Effect of dietary fiber levels on bacterial composition with age in the cecum of meat rabbits

Abstract This study investigated the influence of dietary fiber levels on the growth performance, digestion, metabolism, and cecal microbial community of rabbits with different diets at different age. The different levels of dietary natural detergent fiber (NDF) were formulated accordingly: 400(A), 350(B), 300(C), 250(D) g/kg original matter basis, respectively; the different ages were 52, 62, and 72 days. With NDF increasing, the average daily feed intake (ADFI) and feed conversion rate (FCR) were increased, whereas average daily gain (ADG) and mortality were decreased (p < 0.05). The stomach relative weight, stomach content relative weight, cecal relative weight, and cecal content weight increased with increasing NDF (p < 0.05). The NH 3‐N concentration of cecum dropped when the dietary NDF increased (p < 0.05). The diversity of the total microbiota increased significantly in Diets B, C (p = 0.011), and reached the lowest in 52 days for all diet groups. The richness index was decreased significantly in Diet A, D (p < 0.05) and in 62 days (p < 0.001), respectively. The phylum Firmicutes was higher (p < 0.01) in rabbits fed Diets B, C than Diets A, D and Bacteroidetes was highest in Diets C, D, and Proteobacteria was the highest in Diet A (p < 0.001). Among the classified genera, there were 14 that had levels of abundance of more than 1% and were commonly shared by all samples. Ruminococcus spp. that produced volatile fatty acid (VFA) abundance was highest from Diets B, C at 52 and 62 days. It is interesting to note that Bifidobacterium from Diet C was the most abundant genus during the entire experimental period (p < 0.01). The data from Venn diagrams, principal component analysis (PCA), and heat map plots of the bacterial communities showed that there were more groups of shared microbiota with aging. The above results indicate the cecal microbiota controlled by the 350 g/kg NDF diet can prevent gastrointestinal distress and exhibit good production performance.

Because of the main energy substrate for the microbiota, dietary fiber levels significantly impact of the composition of the intestinal microbiota (Combes, Fortun-Lamothe, Cauquil, & Gidenne, 2013;Sawicki et al., 2015), and most of their beneficial effects are associated with bacterial fermentation (Lattimer & Haub, 2010;Verbeke et al., 2015). The understanding of cecal microbiota structure is considered to have an important role in the nutrition and health of rabbits.
The objectives of the research were to investigate and discuss the effects of different dietary NDF levels on the cecal ecosystem of growing meat rabbits and to determine the appropriate dietary NDF level in growing meat rabbits.

| Animals, housing, and sampling
Two hundred weanling New Zealand rabbits were assigned into four groups according to the mean body weight (1.46 ± 0.16 kg).
They were fed each diet with 400 (Diet A), 350 (Diet B), 300 (Diet C), and 250 (Diet D) g/kg NDF (original matter basis), respectively (Table 1). Rabbits were provided with free access to feed and a water nipple.

| Production trial
All rabbits were weighed at the beginning (day 32) and the end (day 72) of the experimental period and the average daily gain (ADG), the average daily feed intake (ADFI), feed conversion rate (FCR), and mortality were recorded and calculated for the whole experimental period.
The stomach weight, stomach content, cecal weight, cecal content weight, small intestine length, and rabbit body length were measured at 72 days, which were used for computing relative stomach weight (stomach weight/body weight), stomach content relative weight (stomach content weight/body weight), small intestine relative length (small intestine length/body length), cecal relative weight (cecal weight/ body weight), cecal content relative weight (cecal content weight/body weight), mortality percentage (mortality number/rabbits total numbers per group). After slaughter, the cecum were removed immediately, cecal content (n = 8) used for measuring the pH value, NH 3 -N concentration, VFA by the technique of Weatherburn (1967) and Tao and Li (2006).

| Intestinal microbiota trial
Five healthy rabbits randomly selected from each diet treatment group were euthanized at 52, 62, and 72 days of age and harvested their cecal contents from the middle of the ccum, and stored at −80°C until microbial analyses.

| Chemical analysis of experimental diets
The diets were formulated according to the values of the National Research Council (NRC; 1977) and recommendations in "The Nutrition of the Rabbit" (De Blas et al., 1998), and the food was formed the as pellets of 4 mm diameter (Table 1)  MiSeq platform according to protocols described by previous studies (Caporaso et al., 2010).

| Statistical and bioinformatic analyses
The data of production trial were subjected to one-way analysis of variance (ANOVA) using the general linear models (GLM) procedure in SAS 9.1 (SAS institute Inc., Cary, NC). Intestinal microbiota richness and diversity indexes (Tables 5,6) were analyzed by one-way or two-way analysis of variance to examine the effects of diet, age, and diet×age. p < 0.05 and p < 0.01 indicates significant difference and highly significant difference among 16S sequences from different treatments, respectively. The valid sequences were filtered from the high-throughput sequencing with standards previously reported . Briefly, an average quality score<25 and min length <200 bp were considered as low-quality sequences, and any mismatches to the primers, or a homopolymer longer than 8 bases were excluded. The passing sequences were assigned to the individual sample according to the 7-bp barcodes, and the remaining sequences were clustered into operational taxonomic units (OTUs) with a cutoff of 97% identity. Rarefaction curves, abundance-based coverage estimates (ACE), Chao1 (Chao, Chazdon, Colwell, & Shen, 2005), network analysis, heat map analysis, and beta diversity analysis indexes were generated at 97% sequence identity level using RDP classifier. In addition, a principal component analysis (

| Effects of different dietary NDF on growth performance and total tract apparent digestibility and nitrogen balance
ADFI, ADG, FCR, and mortality during the growth trial are summarized in Table 2. ADFI and FCR were increased when NDF increasing (p < 0.05). ADG trend is contrast with above. Mortality reached minimum levels in diet A and diet B (p < 0.01). The stomach relative weight, stomach content relative weight, cecal relative weight, and cecal content weight increased with increasing NDF (p < 0.05). The level of NDF did not affect small intestine relative length (Table 3).
The effect of different dietary NDF on cecal fermentation is shown in Table 4. The only NH 3 -N concentration of cecum dropped when the dietary NDF increased (p < 0.05).

| Illumina MiSeq derived metadata
The raw sequences with an average length of 402 bp were obtained from the Illumina Miseq platform. By quality filtering, the obtained valid sequences per sample (based on 97% sequence identity) were aligned in accordance with the RDP classifier alignment and TA B L E 2 Effects of NDF level on growth performance of growing meat rabbits (n = 50)

| Microbial diversity and abundance
The Shannon-Weaver diversity index was calculated to assess the diversity of the microbial communities. Using two-factor analysis, the DNA sequences from all five rabbits in each diet group were used individually for these calculations, and diversity indexes were compared over time among different diets (

| Taxon-based analysis and Phylum-level difference in microbial composition among group
To describe the bacterial composition of the different dietary treatment groups and how they changed during total experimental period, we conducted a taxon-dependent analysis (Cole et al., 2013). Describing the distribution of DNA sequences at phylum level in Figure 1, the results showed that bacterial communities of all samples were composed primarily of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, which had overall majority of the total sequences. Significant fluctuations of Firmicutes, Bacterioidetes, Proteobacteria, and Actinobacteria were detected among the four diets and three ages ( Table 6). The phylum Firmicutes was higher (p < 0.01) in rabbits fed Diets B and C when compared with the other two diets (Diets A and D). The phylum Bacteroidetes was higher when rabbits were fed with Diets C and D than the other diets (p < 0.001). The highest level of NDF (Diet A) increased the Firmicutes/Bacteroidetes ratio (p < 0.001), and a higher Firmicutes/Bacteroidetes ratio occurred in the 52-d age group (p < 0.001). However, phylum Proteobacteria was the highest in Diet A (p < 0.001). Similarly, means from each age group among the four diets were compared by an analysis of diet × age (Table 6).
For all of the above bacterial phyla, the interaction between diet and age was highly significant (p < 0.001).

| Genus-level differences in microbial composition among groups
As shown Figure 2,

| Relationships of cecal bacterial communities feeding different dietary fiber levels
The principal component analysis of the weighted UniFrac distance matrix and heat map analysis were conducted to further evaluate the pyrosequencing data. The PCA score plot revealed that the majority of the samples harbored characteristic bacterial communities, and the samples from Diet C at 52 days (S1C1, S1C2, S1C3, S1C4, S1C5), Diets C and D at 62 days (S2C1, S2C2, S2C3, S2C4, S2C5),

| D ISCUSS I ON
Although the rabbit relies on the intake of large quantities of fiber that can be fermented by the microbiota found predominantly within the cecum, the change in NDF level does not affect the cecal pH and VFA concentration, which indicates no relation between quantity of NDF and concentration of VFA in the cecum (Bellier & Gidenne, 1996).
Previous studies focused particularly on the analysis of the effects of the dietary fiber level on the whole cecal bacterial community in rabbits using molecular profiles (Michelland et al., 2010Monteils et al., 2008;Rodríguez-Romero, Abecia, & Fondevila, 2013). Our study showed that the cecal microbiota abundance increased significantly when NDF levels reached 350-400 g/kg (original matter basis) (p < 0.001). However, 400 g/kg and 250 g/kg NDF groups decreased the diversity of the cecal microbiota (p = 0.01). Therefore, feeding rabbits with different dietary fiber levels resulted in alteration of the structure of the cecal bacteria community (diversity and relative abundance) (Crowley et al., 2017). We also noticed that the cecal bacterial diversity increases with age, whereas bacterial abundance maintains a dynamic balance (in this case, increased first and then decreased).
We found that more than 90% of DNA sequences of the cecal contents from all samples in the total experiment period belong to the Firmicutes, Bacteroides, Actinobacteria, and Proteobacteria, which is consistent with the findings of Cauquil and Gidenne (2012).
In this study, rabbits feeding on high-fiber diets (Diet A) required high cecal ratio of Firmicutes/Bacteroidetes (p < 0.001). Combes et al. (2011) reported ratio Firmicutes/Bacteroidetes was proposed as an indicator for microbiota maturity. Thus, our results indicated that the higher NDF level diet can accelerate the maturation of intestinal microbiota. In addition, the mice and human studies suggest that a higher Firmicutes/Bacteroidetes impels the gut microbiota to extract efficiently from the diet, which reveal one cause of adiposity (Marcobal et al., 2011;Ley, Lozupone, Hamady, Knight, & Gordon, 2008;Yasuda et al., 2015). However, it is clearly established that an increase in NDF level results in a reduction in the ADG in our study (p < 0.001), which apparently contradicts previous results. Here the result seems to be more related with physiological conditions, such as the rabbit cecum is different significantly from mice and human.  Figure 2). In additional, our study showed that rabbit production performance was elevated, that is, the ADG reached the highest levels in Diets C and D (p = 0.001, Table 2). This result indicated that abundance of Desulfuricans spp. may reflect elevated production (Luo et al., 2018). On the other hand, Bibersteinia is an important pathogen that is associated with serious infection (Bleich, Sargeant, & Wiedmann, 2018;Parr, Smith, Jenks, & Thompson, 2018;Heinse, Hardesty, & Harris, 2016) and is implicated in the high incidence of diarrhea in Diet D at 52 days.

Ruminococcus
Venn diagrams, PCA, and heat map plots of the bacterial communities derived from rabbit cecum showed that a shift of dietary fiber can lead to quick changes in the composition of the microbiota, which is a reflection of a rapid adaptation to reach a new equilibrium in response to a nutritional disturbance Zhu, Sun, Wang, & Li, 2017). Meanwhile, we found that in the cecum, the structure of the bacterial community has no difference between rabbits of the same age or fed the same diet treatment (Michelland et al., 2010).

| CON CLUS ION
We provide clear evidence that the influence of dietary NDF levels on growth performance, digestibility and metabolism, the cecal bacterial community differed between rabbits of different ages. Indeed, better production performance, increasing the cecal microbiota diversity and abundance were all benefited from suitable dietary NDF level in Diet B (350 g/kg NDF, original matter basis).

ACK N OWLED G M ENTS
This work was supported by grants from the Natural Science

DATA ACCE SS I B I LIT Y
MiSeq Illumina sequencing raw sequence reads data: https://www. ncbi.nlm.nih.gov/bioproject/PRJNA291670, which associated with the "Illumina MiSeq derived metadata" section in the article.

CO N FLI C T O F I NTE R E S T
All authors declare that there are no conflicts of interest.