DOI: https://doi.org/10.21203/rs.3.rs-1949857/v3
Context.Our previous study has revealed that micronized bamboo powder (MBP) can promote the growth performance of broilers aged 1-22 days (d) by improving oxidation resistance, balancing intestinal microflora and regulating metabolic pathways.
Aims. This study further evaluates the effects of MBP on the growth performance, intestinal development, cecal chyme microflora and metabolic pathway of broilers aged 24-45 d.
Methods.In this experiment, eight hundred and eighty (880) slow-growing spotted-brown broilers aged 22 d were pre-fed for 2 d, and then randomly divided into two groups according to weight and gender. There were 8 replicates in each group and 55 broilers (23 female and 22 male) in each replicate. The trial lasted for 21 d. The broilers in the control group were fed with an antibiotic-free basal diet (Group CON), and the broilers in the experimental group were supplemented with 1% MBP based on the CON diet to replace 1% corn (Group MBP).
Key results. For the growth performance, during 24-45 d, no significant difference was observed between Group MBP and Group CON, in average daily gain, average daily feed intake, and the weight gain and feed consumption ratio (P > 0.05). For intestinal development, the broilers in Group MBP exhibited a significantly higher organ index of the cecum, jejunum villus height, and ratio of villi to crypt, compared to Group CON (P < 0.05). For the cecal chyme microflora, the abundance ratio of Firmicutes was higher, while the abundance ratio of Bacterodies was relatively lower than that of Group CON. The addition of MBP significantly up-regulated the abundance of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus, f_Enterococcasea, whiledown-regulating the abundance of p_Bacteroidetes, f_Bacteroidaceae, g_Bacteroides, o_Bacteroidales and c_Bacteroidia (P < 0.05). For the metabolic pathways, 66 different pathways were observed between Group MBP and Group CON, including alanine, aspartic acid and glutamic acid metabolism, butyric acid metabolism, arginine synthesis, linoleic acid metabolism and β-alanine metabolism. The correlation analysis revealed that Firmicutesin cecal chyme were significantly positively correlated with some fatty acids, including syringic acid, 3-methyl-2-oxovaleric acid, 3-(2-hydroxyphenyl) propanoic acid, and butyric acid (P < 0.05). The Bacterodieswere positively correlated with some amino acids, including L-Alanine, L-Threonine, 3-Methylthiopropionic acid and L-Glutamic acid (P < 0.05). MBP might be beneficial forcertain fatty acid metabolismand harmful for certain amino acid metabolism by regulating microflora.
Conclusions. Taken together, adding 1% MBP to replace corn equivalently has no negative effect on the growth performance of broilers. This may be related to the fact that MBP can improve intestinal development, and increase the content of bacteria that promote fatty acid metabolism and fiber degradation.
Implications.MBP can be used as beneficial fiber for broilers. It is necessary to further study the appropriate addition level or alternative of MBP in diet of broilers.
Recently, the supply shortage of corn, soybean and other feed raw materials has become a key factor for restricting the sustainable and efficient development of the poultry industry. Feed manufacturers have to consider other unconventional feed resources (Makkar 2018). Since the policies of “Prohibition of growth-promoting antibiotics” implemented in China, seeking unconventional feed raw materials to improve animal intestinal health and growth performance, and promote feed utilization efficiency, has become one of the most effective measures for “Antibiotics replacement” (Jiménez-Moreno et al. 2019).
Bamboo grows fast with a high yield (He et al. 2014). It has been showed that bamboo shoot shell fiber had prebiotic effects, as well as the potential to reduce cholesterol (Wu et al. 2020). However, bamboo pole fiber was the most productive part of bamboo, and the total fiber value of young bamboo culm (> 60%) was more than the powdered bamboo shoot (approximate 24.44% crude fiber) (Mustafa et al. 2016; Felisberto et al. 2017). Compared with bamboo shoot shells, the bamboo powder for feed after processing bamboo poles contains a growing trend of lignin (26-36%) (Zhu et al. 2020) and higher content of insoluble dietary fiber (IDF), which might reduce its nutritional value. Through micronization, other plant powder such as olive pomace powder had higher content of soluble dietary fiber (SDF) and lower content of lignin (Speroni et al. 2020). Superfine grinding can improve the functional properties of rice bran IDF, including higher water capacity, swelling capacity and nitrite ion adsorption capacity (Zhao et al. 2018). In addition, reducing particle size of wheat increased the digestibility of dry matter and crude protein (Bao et al. 2016), the apparent ileal digestibility of total fiber, total energy and insoluble fiber can also be significantly increased (Zhao et al. 2019). These above reports show that micronized treatment can improve fiber properties and nutritional value. Bamboo powder was used to partially replace alfalfa in the feed of ruminants after fermentation (Okano et al. 2009; Oguri et al. 2013), while few report has been performed on its feeding value for Monogastric animals. Our previous study revealed that adding 1% of micronized bamboo powder (MBP) from 5~6 years bamboo poles to replace equivalent corn can improve the growth performance of weaned piglets, and the improvement effect of super-MBP was higher than that of general-MBP (Dai et al. 2021). Above reports show that bamboo powder can partially replace corn or alfalfa after proper treatment, indicating that bamboo is potential to alleviate the global shortage of feed.
Meanwhile, MBP is expected to be used as a functional dietary fiber raw material for broiler to reduce feed costs and improve breeding efficiency. Recent research on dietary fiber for broilers has focused on the regulation mechanism of the fiber on growth performance (Kheravii et al. 2017). According to the traditional views, substantially elevating the cellulose level may dilute the nutrition of broiler obtained from diets. It may affect the energy digestibility (Jiménez-Moreno et al. 2016) and reduce the organic matter digestibility of broilers (Röhe et al. 2020). However, some studies have proved that supplementation of IDF in a proper manner also exhibited several advantages for promoting the growth of young broilers, such as increasing gizzard weight, lowering gizzard pH (Jiménez-Moreno et al. 2019), balancing beneficial gut bacteria (Pourazadi et al. 2020), improving villus growth in jejunum (Sozcu 2019), and enhancing nutrient digestibility and utilization (Nassar et al. 2019). Owing to the fact that MBP is rich in IDF, it is very interesting to study whether MBP can improve growth performance by regulating intestinal development and microbial flora.
MBP is considered as a promising and high-quality raw material for obtaining pro-biotic insoluble dietary fiber because of its beneficial effect in regulating intestinal microbial flora. Our previous report has found that adding 1% MBP to replace equivalent corn increased the relative abundance of Methanobrevibacter in weaned piglets while decreased the relative abundance of Clostridium (Dai et al. 2021). It has been found that adding 1% MBP to replace equivalent corn in the diet of young broilers at age of 1-22 days (d) could promote growth performance by improving chyme microflora composition, increasing the abundance ratio of Firmicutes, and regulating the metabolism pathways responsible for intestinal fatty acids, amino acids, and immunity (Dai et al. 2022). This study will further evaluate whether the MBP has similar growth-promoting and pro-biotic effects on the broilers of the middle growth period (aged 24-45 d). The study will focuse on intestinal tissue development, microflora balance and metabolic pathways, to provide reference for the scientific applications of MBP.
The preparation of MBP
The bamboo poles of Phyllostachys pubescens (from Sichuan Province, China) aged 5 ~ 6 years were collected and processed into MBP with a previous reported method (Dai et al. 2022). The contents of moisture, crude protein (CP), and ash were tested according to AOAC method (AOAC 2007), which were 7.83%, 1.51%, and 1.01%, respectively. The contents of IDF, SDF and total fiber (TF) were determined according to AOAC 991.43, which were 82.33%, 3.30% and 85.63%, respectively. The contents of acid detergent fiber (ADF) and neutral detergent fiber (NDF) were determined with the reference method (Van Soest et al. 1991), which were 74.80% and 82.84%, respectively.
Experimental animals and groups
Eight hundred and eight (880) slow-growing spotted-brown broilers aged 22 d were pre-fed with the same diet (mixed diet with half of treatment and control) for 2 d, and then randomly classified as two groups according to weight and gender. There were 8 replicates in each group, with 55 broilers (23 female and 22 male) in each replicate. The broilers in the control group were fed with an antibiotic-free basal diet (Group CON), the broilers in the experimental group were supplemented with 1% MBP to replace equivalent corn (Group MBP) based on antibiotic-free basal diet. The broilers were housed in floor pens littered with rice hull and fed in a routine procedure. The experiment lasted for 21 d, corresponding to broilers aged 24-45 d.
Diet ingredients and nutrition level
The ingredients and nutrition level of the diet in this study were prepared according to the standard (NY/T3645-2020 Nutrient requirements of yellow chickens). The composition and nutrition level were shown in Table 1. In the diet of Group CON, the same amount of MBP (1%) was used to replace equivalent of corn. The value of metabolic energy (ME) in Group CON and Group MBP was the same, according to the assumption that the energy value of MBP was equivalent to that of corn under the condition of appropriate addition.
Slaughtering and sampling
On the 45 d, two broilers (one male and one female) in each replicate, who had been fasting for 12 h, were randomly selected and sacrificed by cervical dislocation. After euthanasia, the broilers were dissected for collecting internal organs, including heart, liver, spleen, bursa of fabricius, gizzard, and glandular stomach. The chyme in the caecum was put in a 1.5 mL sterile centrifuge tube, frozen with liquid nitrogen, and stored at -80 ℃ for further tests. Intestinal tissues with a length of 0.5 cm were collected from the duodenum, jejunum, and ileum, whose chyme was washed with PBS. These tissues were then fixed with 4% formalin for the following assays.
Indicators and determination methods
Growth performance: At the beginning and end of the experiment, the broilers in one replicate were weighed together to obtain the average initial weight (IW) and final weight (FW). The feed intake was recorded every day for calculating the average daily feed intake (ADFI). The average daily gain (ADG) was calculated as difference between IW and FW, and the ratio of gain to feed consumption (G: F) was calculated as ADG/ADFI.
Organ index: The contents in the gizzard and glandular stomach were removed. The tissues were rinsed with PBS and dried with filter paper for absorbing water. Other tissues and fat were removed for weighing. The duodenum, jejunum, ileum, colorectum, and caecum were separated. The length of each intestinal tract was measured to the accuracy of 0.1 cm. The calculation formula for Organ index (%) = Weight of organ/Live broiler weight * 100%. Intestinal organ index (cm/g) = Length of intestine/Live broiler weight.
HE staining of intestinal tissue sections: The duodenum, jejunum, and ileum of broilers were embedded in paraffin for preparing sections. Samples were cut at a thickness of 5 μm and stained with hematoxylin and eosin. The values of villus height (VH) and crypt depth (CD) were observed with optical microscopy (Leica Microsystems, Wetzlar, German). Twenty spots of villus and crypt from the intestinal samples were measured and calculated for the ratio of the villus height to the crypt depth (VH:CD).
Cecal chyme microflora analysis: The cecal chyme microflora analysis was performed according to our previous report (Dai et al. 2022). The genome DNA of cecal chyme microflora was extracted and the V3-V4 gene region of 16S rRNA was amplified with PCR. The amplification products were purified and the 16S rRNA was sequenced with Illumina MiSeq platform at the BioNovoGene Co., Ltd. (Suzhou, China). The raw reads were deposited in the European Nucleotide Archive database (Accession Number: ERP131138).
The raw sequence data generated from 16S rRNA Miseq sequencing was processed according to the analysis flow of QIIME 2 (2019.4) DADA2 method, and sequence denoising was performed. The low-quality sequences were filtered through following criteria: sequences that had a length of <150 bp, sequences that had average Phred scores of <20, sequences that had contained ambiguous bases, and sequences that contained mononucleotide repats of >8 bp. Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using Vsearch software (Rognes et al. 2016). Greengenes database, Silva database, UNITE database, and nt database were involved in the analysis. The microbial species were annotated with QIIME2 classify-sklearn algorithm and BROCC algorithm.
For the analysis of 16S rRNA, gene sequencing data were normalized by copy number. Phylum and genus at <1.0% relative abundance for both groups were excluded from all analyses. Alpha diversity analyses were performed using MOTHUR. Community richness was identified using the Chao1, Observed_species and Faith_pd. Community diversity was identified using the Shannon, Simpson and Pielou_e. Coverage estimator was characterized by Good’s coverage.
The cecal chyme metabolomics analysis: 100 mg of sample was accurately weighed, and 0.6 mL of 2-chlorophenylalanine in methanol (4 ppm, -20 ℃) was added and mixed for 30 s. After adding 100 mg of glass beads, the solution was ground for 90 s at 60 Hz in an issue grinder, and then sonicated for 10 min at room temperature. After centrifuging at 12000 rpm for 10 min, 300 μL of supernatant was filtered by a 0.22 μm membrane. The filtrate was stored for the following detection. 20 µL of sample was taken for mixing with quality control (QC) samples. The remaining samples were applied for Liquid Chromatograph-Mass Spectrometer (LC-MS) detection. The LC was performed with Thermo Ultimate 3000 and the ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) column, and MS analysis was performed with Thermo Q Exactive HF-X, electrospray ion source (ESI), positive and negative ion ionization modes. The detection conditions were set according to our previous report (Dai et al. 2022).
LC-MS data processing and differential metabolite identification: The raw data were collected and converted into mzXML format by Proteowizard software (v3.0.8789). XCMS package of R(v3.3.2) was applied for peak identification, peak filtration, and peak alignment. The information including mass to charge ratio (m/z), retention time, and peak intensity was obtained.
To find biomarkers, the relative standard deviation (RSD) of potential characteristic peaks in QC samples should be less than 30%. The characteristic peak with RSD < 30% in this study was 77.1% in positive ion mode and 81.5% in negative ion mode, indicating that the data was good.
The accurate molecular weight of the metabolite (error < 30 ppm) was obtained. Then, the fragment information obtained according to the MS/MS mode was retrieved in the Human Metabolome Database (HMDB), Metlin, Massbank, LipidMaps, and mzclound. The information was matched and annotated to obtain accurate information of metabolites.
Data processing and statistical analysis
SPSS 25.0 was applied for statistical analysis. Independent Sample t-test was applied for analyzing the differences in growth performance, organ indexes, intestinal histology, volatile fatty acids, and microflora diversity index. P < 0.05 indicated that the difference was significant.
The microbial differential species markers in cecal chyme were analyzed with Linear discriminant analysis (LDA) Effect size, combined with nonparametric Kruskal-Wallis and Wilcoxon rank sum test. The differential metabolites were screened according to the following conditions: Mann-Whitney-Wilcoxon Test was applied for statistical comparison between the two groups, and the screening condition was Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), with first principal component variable importance value projection (VIP) > 1 and P < 0.5; Kruskal-Walis Test was applied for the statistical comparison among multiple groups, and the screening condition was VIP > 1 and P < 0.5. The correlation between microorganisms and metabolites at different phyla and corresponding P values were calculated with Spearman correlation analysis. P < 0.05 indicated a significant difference.
Effects of MBP on growth performance of broilers
The effects of adding 1% MBP on the growth performance of broilers aged 24-45 d are shown in Table 2. No significant difference was observed in ADG, AFDI, and G: F between Group MBP and Group CON. It indicated that adding 1% MBP to replace corn had no negative effect on the growth performance of broilers aged 24-45 d.
Effects of MBP on organic indexes of broilers
At the end of the experiment, the broilers were slaughtered for determining the effects of adding 1% MBP on the organ indexes of broilers. The results are shown in Figure 1. No significant difference was found in the organ indexes of the heart, liver, spleen, bursa of fabricius, gizzard, glandular stomach, duodenum, jejunum, ileum, and colorectum between Group MBP and Group CON. The organ index of the caecum in Group MBP was significantly higher than that in Group CON (P < 0.05), with an increase of about 15.79%. This indicated that adding 1% MBP could promote the development of caecum.
Effects of MBP on intestinal histology of broilers
At the end of the experiment, the effects of MBP on intestinal histology of the duodenum, jejunum, and ileum of broilers were determined (Figure 2). For the broilers in Group MBP, the villus height (VH) of jejunum and the ratio of villus height to crypt depth (VH:CD) were significantly higher than those of Group CON (P < 0.05), which were increased by 29.66% and 28.77%, respectively. No significant difference was found in VH, crypt depth (CD) and ratio of VH:CD in duodenum and ileum between Group MBP and Group CON.
Effects of MBP on the diversity and abundance of microflora in cecal chyme
The effects of MBP on alpha-diversity and abundance of microflora in cecal chyme of broilers were determined with PCR amplification and Illumina Miseq high-throughput sequencing performed on the bacterial 16S rRNA V3-V4 region. There were 15,630 and 15,464 OTUs in Group CON and Group MBP, respectively. The two groups shared 5,100 OTUs, accounting for 19.62% of the total OTUs. The number of OTUs in Group MBP was less than that of Group CON, with 10,364 unique OTUs, accounting for 39.87% of the total OTUs. Bacterial diversity estimators (Shannon, Simpson and Pielou_e), richness estimators (Chao1, Observed_species and Faith_pd), and coverage estimator (Good's coverage) are shown in Table 3. No significant difference was found in all the indexes of Shannon, Simpson, Pielou_e, Chao1, Observed_species, Faith_pd and Good's coverage between Group MBP and Group CON.
Combined with the results of data alignment in the database and OTU species classification, the top 20 ones in cecal chyme microbiota composition was plotted in different groups at the level of phylum and genus (Figure 3). It can be seen that, in both Group MBP and Group CON, the dominant microflora at the phylum level were Firmicutes, Bacteroidetes, Proteobacteria, etc. At the genus level, Bacterodies, Megamonas and Faecalibacterium were the dominant bacteria. However, the abundance of microflora was different between the two groups. In Group MBP, the abundance ratio of Firmicutes was higher than that of Group CON while the abundance ratio of Bacterodies was lower at the phylum level. MBP reduced the abundance ratio of Bacterodies at the genus level.
The bacteria with significant differences between Group MBP and Group CON were analyzed by LEfSe (LDA Effect Size), of which the marker species shared in both groups were selected (Figure 4). Adding 1% MBP to broiler diet significantly up-regulated the expression abundances of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus and f_Enterococcaseae (P<0.05), and it significantly down-regulated the expression abundances of p_Bacteroidetes, f_Bacteroidaceae, g_Bacteroides, o_Bacteroidales and c_Bacteroidia in cecal chyme of broilers (P < 0.05).
Effects of MBP on chyme metabolic pathways in broilers
The typical chromatograms of metabolites in the broiler cecum of different groups were analyzed with the LC-MS platform. Partial least squares discriminant analysis (PLS-DA) and OPLS-DA were performed on the detected metabolites (Figure 5). The total ion intensity of metabolites in cecal chyme of broilers from the two groups showed great variations. Both the species and concentrations of metabolites in Group MBP were greatly changed, without obvious overlap with Group CON. The significant difference in chyme metabolites between the two groups indicated the obvious regulatory effects of MBP on cecal chyme metabolites of broilers aged 24-45 d.
The differential metabolites were screened with the conditions of VIP > 1 and P < 0.05, since the typical chromatograms of metabolites obtained with the LC-MS platform involved a lot of small molecule substances, and the spectra could not be directly identified and analyzed. Then, the metabolic pathways of different metabolites were analyzed with MetPA database (Figure 6). It can be seen that, compared with Group CON, there were 1,635 up-regulated and 837 down-regulated primary metabolites in Group MBP. A total of 66 different metabolic pathways were identified between Group MBP and Group CON, mainly including alanine, aspartic acid and glutamic acid metabolism, butyric acid metabolism, arginine synthesis, linoleic acid metabolism, and β-Alanine metabolism. It indicated that MBP played a significant role in regulating the metabolism of certain amino acids and fatty acids in broilers aged 24-45 d.
Correlation between cecal microflora at phylum level and metabolites in broilers
The correlation between microflora at phylum level and metabolites in cecal chyme of broilers was analyzed (Figure 7). The results showed that Firmicutes were positively correlated with syringic acid, 3-Methyl-2-oxovaleric acid, 3-(2-Hydroxyphenyl) propanoic acid, and butyric acid (P < 0.05), while negatively correlated with 3-Methylthiopropionic acid, L-Alanine, γ-Aminobutyric acid, L-Glutamic acid and Ornithine (P < 0.05). Bacterodies were positively correlated with L-Alanine, L-Threonine, L-Threone, 3- Methylthiopropionic acid, and L-Glutamic acid, etc. (P < 0.05), while negatively correlated with butyric acid, 3-Methyl-2-oxovaleric acid, 3-(2-Hydroxyphenyl) propanoic acid, syringic acid, etc. (P < 0.05).
Effects of MBP on growth performance
The previous studies with broilers have proved that the supplementation of insoluble fiber can promote growth of broilers (Jiménez-Moreno et al. 2016; Jiménez-Moreno et al. 2019; Nassar et al. 2019). The effects of fiber on the growth performance of broilers have depended on several factors, including the fiber resources (Tejeda and Kim 2020; Pourazadi et al. 2020), addition level (Tejeda and Kim 2021), particle size (Tejeda and Kim 2021) and feeding stage (Berrocoso et al. 2020). In our study, it was found that the regulatory effect of MBP on the growth performance of broilers aged 24-45 d was not as good as that of young broilers aged 1-22 d (Dai et al. 2022). There was no significant effect on ADG, ADFI, and G: F, which may be related to the application stage of MBP. However, adding 1% MBP to replace an equivalent amount of corn had no negative effect on the performance of broilers in the middle growth stage. It indicated that MBP might partially replace the corn in the feed of broilers, which supported the assumption that the ME of the diets in two groups was similar.
Effects of MBP on the development of the gastrointestinal tract
Intestinal structural integrity can reflect the digestive ability of animals. As important indicators for intestinal structure integrity, the villus height and the crypt depth of the small intestine can reflect the absorption capacity of the intestinal mucosa (Li et al. 2016) and the renewal rate of intestinal epithelial cells, respectively (Wang et al. 2021). Several studies have reported that adding insoluble fibers to the diet can promote the gastrointestinal development of broilers, and then improve the digestion and absorption of nutrients, as well as the ability to repair intestinal damage. The application of 0.5-2 kg/T processed lignocellulose can promote the development of jejunum villus in broilers (Sozcu 2019). It has been showed that 1% IDF (Arbocel RC) can improve digestive organ weights and enzyme activities in pullets, which may contribute to an improvement in feed utilization (Yokhana et al. 2016). Adding 4% soybean hull to the corn-soybean meal diet can increase the villus height in the duodenum and jejunum of broilers, thus improving the amino acid digestibility (Tejeda et al. 2020). Supplementing 3% wheat bran in the diet of broilers was beneficial to promote gizzard development, intestinal digestive enzyme activities and morphology of broilers (Shang et al. 2020).
Bamboo powder is rich in insoluble fiber, with a content of approximately 62.54%-89.79% (Felisberto et al., 2017). MBP is a concentrated fiber raw material. As is reported above, the addition level of conventional fiber raw materials is higher than that of processing concentrated fiber raw materials due to the lower content of IDF, which may affect the nutrient level of the formula. Similarly, our study has found that adding only 1% MBP to the diet significantly increased the villus height of jejunum, the ratio of villus height to crypt depth, and the index of digestive organs (caecum). This might be related to the physicochemical properties of IDF, another study found that the consumption of Tea-IDF significantly promoted defecation in slow transit intestinal dyskinesia mice and enhanced the production of short chain fatty acids (Bai et al. 2022). MBP may similarly improve the digestion and absorption ability of broilers through promoting the intestinal development, which may be one of the reasons why the growth performance was not reduced after replacing corn with equivalent MBP (1%).
Effects of MBP on cecal chyme microflora
The caecum was the main organ for the degradation and fermentation of microbial fibers in layer chickens (Sun et al. 2021). The cecal microbial community played an essential role in the nutrition metabolism and immunity system of the host (Ji et al. 2019). Our study found that the microflora of broilers cecal chyme exhibited typical features at the phylum level and genus level. At the phylum level, the main bacteria were Firmicutes, followed by Bacteroidetes and Proteobacteria, while at the genus level, the dominant bacteria were Bacteroides, followed by Megamonas and Faecalibacterium. The results were consistent with a previous study, which reported that the Firmicutes ranked first among the dominant microorganisms in the caecum of broilers, followed by Proteobacteria and bacteroidetes (Sergeant et al. 2014). It indicated that MBP at 1% might not break the structural balance of major cecal microflora of broilers, instead, it was more likely to adjust the proportion of the microbial community.
Firmicutes specialized in fiber degradation (Sun et al. 2021), whose cellulase and cellulosome can degrade dietary fibers into monosaccharides that could be used as energy source (Artzi et al. 2017). This study revealed that adding 1% MBP had no significant effect on the diversity of cecal microorganisms, but the abundance proportion of Firmicutes in cecal chyme was increased, leading to the lower abundance proportion of other bacteria such as Bacterodies. Specifically, MBP can significantly up-regulate the abundance of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus, and f_Enterococcasea, all of which were closely related to fiber degradation (Suen et al. 2011; Leth et al. 2018). Another study also found that adding lignocellulose increased the relative abundance of cellulose-degrading Prevotellaceae_UCG-001 and Alloprevotella, without improving the diversity of microorganisms (Sun et al. 2021). The sequencing studies for various IDF and SDF showed broader effects with more and different types of gut microbial species, which may be due to the type and dose of fibers (Swanson et al. 2020). It indicated that the addition of MBP may improve fiber metabolism by regulating the microflora composition, and MBP was expected to be a high-quality raw material for providing probiotic lignocellulose.
Effects of MBP on metabolic pathways of cecum chyme
There is a symbiotic relationship between microorganisms and their host. Intestinal microorganisms participate in the metabolism of nutrients, and the microbial structure is directly related to its metabolites. Through principal component analysis, this study found that the composition of metabolites in cecum chyme had been considerably changed due to MBP. The results showed that MBP significantly changed the composition of cecal metabolites with partially regulating the amino acid and fatty acid metabolism of broilers. Another study also found that dietary fiber exhibited an improvement in gut microbiota and short-chain fatty acids (SCFAs), and improved hepatic and serum fatty acid composition (Zhai et al. 2018).
SCFAs were produced by intestinal microorganisms through the fermentation of undigested carbohydrates (Li et al. 2018). The caecum was the main organ for producing SCFAs, with the highest abundance of SCFAs, which improved villous height (VH), crypt depth (CD), and their ratio of VH:CD in broilers (Liao et al. 2020). Butyrate can inhibit the inflammation of the large intestine and keep intestinal health (Koh et al. 2016). The addition of rice bran from specific rice cultivars in the feed of broilers resulted in an increased number of metabolites associated with fatty acid metabolism (Rubinelli et al. 2017). Another study found that adding 10% dietary lignocellulose significantly lowered cecal counts of Escherichia/Hafnia/Shigella and reduced the total concentration of SCFAs when compared with the levels of 0.8% and 5%, while there was no significant difference between group 0.8% and 5% (Röhe et al. 2020). These above reports indicated that addition of fiber raw materials mainly with IDF may be closely associated with the regulation of intestinal microflora composition and fatty acid metabolism. Similarly, MBP is rich in IDF, and this conclusion has also been confirmed in this study. The addition of 1% MBP increased the abundance ratio of Firmicutes in the cecal chyme of broilers. Firmicutes were positively correlated with Syringic acid, 3-Methyl-2-oxovalenic acid, 3-(2-Hydroxyphenyl) propanoic acid, Butyric acid, etc. It indicated that MBP can improve the metabolism of fatty acids by regulating the microflora, and then promote the development of caecum and intestinal health.
Besides of fatty acids, the intestinal microflora is also involved in the digestion, absorption, metabolism, and transformation of protein in the gastrointestinal tract. The gut microbiota could synthesize several nutritionally essential amino acids de novo, which was a potential regulatory factor in amino acid homeostasis, such as bacteria of the genera Fusobacterium, Bacteroides, and Veillonella (Lin et al. 2017). As a dietary energy and fiber source, the prime hulls at 9% could increase the population of certain bacteria in the genera Clostridium and Oscillospira, and decrease ileal dry matter and ileal nitrogen digestibility of broilers (Wang et al. 2021). Another study revealed that the high-fiber diet increased the relative abundance of Clostridiaceae_Clostridium and Coprococcus and reduced apparent total tract digestibility of crude protein and 18 amino acids in finishing pigs (Hu et al. 2021). The above research showed that there were correlations between dietary fiber, amino acid metabolism, and microflora. This study analyzed the differential metabolites and found that the MBP was involved in the amino acid metabolism in cecum chyme of broilers aged 24-45 d. Specifically, adding 1% MBP significantly reduced the abundance of Bacteroides in chyme, while Bacterodies were positively correlated with the production of L-Alanine, L-Threonine, 3-Methylthiopropionic acid, L-Glutamic acid, etc. It indicated that MBP had negative effects on amino acid metabolism with the decreased proportion of Bacteroides, and might limit the improvement of growth performance. Further study is still required to explore whether adding MBP would affect the digestion and utilization rate of related amino acids in broilers of the middle growth stage.
This study demonstrated that adding 1% MBP to replace corn in the diet of broilers aged 24-45 d had no significant effect on the growth performance. However, the organ index of the caecum, the height of jejunal villus, and the abundance of cellulolytic bacteria in cecal chyme have been significantly improved. MBP significantly changed metabolite composition of cecum chyme, and regulated the metabolism of certain amino acids and fatty acids with the abundance of Firmicutes and Bacteroides. The above results have indicated that MBP is expected to become a probiotic lignocellulose suitable for broilers. Further research will more focus on the optimal amount of corn that can be replaced with MBP to alleviate the shortage of feed.
Funding
This work was supported by the key Research Program (NO.2021YFN0106) and the Educational Reform Project (JG2021-1250) of Sichuan Province, and the Research Start-up Foundation of Leshan Normal University (No. RC202004).
Conflicts of interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. All results were strictly in accordance with the experimental data.
Ethics approval
All animal care and handling were approved by the Ethics Committee for Animal Experimentation, science and technology department, Leshan Normal University, Sichuan, China. The protocols were supported by the regulations for animal experiments established by the Ministry of Science and Technology in China (2014).
Consent to participate
We declare that all authors confirm the content in the paper and participate in the relevant research work.
Consent for publication
We declare that all authors agree to submit the manuscript and pay for the publication if it is accepted.
Availability of data and material
The data that support this study will be shared upon reasonable request to the corresponding author.
Code availability
The software SPSS25.0, QIIME2(2019.4), Greengenes database, Silva database, UNITE database, and nt database in the current study were open to researchers around the world.
Author contributions
Fawen Dai initiated the idea, finished the experiment design. Fawen Dai, Tao Lin, Xia Huang and Yaojun Yang conducted the animal trial and laboratory index determination. Tao Lin and Fawen Dai prepared the initial manuscript in English. Xiang Nong, Jianjun Zuo and Dingyuan Feng made the final revision. All authors read and approved the final manuscript.
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