Ganoderma lucidum residue (GLR) contains a variety of medicinal and nutritional components and is a potentially high quality roughage. The purpose of the experiment was to study the effects of GLR on the growth performance, immune function and intestinal flora of broilers. The study was conducted in a 90-day indoor cage experiment with broilers, and three feeding groups were set up with the addition of GLR to the basal diet at 0 (A), 10% (B) and 15% (C), with three replicates of 10 broilers in each group. The growth performance, immune organ index, and fecal microbial community structure of broiler chickens in different feeding groups were compared. The results showed that (1) there were no significant differences in the average daily weight gains of broilers in the different feeding groups. (2) The immune organ indices of broilers in Group B were higher than those in Groups A and C, with the spleen index in Group B being significantly higher than that in Group A. (3) The dominant phyla in the broiler faecal bacterial communities in all feeding groups were Firmicutes and Bacteroidetes, and the dominant genera were Lactobacillus, Bacteroides and Alistipes; however, the relative abundance of Bacteroidota was significantly higher in Group B than in Group A, and the relative abundances of Lactobacillus, Bacteroides and Alistipes were higher in Groups B and C than in Group A. (4) Manure microbial FAPROTAX functions predicted that GLR increased the abundances of heterotrophic and fermentative broiler manure microorganisms and significantly reduced the abundances of animal parasites or symbionts. In summary, group B was superior to other groups in terms of growth performance, spleen index and probiotic level. Adding GLR to the base feed has a positive effect on enhancing the immunity of broiler chickens, increasing the abundance of beneficial bacteria in the intestine, and maintaining the ecological balance of the intestinal flora. These findings reveal the effect of GLR on the fecal microorganisms of broiler chickens and provide a new development direction for improving poultry health.
Research Article
Effect of dietary supplementation of Ganoderma lucidum residue on growth performance, immune organ index and faecal microbial community diversity in broiler chickens
https://doi.org/10.21203/rs.3.rs-2976135/v1
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broiler
Ganoderma lucidum residue
growth performance
gut microbiota
immune organ
G. lucidum, a fungus of the family Polyporaceae, has been used in China for thousands of years and is of great interest for its excellent edible and medicinal properties [1]. Ganoderma lucidum residue (GLR) is a natural by-product of the extraction of medicinal components from G. lucidum. GLR is rich in nutrients such as polysaccharides, proteins, and fibers, as well as flavonoids, terpenoids, phenols, and a series of unknown growth-promoting substances with medicinal bioactivities [2]. These substances usually have antibacterial, anti-inflammatory and antiviral drug effects.
With the development of the Chinese medicine industry, a large amount of GLR is produced, yet the industrial process is incomplete in extracting the active ingredients of G. lucidum and a large amount of nutrients remain. Nowadays, the main way to dispose of GLR is still the traditional incineration, pile storage, and burial, which not only causes a large amount of GLR waste but also damages the ecological environment[3, 4]. GLR is reported to be prepared as bio-organic fertilizer [2], but its effect as a feed additive is less studied.
With the development of livestock industry toward safe, green, environmentally friendly, high quality, efficient, ecological and sustainable production, traditional herbs have been used as alternatives to antibiotics, and herbal residues and herbal feeds have become a major trend in today's feed industry, playing an increasingly important role in livestock production. Chen et al[5] showed that the addition of turmeric residues to basal feed increased its in vivo Superoxide dismutase (SOD) activity The addition of turmeric residue to the basal feed increased the activity of SOD, decreased the content of Malondialdehyde (MDA), and enhanced the immune function of the Chinese soft-shelled turtle. Gui zhi Li-Zhong Tang has a high antioxidant capacity and low cytotoxicity. It has antioxidant, anti-inflammatory and anti-apoptotic effects [6]. It can prevent respiratory diseases in piglets, such as pneumonia. Studies have shown that Ganoderma lucidum is valuable not only for improving animal performance, but also for enhancing the immune system and improving the intestinal flora [7–9]. Therefore, GLR may have potential as a feed additive. In this paper, we investigated the feasibility of GLR as a feed additive and its effects on growth performance, immune organ index and intestinal microbial community of broiler chickens.
Broiler Growth Performance and Immune Organ Indices
As shown in Table 2, there was no significant difference between the different GLR addition levels on the initial(p=0.287) and final(p=0.178) body weight and mean daily gain of broilers. After 90 days of feeding, the final body weights and average daily weight gains of the broilers in Group B were higher than those in the other two groups. No abnormalities were observed in the immune organs of the broilers on au-topsy. The effects of GLR on the immune organs of broiler chickens are shown in Table 3. The spleen index was significantly higher in group B when compared to group A (P <0.05). GLR fed Groups B and C exhibited higher values than the control group for all indices.
Structural compositions of broiler manure microbial communities
In this study, nine phyla were identified. Among them, 8 phyla were common phyla among all three groups (see Fig. 1a). The dominant phyla were Firmicutes and Bacteroidetes, as shown in Figure 1b. Addition of GLR to the feed reduced the relative abundance of the phylum Firmicutes and increased the relative abundance of the phylum Bacteroidetes.
A total of 187 genera were identified at the genus level, with 133 genera unique to the three groups, 156 genera unique to Group A, 157 genera unique to Group B and 160 genera unique to Group C (see Fig. 1c). The dominant genera with greater than 5% abundances were, in descending order, Lactobacillus, Christensenellaceae_R-7_group, norank_f__norank_o__Clostridia_UCG-014, Alistipes, Bacteroides, Ruminococ-cus_torques_group, and Rikenellaceae_RC9_gut_group (see Fig. 1d). Additions of GLR to the feed increased the relative abundances of Lactobacillus, nor-ank_f__norank_o__Clostridia_UCG-014, and Bacteroides in broiler manure and de-creased the relative abundances of Christensenellaceae_R-7_group and Ruminococ-cus_torques_group.
The results of the PCoA analysis at the OTU level are shown in Figure 2, where the horizontal principal coordinate component 1 (pc1) and vertical principal coordinate component 2 (pc2) show the explained degree values for the differences in sample compositions, which contribute 60.58% and 25.49%, respectively. By using the hori-zontal coordinate pc1 as the basis for division, Group A is distributed to the right, and Groups B and C are distributed to the left; when using the vertical coordinate pc2 as the basis for division, Groups A and B are distributed above, and Group C is located below. By using principal coordinate analysis, it was determined that the abundances of the faecal bacterial communities in the different GLR broiler feeding groups were signifi-cantly different at the OTU level (p=0.004). The results of the heatmap and sample cluster tree analysis for the genus-level compositions of the broiler faecal communities in the different GLR treatment groups (Figure 3) showed that the genus-level compo-sitions of the faeces in B and C were similar to each other and were different from those of Group A.
Prediction of microbial function
Based on the PICRUSt2 functional predictions, functional prediction information for different bacterial samples was obtained. The relative abundances of carbohydrate metabolism, amino acid metabolism, energy metabolism and other functions at KEGG pathway level 2 were higher in the three groups, as shown in Figure 4a. The functional abundance of the secondary functional laminae did not change significantly among the different groups, except for the carbohydrate metabolism function（p=0.0443）. As shown in Figure 4b, there was significant variability in the carbohydrate metabolism function among the three Groups, A, B and C, and their carbohydrate generation ca-pacities gradually strengthened with increases in the amount of GLR added to the feed.
The FAPROTAX functional prediction results (see Figure 5) show that the abun-dances of chemoheterotrophy (p=0.03899) and fermentation (p=0.03899) function genes in Groups B and C were significantly higher than those in the control group. The abundances of animal_parasites or_symbionts (p=0.03899), mammal_gut (p=0.03899) and human_gut (p=0.03899) genes in Groups B and C were significantly lower than those in the control group.
Effects of GLR on the growth performance and immune organs of broiler chickens
Other researchers have found significant effects from herbs on the growth performance of experimental animals. For example, Shawle et al. [10] found that the addition of certain amounts of Lepidium sativum L. to feed significantly increased the average daily weight gain of broiler chickens, but when the addition amount was too high, this led to a decrease in the average daily weight gain of broiler chickens. Zhou et al. [11] confirmed that baicalin-supplemented diet groups showed significant increases in body weights, average daily weight gains and feed conversion efficiencies in broilers at 21-42 days and 7-42 days. Tan et al. [12] showed that LBP extracts could improve the growth performance of fish. Numerous literature reports have confirmed that herbs improve feed utilization and thus, animal growth performance by regulating the efficiency of rumen fermentation [13, 14] and by influencing animal feed intake and digestive enzyme secretion [15].
However, the results of this study showed that GLR did not have a significant effect on the growth performance of broiler chickens, with a greatest average daily weight gain of 10%. This may be because when the amount of GLR additive is too high, the excessive amount of GLR affects the nutritional level of the feed ration, which results in imbalances in the nutritional intake of broilers and thus reductions in body weight. The effect of GLR on the growth performance of broiler chickens may also be correlated with the breed, period and sex of the experimental animals.
The thymus, spleen and bursa are the immune organs in broilers. Among these, the thymus has an important role in the preparation and development of T lymphocytes and T cells and is the central immune organ of the animal body [16]. The spleen is the largest peripheral immune organ in poultry, is rich in lymphocytes and is involved in systemic immunity [17]. The size of the spleen can reflect the immune status of the body [18]. The value of the immune organ index can indicate the level of immune function [19]. In this experiment, the immune organ indices of broiler chickens increased with addition of GLR to the diet. With increases in the amounts of GLR added, the change trend of the immune organ index exhibited a trend of first increasing and then decreasing. The immune organ index was greatest when the GLR addition amount was 10%. The differences in the spleen index were significant. Additions of GLR to feed could improve the spleen index of broiler chickens, and the spleen index for Group B was significantly higher than for Groups A. This indicates that GLR additions at appropriate levels have stimulating effects on the development of immune organs of broiler chickens.
Effect of GLR on the structural diversity of broiler manure microbial communities
The gut contains the highest concentrations of microorganisms in animals and contains a wide range of commensal flora, including bacteria, archaea, fungi, protozoa and viruses [20]. In recent years, it has been found that large numbers of microorganisms in the gastrointestinal tracts of animals have strong metabolic activities and play important roles in regulating the nutrient utilization and physiological metabolic functions of their hosts [21–23]. In this study, we applied 16S rRNA gene sequencing technology to analyse the compositions and structural characteristics of the faecal microbial communities of broiler chickens for different levels of GLR addition and in the control group. This technology was used to further investigate the relationships among broiler intestinal microorganisms and GLR.
It has been demonstrated that the intestinal flora of chickens are dominated by Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria [24, 25]. The results of this study also showed that the dominant phyla in all feeding groups were Firmicutes and Bacteroidetes. The results of the intergroup variability analysis of the microorganisms in the faecal samples showed that GLR significantly increased the relative abundance of Bacteroidetes (p=0.0002) and significantly decreased the relative abundance of Firmicutes (p=0.0006) in broiler faecal microorganisms (Figure 6). Ley et al. [26] showed that the relative abundance of Firmicutes was higher in obese mice, while the abundance of the Bacteroidetes phylum was lower. The ratio of Firmicutes to Bacteroidetes showed a negative correlation with obesity in mice, with higher ratios leading to fat accumulation, weight gain and perhaps links to certain diseases. Bäckhed et al. [27] showed that the Bacteroides proteome contains a variety of glycosyl hydrolases that can sever most of the glycosidic bonds in animal diets and can thus degrade plant polysaccharides and regulate lipid deposition in organisms. The results of this study suggest that GLR is free from the potential risk of lipid deposition when used as a feed additive. The results of this study suggest that GLR as a feed additive may reduce the potential risk of lipid deposition.
At the genus level, GLR significantly reduced the relative abundances of Christensenellaceae_R-7_group and Ruminococcus_torques_group, significantly increased the relative abundance of Bacteroides and increased the relative abundance of Lactobacillus in broiler manure microbial communities (Figs. 6b, 6d). The relative abundances of norank_f__norank_o__Clostridia_UCG-014, Alistipes, Rikenellaceae_RC9_gut_group, and unclassified_f__Lachnospiraceae in Group B with a 10% GLR addition were significantly higher than those in Group A. The relative abundances of norank_f__Ruminococcaceae and Monoglobus in Group C with a 15% GLR addition were significantly lower than those of CK, and those of Romboutsia and Blautia were significantly lower than those of CK.
At the genus level, Lactobacillus, Bacteroides and Alistipes are the dominant genera present in broiler intestines. Bacteroides, Alistipes, Rikenellaceae RC9 gut group, norank_f__norank_o__Clostridia_UCG-014 aggregated in significantly higher abundances in the experimental group than in the control group, and all act as probiotics in the intestine. Research has shown that (1) Lactobacillus is an important probiotic for promoting intestinal health and is important for promoting the growth and development of immune cells, tissues and organs and enhancing the resistance of the small intestine and other tissues to pathogenic bacteria [28]. (2) The main function of Bacteroides is to degrade carbohydrates and proteins and promote the development of the gastrointestinal immune system [29], which improves the host metabolism by participating in protein and fat metabolism and carbohydrate metabolism. (3) Alistipes is not only associated with immune regulation but is also involved in the metabolism of short-chain fatty acids [30]. Short-chain fatty acids in the intestine regulate the health of the organism in the gut, and these two dominant bacteria together maintain the ecological balance of intestinal microorganisms [31]. (4) Rikenellaceae_RC9_gut_group also belongs to the same phylum, and its members are producers of short-chain fatty acids. The higher abundance of the Rikenellaceae_RC9_gut_group ensures a high proportion of propionic acid in the rumen and thus regulates the balance in the intestine [32]. (5) norank_f__norank_o__Clostridia_UCG-014 is tolerant to gastric acid, enters the intestine, inhibits the growth of harmful intestinal bacteria, restores the function of intestinal flora, improves immunity and promotes the absorption, digestion and assimilation of nutrients [33].
Christensenellaceae_R-7_group and Ruminococcus_torques_group were widely present in the intestinal mucosa of the experimental animals, and the abundances of the aggregates were significantly lower in the experimental group than in the control group . Christensenellaceae_R-7_group are probiotic bacteria that have the function of promoting intestinal health, and Christensenellaceae are also one of the five taxa considered to be markers of intestinal health [34]. The decreased abundance of Christensenellaceae_R-7_group in this experiment may provide room for increased abundances of other probiotic bacteria. (2) Ruminococcus_torques_group is involved in the degradation of cellulose and hemicellulose in the rumen of ruminants and plays an important role in carbohydrate metabolic functions [35]. Some researchers [36] found by using a random forest analysis that the Ruminococcus_torques_group abundance was significantly and positively correlated with duck abdominal fat and that low levels of Ruminococcus_torques_group contributed to body fat reductions [37]. In this experiment, the addition of GLR significantly reduced the abundance of Ruminococcus_torques_group and reduced the potential risk of lipid deposition.
The LEfSe multilevel species hierarchical tree of faecal samples from each group (Fig. 7a) revealed that Bacteroidetes were mainly concentrated in Group B and Firmicutes were mainly concentrated in Group A. The LEfSe analysis revealed that the faecal samples from Group B were significantly enriched in Bacteroidia, Bacteroidota, Bacteroidales and Rikenellaceae compared to Group A. In addition, Escherichia-Shigella, Enterobacteriaceae and Enterobacterales were significantly enriched in Group A (Figure 7b). Escherichia-Shigella, Enterobacteriaceae, and Enterobacterales are all harmful bacteria in the intestinal tract and are the most important pathogens that cause infectious intestinal diseases in humans and animals. Therefore, addition of GLR to basic feed can enrich the biodiversity of flora, increase the abundance of probiotic flora, reduce the abundance of harmful bacteria and regulate the structure of intestinal flora, which thus stabilise the microbial ecological balance in the intestinal tract.
Prediction of microbial function
High-carbohydrate metabolism capacity may lead to production of high levels of short-chain fatty acids [38]. Butyric acid in short-chain fatty acids increases the abundance of Lactobacillus and decreases the abundance of E. coli, which thereby decreases the amounts of pathogenic bacteria in the body; thus, short-chain fatty acids are important for maintaining normal intestinal functions and cell morphology.
Based on the FAPROTAX functional predictions, a functional analysis of the flora was conducted to obtain functional prediction information for different bacterial samples. See Fig. 2, which shows that the abundances of chemoheterotrophy and fermentation in broiler manure microorganisms increased in the GLR addition groups. GLR significantly increased the fermentation function of manure microorganisms, and there was a positive correlation between fermentation and heterotrophic function and increases in the trace elements, C, N and P, in microorganisms [39]. The GLR addition groups also exhibited reduced nitrogen respiration in faeces, significantly reduced the abundance of animal parasites or symbionts, reduced the abundances of parasites and pathogenic bacteria in the broiler intestine and had a positive impact on the health of broiler chickens.
Experimental setup
All chickens were divided in cages（150cm×60cm×60cm）with three replicates per group (10 chickens /replicate), while fed with three different diets. The broiler chickens were 7-day-old Luhua chickens (purchased from the Baotou farmers' market); Group A was the blank control group (CK) that was fed a basic diet without GLR, and Groups B and C were GLR-supplemented groups, with 10% and 15% GLR added to the basic diet, respectively. GLR was provided by Baotou Traditional Chinese Medicine Co., LTD., Baotou City, Inner Mongolia Ancient Autonomous Region. The compositions of the base diet and nutritional contents are shown in Table 1. The groups were kept in individual cages under natural light. During this time, they were free to consume feed and water. They are provided with good ventilation and regular cleaning were strict compliance with the rearing and management system, and broiler rearing were monitored for daily observation, fecal and mental condition. The experimental period was 90 days.
Sample Collection
At the beginning and end of the experiment, after 12 hours of fasting, the initial and final body weights during the trial were recorded and their average daily weight gain was calculated [40].
At the end of the experiment, after 12 hours of fasting, electroshock and bloodletting were then performed, immediate aseptic dissection was performed. The thymus, spleen and bursa phalloides were separated, fat was removed and fresh organ weights were measured to calculate the immune organ index. The formula that was used to calculate the immune organ index is as follows[41]:
At the end of the experiment, fresh chicken faeces from each replicate cage were collected in sterile Ep tubes, and stored at -80 ° C for subsequent experiments.
DNA Extraction and PCR Amplification
Microbial DNA was extracted from chicken faeces using FastDNA® Spin Kit for Soil according to MP Biomedicals’s protocols, and its concentration and quality were determined by the NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of 16S rRNA gene was amplified with primers 338F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′). PCR product was purified using the AxyPrep DNA Gel Extraction Kit and quantified using Quantus™ Fluorometer PCR System. Purified amplicons were pooled in an equimolar solution. NEXTFLEX Rapid DNA-Seq Kit was used for library construction and Illumina's NovaSeq PE250 platform was used for sequencing.
Bioinformatics Analysis
Using UPARSE software, OTU clustering sequences based on 97% similarity. All the data were calculated by mothur software for Alpha diversity knowledge Chao 1, Shannon index, etc., and the Wilxocon rank sum test was used to analyze the inter-group differences of alpha diversity; The similarity of microbial community structure between samples was tested by PCoA analysis based on bray-curtis distance algorithm, and whether the difference in microbial community structure between sample groups was analyzed in combination with PERMANOVA nonparametric tests. LEfSe analysis was used to identify bacterial taxa with significant differences in abundance from phylum to genus level between different groups.
Statistical Analysis
The experimental measurements were analysed by using SPSS 25.0 software for analysis of variance, one-way ANOVA for ANOVA and the LSD method for multiple comparisons among groups. P<0.05 indicates a significant difference, and the results are expressed as the mean ± standard deviation
The experiment showed that GLR increased the average daily weight gain of broilers and their immune organ index to some extent, and improved the community composition of broiler fecal microorganisms, in addition to enriching the species and abundance of probiotic bacteria, which are closely related to the immune function in the organism. GLR as a roughage mainly influenced the immune function of broilers by improving the population of fecal microorganisms.
Data Availability Statement
Data Availability Statement: All sequencing data are available through the NCBI Sequence Read Archive under the accession number PRJNA861103.
The raw reads of our data have been deposited into the NCBI Short Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra/) under accession number SRP387518.
Acknowledgements
Thanks to the Inner Mongolia University of Science and Technology Environmental Protection and Comprehensive Utilization of Resources Laboratory and Huang Yiqun for their help.
Author Contributions
Conceptualization, methodology, designed the experiments, C.S.; data curation, analysis, L.G., H.Y.; supervision, investigation and methodology, X.L., P.D., and J.B.; material preparation, supervision, investigation and methodology, Y.W.
Consent for publication
Consent.
Funding
This study was funded by the Science and Technology Program of Inner Mongolia Autonomous Region (Grant No. 2020GG0071).
Ethics approval and consent to participant
This study was approved by the Ethics Committee of Inner Mongolia University of Science and Technology. All schemes and sample collection of all animal experiments in this study were conducted in strict accordance with the national standard of the People's Republic of China, General Requirements for Experimental Animals (GB/T 35823-2018), and the Implementation Rules of the Code of Academic Ethics of the Chinese Society for Experimental Animals.
Conflict of Interest Statement
The authors declare that they have no conflicts of interest.
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Table 1 Compositions and nutrient levels of basal diets
|
Ingredients |
content (g/kg) |
Nutrient levels |
content |
|
Corn |
680 |
Dry matter (g/kg) |
882 |
|
Soybean meal |
180 |
Crude protein (g/kg) |
170 |
|
Premix |
30 |
Metabolic energy（MJ/kg） |
116 |
|
Rapeseed meal |
40 |
Methionine (g/kg) |
3.1 |
|
Wheat bran |
15 |
Lysine (g/kg) |
6.3 |
|
Powder |
30 |
Ca (g/kg) |
20.6 |
|
CaHPO4 |
20 |
P (g/kg) |
6.2 |
|
NaCl |
5 |
|
|
Note: The premix provides the vitamins and minerals of the basic diet and is the same in all groups. Supplied per kilogram of premix: vitamin A, 15000 IU; vitamin D3, 2000 IU; vitamin E, 5500 IU; vitamin K,0.5 mg; niacin, 0.6 mg; pantothenic acid, 15mg; folic acid, 1.5 mg; Ca, 10.6g; P, 8.8g; Fe, 0.05g; Zn, 0.2g; Mn, 0.03g.
Table 2 Broiler growth performance and immune organ indices
|
Item |
Initial body weight |
Final body weight |
Average daily gain |
|
A |
358.33±13.02a |
2193.33±101.71a |
20.39±1.05a |
|
B |
338.33±7.26a |
2446.67±168.36a |
23.42±1.80a |
|
C |
336.67±6.01a |
2380.00±103.92a |
22.70±1.22a |
|
p-Value |
0.287 |
0.178 |
0.217 |
Note: 1) Group A is the blank control group, fed a basal diet without GLR, while Groups B and C are GLR-supplemented groups, with 10% and 15% GLR added to the basal diet, respectively. Different lowercase letters in the same row indicate significant differences (P < 0.05), while the same letters indicate nonsignificant differences (P > 0.05). Same table and figure below. 2) The unit for growth performance is g.
Table 3 Broiler immune organ indices
|
Item |
Spleen index |
Thymus index |
Bursa Index |
|
A |
1.42±0.13b |
3.33±0.25a |
1.56±0.11a |
|
B |
2.14±0.11a |
4.13±0.41a |
2.10±0.43a |
|
C |
2.11±0.04ab |
4.02±0.18a |
1.72±0.20a |
|
p-Value |
0.005 |
0.202 |
0.437 |
Note: immune organ Index = [organ weight (g)/body weight (g)] × 1,000.
No competing interests reported.