. This may be because of the bioactive ingredients contained in Chinese herb residues or the metabolites in its fermentation culture, which can improve the immune performance of the body.

The analysis of intestinal microbial diversity considered α diversity and β diversity. α diversity is used to measure microbial diversity within an individual sample using different indices, such as ACE, Chao 1, Shannon and Simpson (Koester et al. 2022). Recent studies have reported that traditional Chinese medicine extracts, probiotics, fermentation broth and tea residue have significant effects on animal intestinal flora (Zhang et al. 2017; Wang et al. 2022). In this experiment, no significant difference was found between group I and group III in the ACE and Chao1 indices, but they were significantly higher than those of group II and group IV, indicating that the microorganisms in group I and group III had similar and higher richness. No significant difference was found in the Simpson value among the four groups, indicating that there was no significant difference in intestinal microbial diversity. The Shannon value of group III was significantly higher than that of group II, and there was no significant difference between group I and group IV, indicating that the intestinal microbial diversity of beef cattle in the enzyme-fermented residue group did not change. PCoA and intergroup difference analysis can reflect the microbial diversity between groups. The closer the distance between samples is, the more similar the species composition structure is (Wang 2020). In this experiment, there was no significant difference between the four groups, indicating that the species composition and structure of the four groups were very similar and that the community differences were small. This indicated that the species composition of intestinal microorganisms in beef cattle did not change after the use of fermented Chinese herb residues as feed materials. Therefore, fermented Chinese herb residues can be safely applied as alternative feed materials in beef cattle production.

Intestinal microorganisms play an important role in maintaining host physiological function and are closely related to body growth and development. Flora structure and species abundance are affected by many factors, such as diet type, living environment, and drug factors (Li et al. 2021; Liu 2021). Liu et al. (2022) showed that the dominant bacteria in goat intestinal flora were Firmicutes, Bacteroidetes, Spirochaetes and Proteobacteria. Tian et al. (2021) found that Firmicutes and Bacteroidetes accounted for approximately 80% of the total bacteria in the intestinal tract of lambs. Li et al. (2022) found that Bacteroidetes and Firmicutes were the dominant phyla in beef cattle fed herbal tea residue. The results of this experiment are consistent with those reported above. Feeding fermented Chinese herb residue did not change the dominant intestinal microflora in beef cattle. Compared with group I, the abundance of Firmicutes increased in group II, while it decreased in group III and group IV, possibly because Firmicutes are related to the degradation of structural polysaccharides (Fan et al. 2022). The addition of a biological starter made the content of structural polysaccharides in the fermented Chinese herb residue less than that in the fermented residue group without a starter. Studies have shown a positive relationship between Bacteroidetes and promotion of protein decomposition (Yang et al. 2017). In this experiment, the abundance of Bacteroidetes gradually increased among the fermented residue groups, which showed the same increasing trend as the apparent digestibility of crude protein among the three groups.

At the genus level, the dominant bacterial genera of intestinal microorganisms in fattening beef cattle were the Rikenellaceae RC9 gut group, Ruminococcaceae UCG-005 and Ruminococcaceae UCG-010, which was consistent with the results of Pan et al. (2022), Wang et al. (2022) and Zhu et al. (2021). Ruminococcaceae UCG-005 is a high-efficiency fibre-degrading bacteria in ruminants that mainly degrades and transforms fibrous raw materials (Abdelrahman et al. 2022). Ruminococcaceae UCG-010 is a bacterium that is directly related to enhancing the integrity of the epithelial barrier and inhibiting proinflammatory responses (Liao et al. 2021). In this experiment, the abundance of Ruminococcaceae UCG-005 and Ruminococcaceae UCG-010 in intestinal microorganisms in the fermented residue groups (II, III, IV) increased in comparison to the control group. This may be because the location of degradation and transformation of fibrous raw materials in beef cattle was changed from the rumen to the intestine by the use of fermented medicine residues, which inhibited the proinflammatory response under heat stress, but the specific reasons need to be further explored. Rikenellaceae belongs to Bacteroides and plays an important role in the degradation of fibre polysaccharides (Xue et al. 2022). In this experiment, compared with group I, the abundance of the Rikenellaceae RC9 gut group increased in group II and group III, indicating that the degradation rate of cellulose in the beef cattle intestine increased and the body digested and absorbed cellulose better. The abundance of bacteria in group IV decreased but did not show a significant difference, which may be caused by differences between the test animals.

Based on the PICRUSt functional secondary classification results, we also speculate that the functional gene composition in the sample, so as to analyse the functional differences between different samples or groups. The results showed that at the secondary level of the KEGG metabolic pathway and COG metabolic pathway, the metabolic functional genes of carbohydrate metabolism, amino acid metabolism, and other pathways were abundant in the four groups. Compared with the control group, the abundance of related bacteria in the fermented residue groups (II, III, IV) was increased. This is related to carbohydrate degradation, glycogen synthesis, and cellulose metabolism (Nan et al. 2021; Ma et al. 2022). In conclusion, Chinese herb residues or fermented Chinese herb residues may have the potential to regulate carbohydrate metabolism and amino acid metabolism in the intestinal microflora of beef cattle. However, the role of fermented medicine residues in regulating intestinal microbiota needs further study.

Conclusion

In conclusion, under the conditions of this experiment, concentrate containing 10% fermented Chinese herb residues as a corn husk replacement can be used for feedlot cattle and has no adverse effect on faecal microbial diversit.

Author contributions

All authors equally contributed to this work. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

All authors thank their institutions for the support and help.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are openly available in NCBI, reference number [PRJNA1005949].

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

PhD start-up funding (JNHZ(2022)702); Jilin Province Science and Technology Department project (202300702); Jilin Province Science and Technology Department project (YDZJ202203CGZH050); Jilin Province Education Department project (JJKH20230449KJ).