Micronized Bamboo Powder Affects the Growth Performance, Intestinal Development, Cecal Chyme Microflora and Metabolic Pathway of Broilers aged 24-45 days

doi:10.21203/rs.3.rs-1949857/v1

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Micronized Bamboo Powder Affects the Growth Performance, Intestinal Development, Cecal Chyme Microflora and Metabolic Pathway of Broilers aged 24-45 days

https://doi.org/10.21203/rs.3.rs-1949857/v1

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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. This study further evaluates the effects of MBP on broilers aged 24-45 d. In this experiment, eight hundred and eighty (880) slow-growing spotted-brown broilers aged 22 d were pre-fed for 2 days, and then randomly divided into two groups according to weight and gender. There were 8 replicates in each group and 55 broilers in each replicate. The trial lasted for 21 d. The broilers in the control group were fed with an antibiotic-free basal diet (denoted as Group D), and the broilers in the experimental group were supplemented with 1% MBP based on an antibiotic-free basal diet (denoted as Group F). The results reveal that, for the growth performance, during 24-45 d, no significant difference is observed between Group F and the control group, 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 F exhibit a significantly higher organ index of the cecum, jejunum villus height, and ratio of villi to crypt, compared to the control group (P<0.05). For the cecal chyme microflora, the abundance ratio of Firmicutes is higher, while the abundance ratio of Bacterodies is relatively lower than that of the control group. The addition of MBP significantly up-regulates the expression abundance of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus, f_Enterococcasea, while down-regulates the expression abundance of p_Bacteroidetes, f_Bacteroidaceae, g_Bacteroides, o_Bacteroidales and c_Bacteroidia (P<0.05). For the metabolic pathways, 66 different pathways are observed between Group F and control group, including Alanine, aspartic acid and glutamic acid metabolism, Butyric acid metabolism, Arginine synthesis, Linoleic acid metabolism and β-alanine metabolism. The correlation analysis reveals that Firmicutesin cecal chyme are 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). And the Bacterodiesare positively correlated with some amino acids, including L-Alanine, L-Threonine, 3-Methylthiopropionic acid and L-Glutamic acid (P<0.05). To sum up, 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. It is necessary to further study the effects of MBP on amino acid metabolism and broiler growth by regulating the microflora structure in the chyme.

broiler

micronized bamboo powder (MBP)

growth performance

intestinal development

intestinal microflora

metabolic pathway

Recently, the supply shortage of corn, soybean and other feed raw materials has become a key factor for restricting the green and efficient development of the poultry industry. Feed manufacturers have to consider other unconventional feed resources (Makkar H P S, 2017). Since the policies of “Prohibition of growth-promoting antibiotics” implemented in China, seeking for unconventional feed raw materials to improve animal intestinal health and production performance, and promote feed utilization efficiency, has become one of the most effective measures for “Antibiotics replacement” (Jimenez-Moreno et al, 2019).

Bamboo grows fast with a high yield (Huang Y X et al, 2019; He et al, 2014; Lancefield et al, 2017). The content of insoluble dietary fiber (IDF) in bamboo accounted for 62.54% ~ 89.79%, which was rich in flavonoids, polysaccharides, etc. (Felisberto et al, 2017). It provided several functions, including the regulation of animal immunity (Ge Q et al, 2020), the applications as antiseptic, antibacterial, and antioxidant (YU Y et al., 2019), and the improvement of product quality (Nirmala C et al, 2018). There have been studies on the nutritional and functional values of bamboo shoot shell fiber (Wu W et al,2020; Luo et al,2017) and bamboo leaf extract (Li Yi et al, 2021). However, bamboo pole fiber is the most productive part of bamboo, while few report has been performed on its nutritional value. Bamboo shoot shell fiber can be used in the feed of pigs (Chen T et al, 2020), sheep (Okano K et al, 2009; Oguri M et al, 2013) and cows (Li Yi et al, 2021). It was found that supplementation of 2–3 g/kg bamboo leaf extract can significantly improve the average daily gain, gain to feed ratio, breast muscle tenderness, water-holding capacity, pH value and antioxidant capacity of broilers aged 22–42 d (Shen et al., 2019). Compared with the extracts of bamboo shoot shells and leaves, the bamboo powder for feed after processing bamboo poles contains a certain content of lignin. Through ultra-micro-processing, some insoluble fibers in bamboo powder fibers may be converted into soluble fibers, presented with a reduced lignin ratio. The water-holding capacity and fat adsorption of bamboo powder were increased, resulting in improved feeding value (Speroni C S et al, 2020). Reducing the particle size can improve water holding capacity, swelling capacity, phenol extraction rate, and antioxidant performance (Zhao G et al, 2018). In addition, in vitro digestibility of dry matter and crude protein (Bao Z et al, 2016), the apparent ileal digestibility of total fiber, total energy and insoluble fiber can also be significantly increased (Zhao J B et al, 2019). Our previous study revealed that adding 1% of micronized bamboo powder (MBP) to replace equivalent corn can improve the growth performance of young broilers (Dai et al, 2022) and weaned piglets (Dai et al, 2021). It is expected to be used as a functional dietary fiber raw material to reduce feed costs and improve breeding efficiency.

Recent research on dietary fiber for broilers has focused on the regulation mechanism of fiber on growth performance (Kheravii et al., 2017; Zhou et al., 2018; Makivic et al., 2019; Zeitz et al., 2019). 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 (Jimenez-Moreno et al. 2016) and production performance (Janssen and Carré, 1985), as well as reducing the organic matter digestibility of broilers (Farias-Kovac et al, 2020; Rohe et al, 2020). However, some studies have proved that supplementation of insoluble dietary fiber in a proper manner has also exhibited several advantages for promoting the growth of young broilers, such as stimulating gizzard development (Donadelli R. A et al 2019), increasing gizzard weight in young broilers, lowering gizzard pH (Jimenez-Moreno et al, 2019), balancing beneficial gut bacteria (Stanley et al., 2012), improving the activity of digestive enzymes (Sozcu A, 2019; Mateos et al., 2012), reducing abdominal fat deposition (Okrathok and Khempaka, 2020), and enhancing nutrient digestibility and utilization (Nassar et al., 2019). In short, the growth performance of chicken can be promoted (Donadelli R. A et al 2019).

The fiber source, particle size and supplementation level (Pourazadi et al, 2020; Jimenez-Moreno et al, 2016) have played key roles in regulating the fiber digestibility in the posterior intestinal segment (Zhao J B et al, 2019) and improving the growth of broilers (Tejeda and Kim, 2020). However, at present, the research on dietary fiber for broilers have mainly focused on the young broilers, while a few study has been performed on the application of fibers in broilers of later stages. There is also few report on exploring the function mechanism of dietary fiber, from the perspective of the intestinal flora structure of broilers.

Our previous study found that adding MBP to the diet of young broilers could increase the proportion of Firmicutes in cecal chyme, with a significantly higher abundance of Collinsella. Some differential metabolic pathways were investigated, mainly on amino acids and fatty acids (Dai et al, 2022). MBP is considered a promising and high-quality raw material for obtaining pro-biotic insoluble fiber. This study will further evaluate whether the MBP has similar growth-promoting effects on the broilers of the middle growth period (aged 24–45 d). The study has focused on intestinal tissue development, microflora balance and metabolic pathways, to provide reference for the scientific applications of insoluble dietary fiber raw materials.

The preparation of MBP

The bamboo poles of Phyllostachys pubescens (from Sichuan Province, China) after growing for 5 ~ 6 years were collected and processed into MBP as a previous reported method (Dai et al., 2022). The particle size distribution of MBP was determined with Laser particle size distribution analyzer (BT-9300ST, Dandong Bettersize Instrument Co., Ltd., China), with D98 of 93.33 μm and D90 of 55.47 μm. The content of moisture, crude protein (CP), and ash were tested according to Wang’s method (Wang et al., 2018), which were 7.49, 1.63, and 1.0, 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 65.80% and 82.94%, respectively.

Experimental animals and groups

Eight hundred and eight (880) slow-growing spotted-brown broilers aged 22 d were pre-fed for 2 days, and then randomly classified as two groups. There were 8 replicates in each group, with 55 broilers in each replicate. The broilers in control group was fed with an antibiotic-free basal diet (denoted as Group D), the broilers in the experimental group were supplemented with 1% MBP to replace equivalent corn (denoted as Group F) based on antibiotic-free basal diet. The chickens were cultured with ground bedding and fed in a routine procedure. The experiment lasted for 21 days, corresponding to broilers aged 24 d to 45 d.

Diet ingredients and nutrition level

The diet in this study was prepared according to previous reports (Dai et al., 2022). 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). In the diet of Group F, the same amount of MBP (1%) was used to replace corn.

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 humanitarian cervical dislocation. The broilers were dissected for collecting internal organs. The chyme in the cecum 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 the end of the experiment, the broilers in one replicate were weighed together to obtain the total weight. The feed intake was recorded every day. The average daily gain (ADG), average daily feed intake (ADFI), the ratio of gain to feed consumption (G:F, calculated as ADG/ADFI), and mortality of broilers were calculated.

Organ index: The broilers were killed for collecting heart, liver, spleen, bursa of fabricius, gizzard, and glandular stomach. The contents in the gizzard and glandular stomach were removed. The tissues were rinsed and dried with filter paper for absorbing water and blood. Other tissues and fat were removed for weighing. The duodenum, jejunum, ileum, and colorectum 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 animal 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 5um and stained with hematoxylin and eosin. The villi height to crypt depth were observed with light microscopy（Leica Microsystems, Wetzlar, German). Twenty spots of villi and crypt from the intestinal samples were measured and calculated for the ratio of the villi height to the crypt depth.

Cecal chyme microflora analysis: The cecal chyme microflora analysis was performed according to our previous report (Dai et al., 2022), after the genome DNA of cecal chyme microflora was extracted and amplified with PCR, the amplification products is purified and sequenced with Illumina MiSeq6000 platform.

The original data were processed according to the analysis flow of QIIME 2 (2019.4) DADA2 method, sequence denoising was performed (Benjamin et al., 2016), and sequence OUT clustering was performed by the analysis flow of 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.

The specie composition was further analyzed. The ASV/OUT table was flattened by the Rarefaction method, and the flattening depth was set to 95% of the minimum sample sequence. By analyzing and counting the flattened ASV/OUT table, the specific composition of the microbial community in each sample at different classification levels was obtained, as well as the relative abundance composition of different experimental groups at phylum and genus levels.

Alpha and Beta diversity analyses were performed. The α diversity included Chao1 and Observed_species characterized the abundance, Shannon and Simpson characterized the diversity, Faith_pd indicated the diversity of evolution, Pielou’s evenness indicated evenness and Good’s coverage indicated 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 ultrasound 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 sample was taken for mixing with QC samples. The remaining samples were applied for LC-MS detection. The chromatography was performed with Thermo Ultimate 3000 and the ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) column, and mass spectrometry (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 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) first principal component variable importance value projection (VIP) > 1 and P value < 0.5; Kruskal-Walis Test was applied for the statistical comparison among multiple groups, and the screening condition was VIP > 1 and P value < 0.5.

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 value < 0.05 indicated that the difference was significant, and P value < 0.10 indicated a tendency of difference.

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 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 is observed in ADG, AFDI, and G: F between Group F and Group D (P > 0.05). For the broilers feeding with MBP in Group F, the ADG and AFDI are increased by 0.9 g (P > 0.05) and 2 g (P > 0.05), respectively, compared to that of Group D. The G: F is the same. It indicates that adding 1% MBP to replace corn has 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 are slaughtered for determining the effects of adding dietary MBP on the organ indexes of broilers. The results are shown in Figure 1. The organ index of the cecum in Group F is significantly higher than that in Group D (P < 0.05), with an increase of about 15.79%. However, the organ indexes of the heart, liver, spleen, bursa of fabricius, gizzard, glandular stomach, duodenum, jejunum, ileum, and colorectum are not significantly different from those of the control group (P > 0.05). This indicates that adding 1% MBP could promote the development of cecum.

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 are determined (Figure 2). For the broilers in Group F, the villus height of jejunum and the ratio of villi height to crypt depth are significantly higher than those of the control group (P < 0.05), which are increased by 29.66% and 28.77%, respectively. There is no significant difference in villus height, crypt depth, and ratio of villus height to crypt depth in duodenum and ileum between group F and the control group (P > 0.05). The villus height of the duodenum is increased by 8.11%.

Effects of MBP on the diversity and abundance of microflora in cecal chyme

The effects of MBP on diversity and abundance of microflora in cecal chyme of broilers are determined with PCR amplification and Illumina Miseq high-throughput sequencing performed on the bacterial 16SrRNA V3-V4 region. The statistics have been performed on the flattened ASV/OUT table. As can be seen from Figure 3, there are 15,630 and 15,464 OUTs in Group D and Group F, respectively. The two groups share 5,100 OUTs, accounting for 19.62% of the total OUTs. The number of OUTs in Group F is less than that of in Group D, with 10,364 unique OUTs, accounting for 39.87% of the total OUTs.

From the results in Table 3, it can be seen that Chao1, Observed_species, and Faith_pd of broilers in Group F are lower than those in the control group, while the difference for all indexes is not significant (P > 0.05). In addition, the Shannon and Simpson, Pielou_e, and Goods_coverage of cecal chyme in group F are not significantly different from those in the control group (P > 0.05).

Combined with the results of data alignment in the database and OUT species classification, the Venn histogram of ASV/OUT abundance and the heat-map of species composition are plotted in different groups at the level of phylum and genus (Figure 4). It can be seen that, in both Group F and Group D, the dominant microflora at the phylum level are Firmicutes, Bacteroidetes, Proteobacteria, etc. At the genus level, Bacterodies, Megamonas, Faecalibacterium, Flavonifractor, Lachnoclostridium, Blautia, Alistipes, Lactobacillus, Desulfovibrio, and Barnesiella are the dominant bacteria in the cecum chyme of broilers in both two groups. However, the abundance of microflora differs between the two groups. In Group F, the abundance ratio of Firmicutes is higher than that of the control group, while the abundance ratio of Bacterodies is lower.

The bacteria with significant differences between Group F and Group D are analyzed by LEfSe, of which the marker species shared in both groups are selected (Figure 5). Adding 1% MBP to broiler diets 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 (P < 0.05).

Effects of MBP on chyme metabolic pathways in broilers

The typical chromatograms of metabolites in the cecum of broilers in different groups are analyzed with the LC-MS platform. Partial least squares discriminant analysis (PLS-DA) and OPLS-DA are performed on the detected metabolites (Figure 6). The total ion intensity of metabolites in the cecum of broilers from the two groups shows great variations. Both the species and concentrations of metabolites in Group F are greatly changed, without obvious overlap with Group D. The significant difference in chyme metabolites between the two groups indicated the obvious regulatory effects of MBP on cecum chyme metabolites of broilers aged 24-45 d.

The typical chromatograms of metabolites obtained with the LC-MS platform involve a lot of small molecule substances, and the spectra cannot be directly identified and analyzed. Thus, the differential metabolites are screened with the conditions of VIP > 1 and P value < 0.05. Then, the metabolic pathways of different metabolites are analyzed with MetPA database (Figure 7). It can be seen that, compared with Group D, there are 1,635 up-regulated and 837 down-regulated primary metabolites in the Group F. A total of 66 different metabolic pathways are identified between Group F and Group D, including mainly Alanine, aspartic acid and glutamic acid metabolism, Butyric acid metabolism, Arginine synthesis, Linoleic acid metabolism, and β-Alanine metabolism. It indicates that MBP plays a significant role in regulating the metabolism of amino acids and fatty acids in broilers aged 24-45d.

Correlation between cecal microflora at phylum level and metabolites in broilers

The correlation between microflora at different phylum levels and metabolites in cecal chyme of broilers is analyzed (Figure 8). The results show that Firmicutes are 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 are 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). It indicates that MBP can promote the metabolism of volatile fatty acids by increasing the proportion of Firmicutes, while the decreased proportion of Bacteroides may have negative effects on amino acid metabolism.

Effects of MBP on growth performance

The broiler is generally applied as a biological model for investigating poultry growth and development (Vainio and Imhof, 1995; International Chicken Genome Sequencing Consortium, 2004). The previous study on broilers has proved that the supplementation of the appropriate amount of insoluble fiber can provide many benefits for the growth performance of young broilers, such as stimulating the development of gizzard (Donadelli R. A et al 2019), increasing the weight and decreasing the pH value in the gizzard (Jimenez-Moreno et al, 2019), promoting the activity of intestinal probiotics and digestive enzymes (Mateos et al., 2012; Sozcu ，2019). Further, it can also reduce abdominal fat deposition without affecting body weight gain (Okrathok and Khempaka, 2020), increase the apparent digestibility of protein and true digestibility of amino acids (Farranmt et al., 2017), and improve the utilization rate of nutrients (Nassar et al, 2019). Thus, the growth of young broilers can be promoted (Donadelli R.A et al., 2019).

The effects of fiber on the growth performance of broilers have depended on several factors, including the fiber resources (INCHAROEN et al, 2013; Tejeda et al, 2020), addition level (Jiménez-Moreno et al., 2011; Makivic et al., 2019; Sozcu, 2019), particle size (Pourazadi et al, 2020; Jiménez-Moreno et al, 2016) and feeding stage (Pourazadi et al, 2020). In our study, it was found that, the regulatory effect of MBP on the production 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. MBP was an insoluble fiber, whose effects on young broilers may be superior to that of broilers in the middle growth stage. The insoluble fiber of MBP accounted for 62.54% ~ 89.79% of the total fiber amount (Felisberto et al., 2017). In addition, MBP was rich in bamboo leaf flavonoids, bamboo leaf polysaccharides, etc., which was able to regulate the immunity and anti-oxidation of animals (Ge Q et al,2020). In this study, adding 1% MBP to replace an equivalent amount of corn had no negative effect on the performance of broilers. It indicated that the functional fibers in MBP may partially replace the corn, which alleviated its effects on a dietary nutritional level.

Effects of MBP on the development of the gastrointestinal tract

Intestinal structural integrity reflects the digestive ability of animals. As important indicators for intestinal structure integrity, the villi height and the crypt depth of the small intestine can reflect the absorption capacity of the intestinal mucosa (Aitla et al., 2019) and the renewal rate of intestinal epithelial cells, respectively (Shang et al.,2018). Several studies have reported that adding 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 lignocellulose can promote the development of jejunum villi in broilers (Sozcu, 2019). Adding 4% soybean hull to the corn-soybean meal diet can increase the villi height in the duodenum and jejunum of broilers, thus improving the amino acid digestibility (Tejeda et al, 2020). Supplementing 3% wheat bran fibers in the diet of broilers was beneficial to promoting gizzard development, maintaining intestinal integrity, and improving the activity of digestive enzymes (Shang et al., 2020). Adding 1 ~ 1.5% cassava pulp modified fibers to the broiler diet can enhance gastrointestinal function, and improve nutrient digestibility, without making a negative effect on broiler growth performance (Okrathok et al., 2020). The addition of 4% sunflower seed shell can help to repair the intestinal epithelial injury of animals caused by a bacterial infection (Drazbo et al., 2018).

In above literature, the reported fiber materials are mainly IDF. IDF existed in the form of sponge in chyme, which was more conducive to fully mixing chyme with various digestive enzymes (Sarikhan et al. 2010), thus improving the digestion and absorption function, as well as promoting the development of digestive organs. Our study found that adding 1% MBP to the diet significantly increased the villi height of jejunum, the ratio of villi height to crypt depth, and the index of digestive organs (cecum). It can improve the digestion and absorption ability, which may be one of the reasons why the production performance was not reduced after replacing corn with equivalent MBP (1%).

Effects of MBP on cecal chyme microflora

The cecum was the main organ for the degradation and fermentation of microbial fibers in 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; NEISHAS, 2009), which can be applied as the biomarker for the prevention and diagnosis of poultry intestinal diseases (Forte et al., Poultry Science, 2018). Our study found that the microflora exhibited characteristic features in the cecal chyme of broilers. At the phylum level, the main bacteria were Firmicutes, followed by Bacteroidetes and Proteobacteria, while at the generic level, the dominant bacteria were Bacteroides, Megamonas, Faecalibacterium, Flavonifractor, Lachnoclostridium, Blautia, Alistipes, Lactobacillus, Desulfovibrio, and Barnesiella. These bacteria may be closely related to intestinal health, nutrient digestion, and metabolism in broilers. The results were consistent with a previous study, which reported that the Firmicutes ranked first among the dominant microorganisms in the cecum of broilers, followed by Proteobacteria and bacteroidetes (Sergeant et al, 2014).

Fibers can provide a carbon source and energy source for intestinal microbial proliferation (Moeller et al., 2017). The supplementation of the appropriate level of fiber can increase the diversity of intestinal microbes in the host (Gomez et al., 2016), and avoid diseases caused by the loss and imbalance of intestinal microflora (Sonnenburg et al., 2016; Larsen et al., 2010). Dietary fiber can improve the microbial community and its metabolite (mainly SCFAs), in the posterior segment of the intestinal tract of poultry, thus enhancing the intestinal microenvironment (Miyamoto et al., 2018), intestinal tissue structure, and function (Clausen and Mortensen, 1995), finally affecting the nutrition, immunity and physiological state of the host (Ussar et al, 2016; Imhann et al, 2018; Rubio, 2019). It was found that the changes in fermentation end-products were related to the microflora composition in the digestive tract (Awad et al., 2016). Firmicutes specialized in fiber degradation (Sun et al., 2021), whose cellulase and cellulosome can degrade dietary fibers into monosaccharides (Artzi et al., 2016). This study also revealed that the addition of 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, the addition of 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). It indicated that the addition of MBP may improve fiber metabolism by regulating the microflora composition, and the 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 was significantly different between Group F and the control group. The results showed that MBP significantly changed the composition of cecal metabolites, thus regulating the amino acid and fatty acid metabolism of broilers. Increasing dietary fiber levels can promote intestinal development, accelerate the emptying rate of chyme, and elevate the content of volatile fatty acids in the posterior segment of the intestine, thus improving the intestinal health of animals (Wenk et al., 2001). The cecum was the main organ for producing SCFAs, with the highest abundance of SCFAs (Liao et al, 2020).

The production of SCFAs was closely related to the composition difference of microorganisms (Macfarlane et al., 2012). SCFAs were produced by intestinal microorganisms through the fermentation of undigested carbohydrates (Li et al., 2018), and can be absorbed and metabolized by epithelial-related cells in the posterior segment of the intestine (DEN BENTEN G et al., 2013). The contents of SCFAs in chicken intestines were significantly negatively correlated with Salmonella, such as isovaleric acid, butyric acid, and acetic acid (Liao et al, 2020). Butyrate can inhibit the inflammation of the large intestine and keep its health (KOH A et al, 2016). It was found that adding chicory extract to the poultry diet can significantly reduce the number of harmful bacteria in the cecum (Schnorr et al., 2014). After adding inulin fiber, the relative abundance of Firmicutes and Actinobacteria was increased, while the relative abundance of Bacteroides was reduced in the intestinal microbial composition (DEWULF E M et al., 2013). The above research indicated that a fiber diet may be closely associated with the regulation of intestinal microflora composition, fatty acid metabolism, and intestinal health. This conclusion has also been confirmed in this study. The addition of 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 adding MBP can promote the metabolism of volatile fatty acids by regulating the microflora, and then improve the cecum development and intestinal health.

Besides fatty acids, the intestinal microflora is also involved in the digestion, absorption, metabolism, and transformation of protein in the gastrointestinal tract. There are two main pathways for intestinal microflora to participate in amino acid metabolism: (1) The resident species of intestinal microflora utilized amino acids produced by food or host as elements for protein synthesis; (2) the protein is transformed or fermented to drive nutrient metabolism (Lin et al., 2017). However, the source, content, and amino acid balance of dietary protein were the main factors that affect the composition, structure, and function of intestinal microorganisms (Zhao et al., 2019). It was found that adding soluble fiber inulin polysaccharide to the diets of dairy cows could alleviate mastitis by improving the amino acid level and energy metabolism, increasing the number of associated symbiotic bacteria producing SCFAs and reducing the pro-inflammatory bacteria and metabolites (Wang et al., 2021). Another study revealed that feeding a high-fiber diet and multi-bacteria fermented feed can improve the diversity and richness of microorganisms in the feces of finishing pigs. High-fiber diet significantly reduced amino acid and fat digestibility of finishing pigs. Multi-bacteria solid-state fermentation significantly increased the relative abundance of fecal Lactobacillus and Faecal cocci, which could also alleviate the negative effects of fiber on nutrient digestion (Hu et al., 2021). The above research showed that there were correlations between 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 24-45d. Specifically, adding 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. Further study is still required to explore whether adding MBP would affect the digestion and utilization rate of related amino acids.

This study demonstrated that, adding 1% MBP to replace corn in the diet of broilers aged 24- 45d had no significant effect on the production performance. In addition, the organ index of the cecum, the height of jejunal villi, and the abundance of cellulolytic bacteria in cecal chyme have been significantly improved. MBP significantly affected the microbial structure and metabolite composition of cecum chyme. Through correlation analysis, it was found that the number of Firmicutes was positively correlated with some volatile fatty acids, and the number of Bacteroides was positively correlated with some amino acids. MBP has made beneficial effects on regulating the intestinal histology and cecal chyme microflora of broilers. In addition, it can affect the related fatty acid and amino acid metabolic pathways. The above results have indicated that MBP is expected to be a kind of probiotic lignocellulose for broilers.

Funding

This work was supported by the key Research Program of Sichuan Province (NO.2021YFN0106) 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 datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

The software SPSS25.0, QIIME2(2019.4), and Greengenes 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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Table 1

Composition of the experimental diet(g/kg)

Ingredients	Grower diets
	Control	1%MBP
Corn	678.4	668.4
Corn gluten meal	20	20
Soybean oil	16	16
Fermented soybean meal	30	30
Soybean meal (CP46%)	169	169
MBP		10
Cottonseed meal (CP46%)	40	40
Premix*	46.6	46.6
Total	1000	1000
Calculated composition
ME (Kcal/kg)	3000	3000
CP (%)	17.7	17.7
CF (%)	2.8	3.2
NDF (%)	9.5	10.3
Lys (%)	1	1
Met (%)	0.45	0.45
Thr (%)	0.73	0.73
Ca (%)	0.95	0.95
AP (%)	0.44	0.44

*Supplied the following per kilogram of diet: vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,500 IU; vitamin E (dl-α-tocopheryl acetate), 40 IU; vitamin K3 (menadione sodium bisulfite), 2 mg; vitamin B1 (thiamine), 2mg; vitamin B2 (riboflavin), 6 mg; vitamin B3 (niacin), 50mg; vitamin B5(calcium d-pantothenate), 12mg; vitamin B6 (pyridoxine hydrochloride), 5mg; vitamin B12 (cyanocobalamin), 0.02mg; biotin, 0.12 mg; folic acid, 1.5mg; choline chloride, 600 mg; Mn(MnSO₄∙H₂O), 80mg; Fe(FeSO₄∙7H₂O), 80 mg; Zn (sulphate and oxide) 60mg; Cu(CuSO₄∙5H₂O), 8 mg; I (iodide), 1mg; Se(Na₂SeO₃), 0.3 mg; Co (cobalt), 0.3mg;NaCl, 3g; antioxidant, 40mg.

Table 2

Effects of MBP on growth performance of broilers

Items	Group D	Group F	P value
IW, g	483.6 ± 42.2	507.1 ± 36.1	0.429
FW, g	1333.3 ± 74.6	1377.3 ± 88.7	0.477
ADG, g/d	40.5 ± 2.5	41.4 ± 2.6	0.603
FDI, g	94.1 ± 4.9	95.9 ± 3.5	0.562
G: F	0.43 ± 0.02	0.43 ± 0.02	0.620

Table 3

Effects of dietary fiber on alpha-diversity of cecal chyme bacteria of broilers

Items	Group D	Group F	P-Value
Chao1	3864 ± 771	3567 ± 800	0.491
Simpson	0.93 ± 0.06	0.93 ± 0.04	0.809
Shannon	7.73 ± 1.54	7.47 ± 1.08	0.725
Pielou_e	0.66 ± 0.11	0.65 ± 0.07	0.795
Observed_species	3344 ± 776	3066 ± 755	0.509
Faith_pd	118.58 ± 25.20	112.99 ± 24.89	0.682
Goods_coverage	0.984 ± 0.004	0.984 ± 0.003	0.793

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Micronized Bamboo Powder Affects the Growth Performance, Intestinal Development, Cecal Chyme Microflora and Metabolic Pathway of Broilers aged 24-45 days

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