Effects of micronised bamboo powder on growth performance, intestinal development, caecal chyme microflora and metabolic pathway of broilers aged 24–45 days

Preparation of MBP

Bamboo culm of Phyllostachys pubescens (from Sichuan province, China) aged 5–6 years was collected and processed into MBP using the previously reported method (Dai et al. 2022). Contents of moisture, crude protein (CP) and ash were tested according to AOAC methods (AOAC 2007), and were 7.83%, 1.51% and 1.01%, respectively. Contents of IDF, soluble dietary fibre (SDF) and total fibre (TF) were determined according to AOAC method 991.43, and were 82.33%, 3.30% and 85.63%, respectively. Contents of acid detergent fibre (ADF) and NDF were determined using the method of Van Soest et al. (1991), and were 74.80% and 82.84%, respectively.

Experimental animals and groups

Slow-growing spotted-brown broilers (Jinling, an improved local breed; WEN’S Group, China; n = 880) aged 22 days were all pre-fed with the same diet (a mixed diet with half each of treatment and control) for 2 days, and then randomly divided to two groups according to weight (495.3 ± 36.0 g) and sex. There were 16 pens, 55 birds per pen and eight pens in each treatment. The broilers in the control group were fed with a maize-based diet that was antibiotic-free. The broilers in the experimental group were supplemented with MBP at 1% to replace the equivalent maize of the control diet. The broilers were housed in floor pens littered with 8 cm of rice hull and offered ad libitum access to feed and water. Each replicate pen was 2 m long by 1.8 m wide, and stocking density was 15 birds/pen. Ambient temperature was lowered to 25°C using a cold-air fan. The experiment lasted for 21 days, corresponding to broiler age 24–45 days.

Diet ingredients and nutrition level

The ingredients and nutrition level of the diet in this study were prepared according to the standard ‘Nutritional Requirements of Yellow Feather Broilers’ (NY/T3645-2020, Ministry of Agriculture and Rural Affairs of China). The composition and nutrition level are shown in Table 1. In the diet of the MBP group, the same amount of MBP (1%) was used to replace the equivalent of maize. The value of metabolisable energy (ME) in the two groups was the same according to our previous study; we found that adding MBP at 1% to replace equivalent maize had no negative effect on the growth performance of broilers (Dai et al. 2022). It was assumed that the energy value of MBP was equivalent to that of maize under the conditions of appropriate addition. The formulated diet was mixed and made into pellets with steam-conditioning at 80°C.

Slaughtering and sampling

On Day 45, two randomly selected broilers (one male and one female) in each replicate were fasted for 12 h and sacrificed by cervical dislocation, after which the broilers were dissected for collection of internal organs including heart, liver, spleen, bursa of Fabricius, gizzard and proventriculus. The chyme in the caecum was put in a 1.5-mL sterile centrifuge tube, frozen with liquid nitrogen and stored at −80°C for further tests. Intestinal tissue lengths of 0.5 cm were collected from the duodenum, jejunum and ileum, from which chyme was washed with phosphate-buffered saline (PBS). These tissues were then fixed with 4% formalin for the following assays.

Indicators and methods of determination

Data processing and statistical analyses

SPSS 25.0 (IBM, Armonk, NY, USA) was applied for statistical analysis. Independent sample t-test was applied for analysing differences in growth performance, organ indexes, intestinal histology and microbiota diversity indexes. Data were reported as means ± s.e.m. Differences were considered significance at P ≤ 0.05, and a tendency was considered to exist at P-values >0.05 and <0.10.

The microbial differential species markers in caecal chyme were analysed with linear discriminant analysis (LDA) effect size (LEfSe), combined with nonparametric Kruskal–Wallis and Wilcoxon rank sum test. Spearman correlation analysis was applied to assess the correlations between microbiota at phylum level and metabolites in caecal chyme of broilers.

Ethics approval

All aspects of 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).

Effects of MBP on growth performance of broilers

Growth performance of broilers aged 24–45 days is shown in Table 2. No significant differences were observed in ADG, AFDI and G:FI between control and MBP groups.

Effects of MBP on organic indexes of broilers

No significant differences were found in organ indexes of the heart, liver, spleen, bursa of Fabricius, gizzard, proventriculus, duodenum, jejunum, ileum and colorectum between control and MBP groups (Fig. 1). The organ index of the caecum was significantly higher (P < 0.05) in the MBP than the control group, with an increase of ~15.79%.

Fig. 1.

Effects of dietary MBP on organ indexes of broilers: (a) organ index (%) = weight of organ/live broiler weight × 100; (b) intestinal organ index (cm/g) = length of intestine/live broiler weight. CON, control group, fed antibiotic-free basal diet; MBP, group fed basal diet with MBP added at 1% to replace equivalent maize. Data are presented as means ± s.e.m., n = 8. *P < 0.05.

Effects of MBP on intestinal histology of broilers

With regard to intestinal histology, the broilers in the MBP group had significantly (P < 0.05) higher VH of the jejunum and ratio VH:CD than the control broilers, with increases of 29.66% and 28.77%, respectively (Fig. 2). No significant differences were found in VH, CD and ratio of VH:CD in the duodenum and ileum between treatment groups.

Fig. 2.

Effects of dietary MBP on intestinal histology of broilers: (a) villus height, (b) crypt depth, (c) villus height/crypt depth. CON, control group, fed antibiotic-free basal diet; MBP, group fed basal diet with MBP added at 1% to replace equivalent maize. Data are presented as means ± s.e.m., n = 8. *P < 0.05.

Effects of MBP on diversity and abundance of microflora in caecal chyme

There were 15 630 OTUs in the control group and 15 464 in the MBP group. The two groups shared 5100 OTUs, accounting for 19.62% of the total OTUs. The MBP group had 10 364 unique OTUs, accounting for 39.87% of the total OTUs found. Bacterial diversity estimators (Shannon, Simpson and Pielou), richness estimators (Chao1, observed species and Faith’s PD), and coverage estimator (Good’s coverage) are shown in Table 3. No significant difference was found in any of the indexes between the treatment groups.

Combined with the results of data alignment in the databases and OTU species classification, the top 20 in caecal chyme microbiota composition were plotted in different groups at the levels of phylum and genus (Fig. 3). It can be seen that, in both MBP and control groups, the dominant microflora at the phylum level were Firmicutes, Bacteroidetes, Proteobacteria, etc. At the genus level, Bacteroides, Megamonas and Faecalibacterium were the dominant bacteria. However, the abundance of microflora was different between the two treatments groups. In the MBP group, the relative abundance of Firmicutes was higher than in the control group, whereas the relative abundance of Bacteroidetes was lower at the phylum level. MBP reduced the relative abundance of Bacteroides at the genus level.

Fig. 3.

Effects of dietary MBP on the caecal chyme microbial composition of broilers. Relative abundance of caecal chyme microbiota at (a) phylum level and (b) genus level. CON, control group, fed antibiotic-free basal diet; MBP, group fed basal diet with MBP added at 1% to replace equivalent maize. Data are presented as mean ± s.d. (n = 8).

Results of the LEfSe algorithm module are presented in Fig. 4. Addition of MBP at 1% to the broiler diet significantly upregulated expression abundances of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus and f_Enterococcaceae (P < 0.05), and significantly downregulated expression abundances of p_Bacteroidetes, f_Bacteroidaceae, g_Bacteroides, o_Bacteroidales and c_Bacteroidia in caecal chyme of broilers (P < 0.05).

Fig. 4.

Comparison of microbiota analysed with LEfSe. (a) Histogram of LDA effect values of marker species. Note: ordinate represents taxa with significant differences between groups (P < 0.05), and abscissa visually displays the logarithmic score of LDA analysis for each taxon in a bar chart. (b–f) Relative abundance distribution of marker species p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae and f_Clostridiaceae in the two treatment groups. CON, control group, fed antibiotic-free basal diet; MBP, group fed basal diet with MBP added at 1% to replace equivalent maize.

Effects of MBP on chyme metabolic pathways in broilers

Typical metabolite chromatograms are presented in Fig. 5. PLS-DA and OPLS-DA models showed an obvious separation in metabolites between treatment groups, suggesting that the fecal metabolic profiles have been greatly changed by addition of MBP.

Fig. 5.

Effects of dietary MBP on chyme metabolites in broilers: (a) chromatogram of basal peaks of typical caecal chyme samples of broilers in the two treatment groups; (b) PLS-DA and OPLS-DA scores of caecal chyme of broilers in the two groups. CON, control group, fed antibiotic-free basal diet; MBP, group fed basal diet with MBP added at 1% to replace equivalent maize.

The metabolic pathways of different metabolites are presented in Fig. 6. Compared with the control group, there were 1635 upregulated and 837 downregulated primary metabolites in the MBP group. In total, 66 different metabolic pathways were identified between treatment groups, mainly including alanine, aspartic acid and glutamic acid metabolism, butyric acid metabolism, arginine synthesis, linoleic acid metabolism, and β-alanine metabolism.

Fig. 6.

Effects of dietary MBP on chyme metabolic pathways in broilers: (a) histogram of differential metabolites; (b) metabolome view map of the differential metabolites (VIP >1, P < 0.05) identified in caecal chyme of young broilers between the MBP and control (CON) groups. The node colour is based on its P-value, and the node radius is determined based on the pathway impact values. Larger sizes and darker colours represent higher pathway enrichment and impact values, respectively.

Correlation between caecal microflora at phylum level and metabolites in broilers

The correlation between microflora at phylum level and metabolites in caecal chyme of broilers was analysed (Fig. 7). Firmicutes were positively correlated (P < 0.05) with syringic acid, 3-methyl-2-oxovaleric acid, 3-(2-hydroxyphenyl) propanoic acid and butyric acid, and negatively correlated (P < 0.05) with 3-(methylthio)propionic acid, L-alanine, γ-aminobutyric acid, L-glutamic acid and ornithine. Bacteroidetes were positively correlated (P < 0.05) with L-alanine, L-threonine, 3-(methylthio)propionic acid and L-glutamic acid, etc., and negatively correlated with butyric acid, 3-methyl-2-oxovaleric acid, 3-(2-hydroxyphenyl) propanoic acid, syringic acid, etc.

Fig. 7.

Spearman correlation analysis between microbiota and metabolites at phylum level in caecal chyme of broilers. Significant correlations: *P < 0.05, **P ≤ 0.01.

Effects of MBP on growth performance

Previous studies with broilers proved that supplementation with insoluble fibre can promote growth of broilers (Jiménez-Moreno et al. 2016, 2019; Nassar et al. 2019). The effects of fibre on the growth performance of broilers is dependent on several factors including fibre resources (Pourazadi et al. 2020; Tejeda and Kim 2020), addition level (Tejeda and Kim 2021), particle size (Tejeda and Kim 2021) and feeding stage (Berrocoso et al. 2020). We found that the regulatory effect of MBP on the growth performance of broilers aged 24–45 days in this study was greater than its effect on growth performance of young broilers aged 1–22 days (Dai et al. 2022). There was no significant effect on ADG, ADFI or G:FI, which may be related to the application stage of MBP. However, adding MBP at 1% to replace an equivalent amount of maize had no negative effect on the performance of broilers in the middle growth stage.

Effects of MBP on the development of the gastrointestinal tract

Several studies have reported that adding insoluble fibres to the diet can promote the gastrointestinal development of broilers, with improvement in digestive organ development and intestinal histology, and then improve the digestion and absorption of nutrients, as well as the ability to repair intestinal damage. Processed lignocellulose added to feed at 0.5–2 kg/t can promote the development of jejunum villi in broilers (Sozcu 2019). Supplementation with IDF (Arbocel RC) at 1% can improve digestive organ weights and enzyme activities in pullets, which may contribute to an improvement in feed utilisation (Yokhana et al. 2016). Adding soybean hull at 4% to the maize–soybean meal diet can increase villus height in the duodenum and jejunum of broilers, thus improving amino acid digestibility (Tejeda and Kim 2020). Supplementing wheat bran at 3% in the diet of broilers was shown to be beneficial to gizzard development, intestinal digestive enzyme activities and morphology of broilers (Shang et al. 2020). The fibre composition of lignocellulose, Arbocel RC, soybean hull and wheat bran in the above studies is similar to that of MBP, which is mainly composed of insoluble fibres. Similarly, bamboo leaf fibre, which is rich in IDF, could increase intestinal length of broilers (Bersalona 2015). Bamboo charcoal containing 1.0% bamboo vinegar could also significantly improve the structure of villi in laying hens (Rattanawut 2014).

Bamboo powder is rich in insoluble fibre, with a content of ~62.54–89.79% (Felisberto et al. 2017). MBP is a type of concentrated fibre raw material, and its IDF content reached 82.33% in the present study. Similar to previous research, our study has found that adding MBP at only 1% to the diet significantly increased the VH of jejunum, the ratio VH:CD, and the index of digestive organs (caecum). This may be related to the physicochemical properties of IDF, such as water-holding capacity, oil-holding capability, swelling capacity and cholesterol absorption capability. These physicochemical properties have a good correlation with the function of the IDF of tea; tea IDF significantly enhanced the production of short chain fatty acids during in vitro fermentation (Bai et al. 2022). MBP may similarly improve the digestion and absorption ability of broilers by promoting intestinal development, which may be one of the reasons why the growth performance was not reduced after replacing maize with equivalent MBP (1%).

Effects of MBP on caecal chyme microflora

Our study found that the composition of the caecal chyme microbiota was essentially stable in broilers of the two groups at the phylum level and genus level. At the phylum level, the main bacteria were Firmicutes, followed by Bacteroidetes and Proteobacteria, whereas at the genus level, the dominant bacteria were Bacteroides, followed by Megamonas and Faecalibacterium. The results are 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). This indicates that MBP at 1% might not interfere with the natural composition of caecal microflora of broilers; instead, it is more likely to regulate the relative abundance of caecal microbiota.

Firmicutes are specialists in fibre degradation (Sun et al. 2021a); their cellulase and cellulosomes can degrade dietary fibres into monosaccharides that could be used as an energy source (Artzi et al. 2017). The present study revealed that adding MBP at 1% had no significant effect on the diversity of caecal microorganisms, but the abundance proportion of Firmicutes in caecal chyme was increased, leading to the lower abundance proportion of other bacteria such as Bacteroidetes. Specifically, MBP can significantly upregulate the abundance of p_Firmicutes, f_Alicyclobacillaceae, g_Acutalibacter, f_Peptococcaceae, f_Clostridiaceae, f_Bacillaceae, g_Enterococcus and f_Enterococcaceae, all of which are key degraders of dietary fibre (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. 2021b). Another study of various IDFs and SDFs showed broader effects with more and different types of gut microbial species, which may be due to the type and dose of fibre (Swanson et al. 2020). It indicated that the addition of MBP may improve fibre metabolism by improving the microflora composition, and MBP was expected to be a high-quality raw material for providing prebiotic lignocellulose.

Effects of MBP on metabolic pathways of caecal chyme

Given the significant differences in chyme microbiota, we next investigated the alterations of chyme metabolic pathways. Through metabolic pathway enrichment analysis, this study found alanine, aspartic acid and glutamic acid metabolism, butyric acid metabolism, arginine synthesis, linoleic acid metabolism and β-alanine metabolism to be considerably affected by MBP. The results show that MBP might have the potential to regulate the metabolism of amino acids and fatty acids in broilers. An alteration in the levels of caecal acetate and iso-butyrate was more pronounced for young broilers with inulin in the diet than with wheat bran (Li et al. 2018). Another study found that dietary mixed fibre (4% insoluble fibre/4% soluble fibre) promoted an improvement in gut microbiota and short-chain fatty acid (SCFA) profile, and the multiplicity of gut microbiota was useful in sustaining colonic integrity by producing SCFAs to some extent (Zhai et al. 2018). Rice bran from specific rice cultivars resulted in significant changes including decreased Firmicutes abundance and an increased number of metabolites associated with fatty acid metabolism (Rubinelli et al. 2017). Another study found that increasing the levels of dietary lignocellulose significantly lowered caecal counts of Escherichia/Hafnia/Shigella and reduced the total concentration of SCFAs (Röhe et al. 2020). The above reports indicated that addition of dietary fibre mainly with IDF may regulate the chyme microbiota and SCFA profile, and is associated with fibre source and addition level. Similarly, MBP is rich in IDF and its addition at 1% increased the relative abundance of Firmicutes in the caecal chyme of broilers. Meanwhile, Firmicutes were positively correlated with syringic acid, 3-methyl-2-oxovalenic acid, 3-(2-hydroxyphenyl) propanoic acid and butyric acid. This indicates that MBP might improve the metabolism of fatty acids by improving relative abundance of specific microbiota, and then promote the development of the caecum and intestinal health.

The gut microbiota perform a crucial function in facilitating the regulation of the amino acid pool and profile. Gut microbiota such as bacteria of the genera Fusobacterium, Bacteroides and Veillonella can synthesise several nutritionally essential amino acids de novo, a potential regulatory factor in amino acid homeostasis (Lin et al. 2017). Thus, the metabolism of amino acids might be regulated by specific microbiota. As a dietary fibre source with 26.4% crude fibre, California-type almond hulls at 9% in the diet 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. 2021b). Another study revealed that a high-fibre diet increased the relative abundance of Clostridium and Coprococcus and reduced apparent total tract digestibility of CP and 18 amino acids in finishing pigs (Hu et al. 2021). The above research showed that there was a strong link between dietary fibre, gut microbiota, and amino acid metabolism. The present study analysed the differential metabolites and found that MBP was involved in amino acid metabolism in caecal chyme of broilers aged 24–45 days. Specifically, adding MBP at 1% significantly reduced the abundance of Bacteroides in chyme, and Bacteroidetes were positively correlated with the production of L-alanine, L-threonine, 3-(methylthio)propionic acid and L-glutamic acid.