Gut Microbiota Related to Fiber Digestibility were Identified by Variation of Apparent Fiber Digestibility in Chinese Suhuai pig

Background: Dietary fiber plays an important role in porcine gut health and welfare. Fiber mainly degraded by the gut microbiota, whereas most gut microbiota related to fiber digestibility of pigs are still unidentified. To reveal gut microbiota associated with apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF), apparent NDF, ADF digestibility of 274 Suhuai female finishing pigs at the age of 160 days were measured. The gut microbiota of Suhuai pigs were analyzed through 16S rRNA gene sequencing, respectively. Results: Large phenotypic variations in apparent NDF and ADF digestibility were separately found among Suhuai pigs. The coefficient of variation of NDF and ADF digestibility was 12.08% and 18.08%, respectively. The mean values of digestibility of H-NDF and H-ADF groups were 30.20% and 33.76% more than those of the L-NDF and L-ADF groups (P0.01), respectively. A total of 927 and 935 operational taxonomic units (OTUs) were confirmed from two types of fecal samples,respectively. There were14 phyla in all samples and the abundances of Spirochaetae, Bacteroidetes and unclassified_k__norank were significantly different between H-NDF and L-NDF groups (P0.05) and the abundances of Spirochaetae, Verrucomicrobia, unclassified_k__norank and Fibrobactere were significantly different between H-ADF and L-ADF group (P0.05).A total of 188, 183, 188 and 185 genera were separately identified in H-NDF, L-NDF, H-ADF and L-ADF groups, while 6, 1, 5 and 2 genera were separately specific to H-NDF, L-NDF, H-ADF and L-ADF groups. The microbiota of H-NDF and H-ADF clustered separately from the microbiota of the L-NDF and L-ADF along principal coordinate 1, respectively. Compared with L-NDF group, 29 genera were found to be potential biomarkers in H-NDF

of high and low fiber digestibility in Suhuai pig. This study aimed to detect phenotypic variation of fiber digestibility within Suhuai pig breed and reveal gut microbiota associated with apparent NDF and ADF digestibility by 16S rRNA gene sequencing of Suhuai pigs with extreme apparent NDF and ADF digestibility, respectively.

Variation of apparent NDF and ADF digestibility within Suhuai pigs
Data regarding the apparent NDF and ADF digestibility of the 274 Suhuai pigs were shown in table 1.
The coefficient of variation (CV) of apparent NDF and ADF digestibility was 12.08% and 18.08%, respectively.

Comparison of apparent NDF and ADF digestibility between high and low groups of Suhuai pigs
Apparent digestibility of NDF and ADF were both significantly different between high and low groups.
The mean values of H-NDF and H-ADF groups were 30.20% and 33.76% more than those of L-NDF and L-ADF groups, respectively (P 0.01, Table 2).

Prediction function of microbial metabolism
Sixty four functions were predicted in the present study. General function prediction only (8.56%), Carbohydrate transport and metabolism (8.25%), Amino acid transport and metabolism (8,23%), Replication (8.16%), Translation (7.85%), Transcription (7.50%), Cell wall/membrane/envelope biogenesis (6.47%), Energy production and conversion (5.51%), Inorganic ion transport and metabolism (5.08%) and Signal transduction mechanisms (4.49%)were the most enrichment functions. At the same time, 39 metabolic pathways were predicted. Membrane Transport (   Oscillibacter, and Clostridium were generally prevalent in pig gastrointestinal tract [17]. The data between the two types of samples had statistical significance was analyzed by Adonis/PERMANOVA [18]. Microbial composition had a strong difference between high and low groups of NDF and ADF (adonis/PERMANOVA P < 0.01). The result illustrated that the gut microbiota between the two types of samples in NDF group and ADF group were statistically significant and all data comparisons made between different groups in this study were meaningful.
Although chao and ace indexes had no different between the two types of samples of this study, shannon and simpson indexes were significantly different between the two types of samples in NDF groups. The PCA of UniFrac distance matrices showed that the variation between H-group and L-group was primarily explained by the apparent NDF or ADF digestibility, respectively. This suggested that the differences of microbialcommunitystructure between high and low groups were related to apparent NDF and ADF digestibility. However, these diversity indexes only showed the overall situation of microbiota in each group, as the objective of the present study was to reveal gut microbiota associated with apparent NDF and ADF digestibility, we needed to know the microbiota with higher abundance in the H-group and then predicted their microbes functions. A total of 29 and 23 genera were found to be potential biomarkers in H-NDF group and H-ADF group, respectively.

Conclusions
A large phenotypic variation in apparent NDF and ADF digestibility among Suhuai pigs, respectively.
Microbial community structures were significantly different between high and low fiber digestibility groups. Twenty nine and 23 genera were found to be potential biomarkers in H-NDF group and H-ADF group, respectively. The most important functions and metabolic pathways of the above different potential biomarkers included carbohydrate transport and metabolism. The biomarkers may be the key functional microbiota related to apparent fiber digestibility.

Sample Collection in Animal
A total of 274 healthy Suhuai pigs were selected to collect fecal and diet samples(no disease or diarrhea happened one week before sampling) at age of 160 days were collected from Huaiyin Pig Breeding Farm, Huaian, China, under same husbandry conditions. All pigs were selected according to a unified breed standard and fed with an antibiotic-free corn-soybean diet (Supplementary Table   1). One month before sampling, antibiotics in the feed or for any therapeutic purposes were not provided for pig. Diet samples and approximately200 g of each fecal sample were collected in plastic bags and fecal samples were mixing with 15 ml 10% sulfuric acid to be fixed on site. These samples were used for analyzing apparent nutrient digestibility. Each sample was individually collected in 2 ml centrifuge tubes without any treatment for 16S rRNA gene sequencing. All samples were kept in ice box for preservation and transportation and then stored at -80℃ in the laboratory [24].

Chemical Analysis
Fecal samples from Suhuai pigs were dried at 65℃ to a constant weight. Determination of NDF and ADF contents was using ANKOM A200 filter bag technique (AOAC 962.09) [25].
Chimera identification and removal were using UCHIME [28]

Statistical Analysis
The apparent fiber digestibility was calculated using SAS 9.4 software [32]. Alpha diversity was calculated using Mothur [29]. Linear discriminate analysis effect size (LEfSe) was used to identify the bacteria enriched [33]. Pair-wise phylogenetic distance was measure by weighted UniFrac [34] to compare community compositions across samples. Principal co-ordinates analysis (PCoA) were used compress dimensionality into 2D principal coordinate analysis plots [35], enabling visualization of sample relationships. PICRUSt was used to explore the functional composition of that bacterial community data might convey [36].

Supplementary Files
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