Response of goose intestinal microflora to the source and level of dietary fiber

Response of goose intestinal microflora to the source and level of dietary fiber ABSTRACT Geese are capable of digesting and making use of a high-fiber diet, but the mechanism is not well understood and would be of great significance for the development and utilization of roughage resources. In this study, we investigated the effect of dietary fiber (source: corn stover and alfalfa, included at 5% or 8%) on microflora in goose intestines. We used 35-day-old Carlos geese in which we first studied the influence of fiber ingestion on diet digestibility and immune organ indices of geese and found that high dietary fiber (8% content) significantly increased feed intake, the digestibility of neutral and acid detergent fiber, and thymus, bursa, and spleen size. Subsequently, we investigated the effect of dietary fiber on the microbial flora in the various intestinal segments by high throughput sequencing. The bacterial diversity and relative abundance were significantly affected by the type and amount of dietary fiber fed, including that of cellulolytic bacteria such as Bacteroides, Ruminococcus, Clostridium, and Pseudomonas spp. Finally, we isolated and identified 8 strains with cellulolytic ability from goose intestine and then analyzed their activities in combination. The optimal combination for cellulase activity was Cerea bacillus and Pseudomonas aeruginosa. This study has laid a theoretical and practical foundation for knowledge of the efficient conversion and utilization of cellulose by geese. INTRODUCTION Dietary fiber has a complex structure and composition and plays a key role in the nutrition and physiology of animals (Girard et al., 1995; Franklin et al., 2002). The digestion and utilization of dietary fiber by geese is a complicated process, which is accomplished by physical digestion, chemical digestion, and microbial digestion (Duke et al., 1972; Clemens et al., 1975). The digestion and utilization of crude fiber by geese occurs principally in the gizzard and cecum: plant cell walls can be broken down in the mature gizzard, which permits digestion of the cell contents (Duke et al., 1972; Clemens et al., 1975), and the cecum contains a large number of microorganisms that can utilize various sources of dietary nutrition, including crude fiber (CF), neutral detergent fiber (NDF), and acidic detergent fiber (ADF) (Ley et al., 2008; Wang et al., 2009). The intestinal microflora, a large and complex microecosystem, is crucial to the health of geese and their digestion of nutrients (Hooper and Gordon, 2001; Manco, 2012; Cryan and Dinan, 2012; Cryan and O’Mahony, 2011). Many studies have been carried out on the species and quantity of, and the nutrient digestion by, goose intestinal microorganisms, especially with regard to the interactions between microorganisms and fiber (Luo and Zhu, 2007; Lumpkins et al., 2010). Previous studies have shown that gut commensals adapt to dietary composition (Wagner and Thomas, 1978; Savory, 1992). Awati et al. (2005) found that the level of dietary fiber can influence the predominant species of bacteria comprising the intestinal flora of healthy animals. In particular, the abundance of Bifidobacteria and Lactobacillus was higher when high-fiber diets were fed, whereas the abundance of Escherichia coli and Salmonella were lower within a specific range of dietary fiber content. To date, more than 200 naturally occurring microorganisms (fungi, bacteria, and actinomycetes) with cellulolytic ability have been identified. There are many microorganisms present in the normal goose intestine that have the capacity to degrade cellulose (Godden et al., 1992; Apj et al., 1994), but many have not been isolated and identified because of a lack of specific isolation and culture methods. However, the MiSeq PE300 sequencing system (Illumina, Inc., San Diego, CA) sequences at enough depth to identify low abundance bacteria accurately (Lumpkins et al., 2010), and this has become an important tool for the conduct of metagenomic studies. In this study, paired-end sequencing on the MiSeq PE300 platform was used to study the effects of various fiber sources and inclusion levels on the intestinal microflora of geese. World population growth has led to international food shortages; therefore, high-fiber resources that cannot be used by humans have attracted more attention for their potential use in animal feed. Improvement in the utilization efficiency of dietary fiber may be one of the more effective means to alleviate the shortages in food and feed resources in China. Although fiber digestion and utilization, and the intestinal microecological environment of geese, have been extensively studied (Ley et al., 2008; Wang et al., 2009; Liu et al., 2011), the microscopic structure of their digestive tract, the diversity of bacteria, and the key bacteria involved in fiber digestion require further elucidation. It has been reported that sources of dietary fiber differ in their digestibility and lead to differences in the performance of animals fed on them (He et al., 2015; Jørgensen et al., 1996; Jiménez-Moreno et al., 2010). Corn stover and alfalfa are distinct fiber sources, and our previous study showed that they have different digestibility (Lou et al., 2010); however, the mechanism of this is unknown. Furthermore, the level of fiber in the diet is known to affect the digestibility of the diet. It has been reported that 5% CF in the diet is the normal level for geese (Wang et al., 2008). Therefore, the present study was conducted to determine the response of goose intestinal microflora to the source (corn stover vs. alfalfa) and level of incorporation (5% vs. 8%) of dietary fiber, and then to identify the species of cellulolytic bacteria affected. MATERIALS AND METHODS Materials One hundred eighty 35-day-old healthy Carlos geese (male) were randomly divided into 4 groups (n = 45). For each group, 3 pens containing 15 geese each were created. The geese were fed a diet containing 5% corn stover CF (the LJ group), 8% corn stover CF (the HJ group), 5% alfalfa CF (the LM group), or 8% alfalfa CF (the HM group). The 6–8/m2 pens were situated in half-open sheds. The geese were raised on the floor and fed 3 times daily, and had free access to water. The composition of diets is shown in Table 1. During the feeding period, 2 healthy geese were randomly selected from each pen to enter a digestion trial using the total collection method. After 42 d of feeding, the geese were killed by decapitation method, and the intestinal mucosa and immune organs were collected. This study was approved by the Institutional Animal Care and Use Committee of Jilin Agricultural University (IACUC-JLAU201503). All procedures were performed according to international guidelines concerning the care and treatment of experimental animals. Table 1. Composition of diets (%). Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Note:1per kg of premix contains: vitamin A, 800,000 IU; vitamin D, 160,000 IU; vitamin E, 500 IU; Zn 8,000 mg; Mn, 6,000 mg; Fe, 6,000 mg; Cu, 800 mg; I, 35 mg; Se, 30 mg; vitamin K, 50 mg; vitamin B1, 80 mg; vitamin B2, 250 mg; vitamin B5, 220 mg; vitamin B3, 2,000 mg; vitamin B6, 300 mg; vitamin H, 10 mg; vitamin B9, 25 mg. 2ME (metabolizable energy) was calculated, whereas the levels of the other nutrients were measured. HJ group: geese fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 1. Composition of diets (%). Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Note:1per kg of premix contains: vitamin A, 800,000 IU; vitamin D, 160,000 IU; vitamin E, 500 IU; Zn 8,000 mg; Mn, 6,000 mg; Fe, 6,000 mg; Cu, 800 mg; I, 35 mg; Se, 30 mg; vitamin K, 50 mg; vitamin B1, 80 mg; vitamin B2, 250 mg; vitamin B5, 220 mg; vitamin B3, 2,000 mg; vitamin B6, 300 mg; vitamin H, 10 mg; vitamin B9, 25 mg. 2ME (metabolizable energy) was calculated, whereas the levels of the other nutrients were measured. HJ group: geese fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Composition of reagents and media used are as follows: 3,5-dinitrosalicylic acid (DNS) reagent: potassium sodium tartrate 200 g, DNS 10 g, NaOH 10 g, phenol 2 g, Na2SO3 5 g, distilled water to 1,000 mL. Beef extract peptone medium: beef extract 3 g, peptone 10 g, NaCl 5 g, agar 15 g, distilled water to 1,000 mL, pH 7.0, autoclaved at 121°C for 20 min. Potato dextrose agar medium (PDA medium): potato 200 g, glucose 20 g, agar 20 g, distilled water to 1,000 mL, autoclaved at 121°C for 20 min. Fermentation medium: carboxymethylcellulose sodium (CMC-Na) 5 g, beef extract 5 g, peptone 10 g, NaCl 5 g, distilled water to 1,000 mL, pH 7.0, autoclaved at 121°C for 20 min. Cellulase screening medium: CMC-Na 20 g, yeast extract 0.5 g, Na2HPO4 2.5 g, KH2PO4 1.5 g, peptone 2.5 g, agar 15 g, distilled water to 1,000 mL, pH 7.0 to 7.2, autoclaved at 121°C for 30 min. The primers used in this study are listed in Table 2. Table 2. Primers used in this study. Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ View Large Table 2. Primers used in this study. Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ View Large Determination of Feed Conversion Ratio and Digestibility During the 42-d feeding period, body mass (BW) and feed intake (FI) were determined, and feed conversion ratio (FCR) was calculated as FCR = FI (kg)/BW gain (BWG, kg). Crude protein (CP), CF, NDF, and ADF were measured according to GB/T6432 (Chinese National Standard: Determination of crude protein in feed), GB/T6434 (Chinese National Standard: Determination of crude fiber in feed), and Van Soest methods, then the digestibility of each nutrient was calculated using the following formula: Digestibility = (a − b) / a × 100%, where a represents the intake of a nutrient, and b represents the amount of a nutrient in the feces. Immune Organ Indices Body mass and the mass of the thymus, bursa, and spleen of 6 geese per group were measured, then the immune organ indices were calculated using the following formula: Immune organ index = immune organ mass / body mass × 100%. Determination of Cellulase Activity The cellulase activity of chyme from the duodenum, jejunum, ileum, and cecum of 6 geese per group was measured according to method of Magro et al. (2016). One unit of cellulase was defined as the amount of enzyme required to produce 1 μmol/min of glucose. High Throughput Sequencing of Intestinal Mucosal Samples The genomic DNA of the intestinal mucosal bacteria was extracted using QIAamp DNA Mini kits (Qiagen, Germantown, MD), according to the manufacturer's instructions. Universal primers (515F and 806R) were used to amplify the V4 region of 16S rDNA. The PCR was performed using 15 μL of 2 × Phusion Master Mix, 3 μM of each primer, and 10 ng of genomic DNA as the template, in a final volume of 30 μL. Thermal cycling was carried out with an initial activation step at 98°C for 1 min, and DNA amplification was achieved using 30 cycles of 99°C for 10 s, 50°C for 30 s, and 72°C for 30 s, with a final extension step of 72°C for 5 min. The amplified products were separated on 2% agarose gel, then recovered. The gene library was constructed using a NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs, Ipswich, MA) and sequenced using a MiSeq PE300 (Illumina, Inc.). Evaluation of the Quality of the Sequencing Data and Species Annotation Forty-eight samples from the 4 groups (HJ, LJ, HM, LM) were sequenced, and 1,913,581 effective tags were acquired. The mean number of effective tags per sample was 39,866 and the mean length of an effective tag was 253 nt. With a similarity of 95% being required to assign a genus and of 97% being required to assign a species, effective tags were clustered into operational taxonomic units (OTUs) using Uparse software, and RDP Classifier software and the GreenGene database were used to assign a species to representative sequence sets. Analysis of Species Diversity Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/) (Edgar, 2013)was used to cluster the effective tags into specific OTUs, based on a phylip-formatted lower triangle matrix and with the furthest neighbor algorithm set at 97% sequence similarity. Representative sequences from each OTU were annotated by the Ribosomal Database Project (RDP) Classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) (Wang et al., 2007) and the GreenGene database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) (Desantis et al., 2006), and the annotation results were used to analyze the species diversity among the diet groups. Screening for Cellulose Degrading Bacteria Under sterile conditions, 5 g goose intestinal mucosa samples were placed in a conical flask, cultured with agitation for 2 h, left to stand for 1 h, then the supernatant was incubated in PDA medium after gradient dilution (10−1, 10−2, 10−3), cultivated at 37°C for 1 to 2 d, in both aerobic and anaerobic conditions. Colonies were selected for streak culture repeatedly, until a single pure culture colony was isolated. The purified strains were incubated on cellulase screening medium and cultured for 12 to 24 h. Sufficient Congo red stain (1 mg/mL) was added to cover the plates for 15 min, and then poured out. Subsequently, 1 mol/L NaCl was added to soak and wash the agar for 15 min, then the plates were examined to identify transparent circles around the colonies. Morphological Identification Gram staining and spore staining were performed, and colony morphology and cultural characteristics of the strains were determined, with reference to Bergey's Manual of Systematic Bacteriology (Buchanan and Gibbons, 1984). Molecular Identification The genomic DNA of the strains was extracted and used as the template for PCR. Bacterial 16S rRNA universal primers (P1 and P2, 10 pmol) were used for PCR reactions in a total volume of 25 μL, containing gDNA 1.5 μL, dNTP (2.5 mM) 2 μL, 10 × Ex Taq buffer 2.5 μL, and Ex Taq polymerase 2 U. Thermal cycling was carried out with using initial heat action step of 94°C for 5 min, followed by 30 cycles of 94°C for 60 s, 50°C for 45 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. The amplified products were separated on 1% agarose gels, then recovered and sequenced. BLAST was used to determine the homology of the PCR products with known bacterial sequences. Statistical Analysis Data are expressed as mean ± standard deviation (SD). SPSS 17.5 statistical software was used to analyze the data. One-way analysis of variance (ANOVA) was used to compare differences between the experimental groups. P < 0.05 was considered to represent statistical significance. RESULTS Effect of Dietary Fiber on Digestibility Comparing diets with the same fiber source, the effects of fiber content on digestibility was as follows: for the corn straw group, feed intake, FCR, and the digestibility of CF, NDF, and ADF in the HJ group were significantly higher than for the LJ group; for the alfalfa group, the digestibility of CF, NDF, and ADF in the HM group were significantly higher than in the LM group (Table 3). Table 3. Daily feed intake, feed efficiency, and nutrient digestibility for geese fed diets of contrasting fiber level and source. HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 3. Daily feed intake, feed efficiency, and nutrient digestibility for geese fed diets of contrasting fiber level and source. HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing diets with the same fiber content, the effects of fiber source on digestibility were as follows: digestibility of NDF in the LJ group was significantly higher than in the LM group, and the digestibility of CP, CF, NDF, and ADF in the HJ group was significantly higher than in the HM group. Other values were not significantly different (Table 3). Effect of Dietary Fiber on Immune Organ Indices The bursa, thymus, and spleen indices in the 8% fiber groups (HJ and HM) were significantly higher than those of the 5% fiber groups (LJ and LM). In particular the thymus index of the HJ group was 2.76 times that of the LJ group (Table 4). Table 4. Immune organ indices for geese fed diets of contrasting fiber level and source. HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 4. Immune organ indices for geese fed diets of contrasting fiber level and source. HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing groups being fed the same amounts of fiber, the thymus index of the LM group was significantly higher than that of LJ group, but that of HM group was significantly lower than that of HJ group (Table 4). Effect of Dietary Fiber on Cellulase Activity As shown in Table 5, in the corn straw groups higher dietary fiber levels were associated with a trend towards greater cellulase activities in the duodenum, ileum, and cecum. In the alfalfa groups, fiber level affected cellulase activity differently; compared with the LM group, it was significantly lower in the jejunum and higher in the ileum and cecum of the HM group. Table 5. Cellulase activity in intestine for geese fed diets of contrasting fiber level and source. Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b a,bIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 5. Cellulase activity in intestine for geese fed diets of contrasting fiber level and source. Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b a,bIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing groups fed the same amount of fiber, fiber source also had an effect on cellulase activity. There was no significant difference in cellulase activity between groups LM and LJ, but cellulase activity in the jejunum of the HM group was significantly lower than that of HJ group, whereas those of the cecum of the HM group were significantly higher than that of the HJ group. Analysis of Microflora at the Genus Level To compare the differences in composition of the intestinal microflora at the genus level, bacterial genera with relative abundances of >0.1% were counted, and then the dominant bacteria (relative abundance ≥ 1%) were analyzed by variance analysis. Analysis of Duodenal Microflora In the duodenum of the HJ group, there were 20 phyla and 298 genera, of which 34 genera had a relative abundance of >0.1%; in the LJ group, there were 21 phyla and 312 genera, of which 32 genera had a relative abundance of >0.1%; in the HM group, there were 18 phyla and 287 genera, of which 31 genera had a relative abundance of >0.1%; and in the LM group, there were 17 phyla and 312 genera, of which 33 genera had a relative abundance of >0.1%. The differences in the dominant flora among the groups are shown in Table 6. Table 6. Analysis of duodenal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 6. Analysis of duodenal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large When a higher-fiber diet was fed, the abundance of the Lactobacillus, Pediococcus, and Ruminococcus genera were significantly higher, and that of the Pseudomonas, Escherichia, Phyllobacterium, and Proteus genera were significantly lower. In addition, the abundance of the Escherichia, Clostridium, Corynebacterium, and Megasphaera genera were significantly higher in the HJ group than in the HM group. Analysis of Jejunal Microflora In the jejunum of the HJ group, there were 25 phyla and 308 genera, of which 34 genera had a relative abundance of >0.1%; in the LJ group, there were 21 phyla and 287 genera, of which 30 genera had a relative abundance of >0.1%; in the HM group, there were 21 phyla and 266 genera, of which 23 genera had a relative abundance of >0.1%; and in the LM group, there were 20 phyla and 289 genera, of which 24 genera had a relative abundance of >0.1%. The differences in the dominant flora and in some cellulolytic bacteria among the treatment groups are shown in Table 7. Table 7. Analysis of jejunal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 7. Analysis of jejunal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Feeding a larger amount of dietary fiber led to a decrease in abundance of the genus Brevundimonas; this genus was most abundant in the LJ group. Ruminococcus showed a similar trend, but the difference was not significant. In groups fed corn stover, the abundance of the genus Helicobacter was significantly lower when high fiber levels were fed, but the opposite association was shown in the alfalfa groups. In addition, the abundance of the genus Tolumonas was significantly higher than in the corn stover groups, but there were no significant differences among the other genera. Analysis of Ileal Microflora In the ileum of the HJ group there were 25 phyla and 348 genera, of which 36 genera had a relative abundance of >0.1%; in the LJ group, there were 22 phyla and 295 genera, of which 24 genera had a relative abundance of >0.1%; in the HM group, there were 19 phyla and 309 genera, of which 29 genera had a relative abundance of >0.1%; and in the LM group, there were 24 phyla and 303 genera, of which 27 genera had a relative abundance of >0.1%. The differences in the dominant flora and some cellulolytic bacteria among the treatment groups are shown in Table 8. Table 8. Analysis of ileal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 8. Analysis of ileal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large The abundance of the genera Clostridium and Ruminococcus was significantly higher when dietary fiber content was higher, but there were no significant differences among the other genera. Analysis of Cecal Microflora In the cecum of the HJ group there were 20 phyla and 225 genera, of which 25 genera had a relative abundance of >0.1%; in the LJ group, there were 18 phyla and 212 genera, of which 24 genera had a relative abundance of >0.1%; in the HM group, there were 17 phyla and 199 genera, of which 22 genera had a relative abundance of >0.1%; and in the LM group, there were 19 phyla and 226 genera, of which 24 genera had a relative abundance of >0.1%. The differences in the dominant flora and some cellulolytic bacteria among the treatment groups are shown in Table 9. Table 9. Analysis of cecal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 9. Analysis of cecal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large The abundance of the genera Desulfovibrio and Akkermansia was affected differently by fiber level between the corn stover and alfalfa groups. The abundance of the genus Bacteroides in the LM group was significantly higher than in the other 3 groups, but the differences between the other 3 groups were not significant. When dietary fiber content was higher, the abundance of the genera Prevotella and Helicobacter was significantly greater. Screening for Cellulose Degrading Bacteria Using the CMC-Na plate and the Congo red staining methods, 8 strains with cellulolytic ability were isolated from the intestinal mucosa of the geese, comprising M4, M5, and M8 from the cecum, H2, H4, and H6 from the ileum, and S2 and S6 from the duodenum. Morphological and Biochemical Identification of 8 Strains As shown in Table 10, all 8 strains could use D-glucose and sucrose, and liquefy gelatin. Strains H2, H4, M5, and S6 were positive on the methyl red test, and only strain S6 was negative for the Voges-Proskauer (V-P) and citrate utilization tests. Strains S2 and S6 were negative on the indole test, whereas the others were positive. Strains M8 and S6 could not hydrolyze starch and only strain S2 was negative on the catalase test. Strains H6, M5, and M8 were positive on the hydrogen sulfide test, whereas the others were negative. Only strain M8 was negative on the urease test, strains M5 and S2 were negative for nitrate reduction, and strains M8 and S6 were negative for gram staining, whereas the others were positive. By reference to Microbial Taxonomy and the Berger Bacterial Identification Manual, strains H2, H4, M4, and M5 were preliminarily identified as Bacillus, strain H6 as Clostridium, strain M8 as Enterococcus, strain S2 as Staphylococcus, and strain S6 as Pseudomonas. Table 10. Physical and biochemical properties of functional strain. H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − Note: “+”represents positive, “−”represents negative. View Large Table 10. Physical and biochemical properties of functional strain. H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − Note: “+”represents positive, “−”represents negative. View Large Molecular Identification of the 8 Strains The genomic DNA of these 8 strains was extracted and used as templates for PCR. The sequencing results for each strain were analyzed using BLAST, which showed that the H2, H4, H6, M4, M5, M8, S2, and S6 sequences were most homologous with Paenibacillus cookii (99%), Bacillus subtilis strain CJ2 (100%), Clostridium lentocellum DSM 5427 (99%), Bacillus licheniformis strain C1-5-8 (98%), Bacillus cereus strain P14 (99%), Clostridium cellulolyticum H10 (99%), Staphylococcus haemolyticus strain CIFRI P-TSB-72 (99%), and Pseudomonas aeruginosa strain F1 (99%), respectively. When combined with bacterial morphology, strain H2 was identified as Paeniacillus mucilaginosus, H4 as Bacillus subtilis, H6 as Clostridium, M4 as Bacillus licheniformis, M5 as Bacillus cereus, M8 as Clostridium cellulolyticum, S2 as hemolytic Staphylococcus, and S6 as Pseudomonas aeruginosa. Orthogonal Combination Analysis of Strains for Cellulase Production Because strains H2 and S2 were antagonistic to most other strains, the other 6 strains were isolated and tested for cellulase production. As shown in Table 11, the enzyme activity of strain S6 was the highest, at 7.52 ± 0.24 U/mL. The combined enzyme activity of 2 strains was mostly additive, but some strains also showed synergistic effects. The combined enzyme activity of strains M5 and S6 was 9.86 ± 0.22 U/mL, and this was therefore considered to be the optimal combination. Table 11. Orthogonal combination analysis of cellulase activity (U/mL). H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 View Large Table 11. Orthogonal combination analysis of cellulase activity (U/mL). H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 View Large DISCUSSION In this study, we aimed to investigate the effect of dietary fiber quantity and source on the microflora of goose intestines. We first studied the influence of dietary fiber on digestibility and immune organ indices, then analyzed the effects on the flora in the various intestinal segments by high-throughput sequencing. Finally, we isolated and identified 8 strains of bacteria with cellulose-degrading ability from the geese. Dietary fiber plays a vital role in the nutrition and physiology of animals. It can promote intestinal movement, increase the emptying rate of chyme (Franklin et al., 2002; Girard et al., 1995), improve gastrointestinal function, promote the digestion, absorption, and utilization of nutrients, and enhance immune function. In this study, a greater quantity of fiber in the diet increased feed intake, the digestibility of NDF, ADF, and CF, and immune organ indices, findings that are consistent with those of Gao et al. (2003) and Wang et al. (2008). However, there were some limitations to this study. In particular, the digestibility measured here was total tract digestibility, which is less specific than ileal digestibility, because it is altered by microbial activity in the hindgut. The intestinal flora is a very complicated system that plays a significant role in the immunity, nutrition, and metabolism of geese (Hooper and Gordon, 2001; Manco, 2012; Cryan and Dinan, 2012; Cryan and O’Mahony, 2011). The function of the microbial flora depends on its composition. Geese are capable of utilizing cellulose because microorganisms in the intestinal tract secrete digestive enzymes, especially cellulase. Yu et al. (1998) found that the addition of fiber to the diet, especially alfalfa powder, could increase the activity of cellulase in the cecum, and we have generated consistent data. In addition, cellulase activity varied between the intestinal segments, and was highest in the cecum (Huang et al., 2010). Dietary composition has a major influence on the composition of the intestinal microflora (Guo et al., 2011; Malenovic et al., 2009), and a dynamic balance in the intestinal microflora is essential for efficient absorption and metabolism of nutrients (Belanche et al., 2012). Variation in intestinal nutrition, metabolism, and immunity leads to contrasting bacterial composition in the various intestinal segments (Finkmann et al., 2000). Awati et al. (2005) showed that the intestinal microflora are affected by dietary composition, and dietary fiber is an important component of this. In our study, we found that the species present and the relative abundance of the dominant bacteria in the various intestinal segments were different, and were affected by the amount and source of dietary fiber. Bacteroides, Ruminococcus, Clostridium, and Pseudomonas spp. are important cellulolytic bacteria (Desvaux et al., 2000). In this study, the abundance of Bacteroides and Ruminococcus in the cecum was much greater than in other intestinal segments (Tables 6 to 9), implying that the cecum plays a significant role in fiber utilization by geese, consistent with previous studies. The Ruminococcus genus includes R. flavefaciens, R. albus, R. bromii, R. callidus, and R. gaavus, which directly participate in the decomposition of cellulose. A high-fiber diet significantly stimulated the growth of Clostridium in the duodenum and ileum, indicating that Clostridium likely has its effects in the duodenum or ileum. When diets containing different fiber sources or quantities were compared, the frequency of many bacterial genera were different, indicating that fiber absorption and utilization by geese are complex processes, and the result of synergism among the species comprising the intestinal microflora. Guo et al. (2010) screened a compound microbial system with the ability to degrade cellulose, which was composed of Clostridium, Bacteroides, Pseudomonas, and Alcaligenes. In our study, Clostridium, Bacteroides, and Pseudomonas existed in most intestinal segments, and they were abundant, implying that they might be an important part of the cellulose-degrading machinery in goose intestine. The homology comparison of 16S rDNA sequences obtained from the microflora is an important method for the identification of bacteria, and when this was combined with physiological and biochemical methods, we successfully identified 8 strains isolated from the gut of the geese, all of which had already been detected by high-throughput sequencing. The decomposition of dietary fiber by geese is a complex process involving multiple microbial interactions. 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Effect of different sources of dietary fibre on growth performance, intestinal morphology and caecal carbohydrases of domestic geese . Br. Poult. Sci. 39 : 560 – 567 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Response of goose intestinal microflora to the source and level of dietary fiber

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey045
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Abstract

ABSTRACT Geese are capable of digesting and making use of a high-fiber diet, but the mechanism is not well understood and would be of great significance for the development and utilization of roughage resources. In this study, we investigated the effect of dietary fiber (source: corn stover and alfalfa, included at 5% or 8%) on microflora in goose intestines. We used 35-day-old Carlos geese in which we first studied the influence of fiber ingestion on diet digestibility and immune organ indices of geese and found that high dietary fiber (8% content) significantly increased feed intake, the digestibility of neutral and acid detergent fiber, and thymus, bursa, and spleen size. Subsequently, we investigated the effect of dietary fiber on the microbial flora in the various intestinal segments by high throughput sequencing. The bacterial diversity and relative abundance were significantly affected by the type and amount of dietary fiber fed, including that of cellulolytic bacteria such as Bacteroides, Ruminococcus, Clostridium, and Pseudomonas spp. Finally, we isolated and identified 8 strains with cellulolytic ability from goose intestine and then analyzed their activities in combination. The optimal combination for cellulase activity was Cerea bacillus and Pseudomonas aeruginosa. This study has laid a theoretical and practical foundation for knowledge of the efficient conversion and utilization of cellulose by geese. INTRODUCTION Dietary fiber has a complex structure and composition and plays a key role in the nutrition and physiology of animals (Girard et al., 1995; Franklin et al., 2002). The digestion and utilization of dietary fiber by geese is a complicated process, which is accomplished by physical digestion, chemical digestion, and microbial digestion (Duke et al., 1972; Clemens et al., 1975). The digestion and utilization of crude fiber by geese occurs principally in the gizzard and cecum: plant cell walls can be broken down in the mature gizzard, which permits digestion of the cell contents (Duke et al., 1972; Clemens et al., 1975), and the cecum contains a large number of microorganisms that can utilize various sources of dietary nutrition, including crude fiber (CF), neutral detergent fiber (NDF), and acidic detergent fiber (ADF) (Ley et al., 2008; Wang et al., 2009). The intestinal microflora, a large and complex microecosystem, is crucial to the health of geese and their digestion of nutrients (Hooper and Gordon, 2001; Manco, 2012; Cryan and Dinan, 2012; Cryan and O’Mahony, 2011). Many studies have been carried out on the species and quantity of, and the nutrient digestion by, goose intestinal microorganisms, especially with regard to the interactions between microorganisms and fiber (Luo and Zhu, 2007; Lumpkins et al., 2010). Previous studies have shown that gut commensals adapt to dietary composition (Wagner and Thomas, 1978; Savory, 1992). Awati et al. (2005) found that the level of dietary fiber can influence the predominant species of bacteria comprising the intestinal flora of healthy animals. In particular, the abundance of Bifidobacteria and Lactobacillus was higher when high-fiber diets were fed, whereas the abundance of Escherichia coli and Salmonella were lower within a specific range of dietary fiber content. To date, more than 200 naturally occurring microorganisms (fungi, bacteria, and actinomycetes) with cellulolytic ability have been identified. There are many microorganisms present in the normal goose intestine that have the capacity to degrade cellulose (Godden et al., 1992; Apj et al., 1994), but many have not been isolated and identified because of a lack of specific isolation and culture methods. However, the MiSeq PE300 sequencing system (Illumina, Inc., San Diego, CA) sequences at enough depth to identify low abundance bacteria accurately (Lumpkins et al., 2010), and this has become an important tool for the conduct of metagenomic studies. In this study, paired-end sequencing on the MiSeq PE300 platform was used to study the effects of various fiber sources and inclusion levels on the intestinal microflora of geese. World population growth has led to international food shortages; therefore, high-fiber resources that cannot be used by humans have attracted more attention for their potential use in animal feed. Improvement in the utilization efficiency of dietary fiber may be one of the more effective means to alleviate the shortages in food and feed resources in China. Although fiber digestion and utilization, and the intestinal microecological environment of geese, have been extensively studied (Ley et al., 2008; Wang et al., 2009; Liu et al., 2011), the microscopic structure of their digestive tract, the diversity of bacteria, and the key bacteria involved in fiber digestion require further elucidation. It has been reported that sources of dietary fiber differ in their digestibility and lead to differences in the performance of animals fed on them (He et al., 2015; Jørgensen et al., 1996; Jiménez-Moreno et al., 2010). Corn stover and alfalfa are distinct fiber sources, and our previous study showed that they have different digestibility (Lou et al., 2010); however, the mechanism of this is unknown. Furthermore, the level of fiber in the diet is known to affect the digestibility of the diet. It has been reported that 5% CF in the diet is the normal level for geese (Wang et al., 2008). Therefore, the present study was conducted to determine the response of goose intestinal microflora to the source (corn stover vs. alfalfa) and level of incorporation (5% vs. 8%) of dietary fiber, and then to identify the species of cellulolytic bacteria affected. MATERIALS AND METHODS Materials One hundred eighty 35-day-old healthy Carlos geese (male) were randomly divided into 4 groups (n = 45). For each group, 3 pens containing 15 geese each were created. The geese were fed a diet containing 5% corn stover CF (the LJ group), 8% corn stover CF (the HJ group), 5% alfalfa CF (the LM group), or 8% alfalfa CF (the HM group). The 6–8/m2 pens were situated in half-open sheds. The geese were raised on the floor and fed 3 times daily, and had free access to water. The composition of diets is shown in Table 1. During the feeding period, 2 healthy geese were randomly selected from each pen to enter a digestion trial using the total collection method. After 42 d of feeding, the geese were killed by decapitation method, and the intestinal mucosa and immune organs were collected. This study was approved by the Institutional Animal Care and Use Committee of Jilin Agricultural University (IACUC-JLAU201503). All procedures were performed according to international guidelines concerning the care and treatment of experimental animals. Table 1. Composition of diets (%). Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Note:1per kg of premix contains: vitamin A, 800,000 IU; vitamin D, 160,000 IU; vitamin E, 500 IU; Zn 8,000 mg; Mn, 6,000 mg; Fe, 6,000 mg; Cu, 800 mg; I, 35 mg; Se, 30 mg; vitamin K, 50 mg; vitamin B1, 80 mg; vitamin B2, 250 mg; vitamin B5, 220 mg; vitamin B3, 2,000 mg; vitamin B6, 300 mg; vitamin H, 10 mg; vitamin B9, 25 mg. 2ME (metabolizable energy) was calculated, whereas the levels of the other nutrients were measured. HJ group: geese fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 1. Composition of diets (%). Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Composition HJ LJ HM LM Corn (%) 50 53.2 53 57.2 Soybean meal (%) 16.2 25 18.2 21 Fish meal (%) 7 5 4 5 Corn protein (%) 2 2 2 2 Corn stover (%) 18 8 — — Alfalfa (%) — — 16 8 Cottonseed oil (%) 3 3 3 3 Dicalcium phosphate (%) 1 1 1 1 Limestone powder (%) 1.5 1.5 1.5 1.5 Salt (%) 0.3 0.3 0.3 0.3 Premix1 (%) 1 1 1 1 Total (%) 100 100 100 100 Nutrient composition2 ME (MJ/kg) 11.19 11.21 11.45 11.48 CP (g/kg) 16.58 16.60 17.31 17.28 CF (g/kg) 8.02 5.02 7.98 4.96 NDF (g/kg) 32.51 22.18 30.45 20.62 ADF (g/kg) 12.14 11.16 13.12 12.14 Note:1per kg of premix contains: vitamin A, 800,000 IU; vitamin D, 160,000 IU; vitamin E, 500 IU; Zn 8,000 mg; Mn, 6,000 mg; Fe, 6,000 mg; Cu, 800 mg; I, 35 mg; Se, 30 mg; vitamin K, 50 mg; vitamin B1, 80 mg; vitamin B2, 250 mg; vitamin B5, 220 mg; vitamin B3, 2,000 mg; vitamin B6, 300 mg; vitamin H, 10 mg; vitamin B9, 25 mg. 2ME (metabolizable energy) was calculated, whereas the levels of the other nutrients were measured. HJ group: geese fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Composition of reagents and media used are as follows: 3,5-dinitrosalicylic acid (DNS) reagent: potassium sodium tartrate 200 g, DNS 10 g, NaOH 10 g, phenol 2 g, Na2SO3 5 g, distilled water to 1,000 mL. Beef extract peptone medium: beef extract 3 g, peptone 10 g, NaCl 5 g, agar 15 g, distilled water to 1,000 mL, pH 7.0, autoclaved at 121°C for 20 min. Potato dextrose agar medium (PDA medium): potato 200 g, glucose 20 g, agar 20 g, distilled water to 1,000 mL, autoclaved at 121°C for 20 min. Fermentation medium: carboxymethylcellulose sodium (CMC-Na) 5 g, beef extract 5 g, peptone 10 g, NaCl 5 g, distilled water to 1,000 mL, pH 7.0, autoclaved at 121°C for 20 min. Cellulase screening medium: CMC-Na 20 g, yeast extract 0.5 g, Na2HPO4 2.5 g, KH2PO4 1.5 g, peptone 2.5 g, agar 15 g, distilled water to 1,000 mL, pH 7.0 to 7.2, autoclaved at 121°C for 30 min. The primers used in this study are listed in Table 2. Table 2. Primers used in this study. Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ View Large Table 2. Primers used in this study. Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ Number Primer 515F 5΄-GTGCCAGCMGCCGCGGTAA-3΄ 806R 5΄-GGACTACHVGGGTWTCTAAT-3΄ P1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ P2 5΄-GGTTACCTTGTTACGA CTT-3΄ View Large Determination of Feed Conversion Ratio and Digestibility During the 42-d feeding period, body mass (BW) and feed intake (FI) were determined, and feed conversion ratio (FCR) was calculated as FCR = FI (kg)/BW gain (BWG, kg). Crude protein (CP), CF, NDF, and ADF were measured according to GB/T6432 (Chinese National Standard: Determination of crude protein in feed), GB/T6434 (Chinese National Standard: Determination of crude fiber in feed), and Van Soest methods, then the digestibility of each nutrient was calculated using the following formula: Digestibility = (a − b) / a × 100%, where a represents the intake of a nutrient, and b represents the amount of a nutrient in the feces. Immune Organ Indices Body mass and the mass of the thymus, bursa, and spleen of 6 geese per group were measured, then the immune organ indices were calculated using the following formula: Immune organ index = immune organ mass / body mass × 100%. Determination of Cellulase Activity The cellulase activity of chyme from the duodenum, jejunum, ileum, and cecum of 6 geese per group was measured according to method of Magro et al. (2016). One unit of cellulase was defined as the amount of enzyme required to produce 1 μmol/min of glucose. High Throughput Sequencing of Intestinal Mucosal Samples The genomic DNA of the intestinal mucosal bacteria was extracted using QIAamp DNA Mini kits (Qiagen, Germantown, MD), according to the manufacturer's instructions. Universal primers (515F and 806R) were used to amplify the V4 region of 16S rDNA. The PCR was performed using 15 μL of 2 × Phusion Master Mix, 3 μM of each primer, and 10 ng of genomic DNA as the template, in a final volume of 30 μL. Thermal cycling was carried out with an initial activation step at 98°C for 1 min, and DNA amplification was achieved using 30 cycles of 99°C for 10 s, 50°C for 30 s, and 72°C for 30 s, with a final extension step of 72°C for 5 min. The amplified products were separated on 2% agarose gel, then recovered. The gene library was constructed using a NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs, Ipswich, MA) and sequenced using a MiSeq PE300 (Illumina, Inc.). Evaluation of the Quality of the Sequencing Data and Species Annotation Forty-eight samples from the 4 groups (HJ, LJ, HM, LM) were sequenced, and 1,913,581 effective tags were acquired. The mean number of effective tags per sample was 39,866 and the mean length of an effective tag was 253 nt. With a similarity of 95% being required to assign a genus and of 97% being required to assign a species, effective tags were clustered into operational taxonomic units (OTUs) using Uparse software, and RDP Classifier software and the GreenGene database were used to assign a species to representative sequence sets. Analysis of Species Diversity Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/) (Edgar, 2013)was used to cluster the effective tags into specific OTUs, based on a phylip-formatted lower triangle matrix and with the furthest neighbor algorithm set at 97% sequence similarity. Representative sequences from each OTU were annotated by the Ribosomal Database Project (RDP) Classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) (Wang et al., 2007) and the GreenGene database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) (Desantis et al., 2006), and the annotation results were used to analyze the species diversity among the diet groups. Screening for Cellulose Degrading Bacteria Under sterile conditions, 5 g goose intestinal mucosa samples were placed in a conical flask, cultured with agitation for 2 h, left to stand for 1 h, then the supernatant was incubated in PDA medium after gradient dilution (10−1, 10−2, 10−3), cultivated at 37°C for 1 to 2 d, in both aerobic and anaerobic conditions. Colonies were selected for streak culture repeatedly, until a single pure culture colony was isolated. The purified strains were incubated on cellulase screening medium and cultured for 12 to 24 h. Sufficient Congo red stain (1 mg/mL) was added to cover the plates for 15 min, and then poured out. Subsequently, 1 mol/L NaCl was added to soak and wash the agar for 15 min, then the plates were examined to identify transparent circles around the colonies. Morphological Identification Gram staining and spore staining were performed, and colony morphology and cultural characteristics of the strains were determined, with reference to Bergey's Manual of Systematic Bacteriology (Buchanan and Gibbons, 1984). Molecular Identification The genomic DNA of the strains was extracted and used as the template for PCR. Bacterial 16S rRNA universal primers (P1 and P2, 10 pmol) were used for PCR reactions in a total volume of 25 μL, containing gDNA 1.5 μL, dNTP (2.5 mM) 2 μL, 10 × Ex Taq buffer 2.5 μL, and Ex Taq polymerase 2 U. Thermal cycling was carried out with using initial heat action step of 94°C for 5 min, followed by 30 cycles of 94°C for 60 s, 50°C for 45 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. The amplified products were separated on 1% agarose gels, then recovered and sequenced. BLAST was used to determine the homology of the PCR products with known bacterial sequences. Statistical Analysis Data are expressed as mean ± standard deviation (SD). SPSS 17.5 statistical software was used to analyze the data. One-way analysis of variance (ANOVA) was used to compare differences between the experimental groups. P < 0.05 was considered to represent statistical significance. RESULTS Effect of Dietary Fiber on Digestibility Comparing diets with the same fiber source, the effects of fiber content on digestibility was as follows: for the corn straw group, feed intake, FCR, and the digestibility of CF, NDF, and ADF in the HJ group were significantly higher than for the LJ group; for the alfalfa group, the digestibility of CF, NDF, and ADF in the HM group were significantly higher than in the LM group (Table 3). Table 3. Daily feed intake, feed efficiency, and nutrient digestibility for geese fed diets of contrasting fiber level and source. HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 3. Daily feed intake, feed efficiency, and nutrient digestibility for geese fed diets of contrasting fiber level and source. HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c HJ LJ HM LM FI (g) 368.50 ± 13.49a 335.99 ± 13.85b 356.13 ± 9.17a 340.74 ± 10.73b FCR 5.18 ± 0.17a 4.54 ± 0.05b 4.83 ± 0.15a,b 4.55 ± 0.10b CP (%) 48.08 ± 13.35a 40.73 ± 8.21a,b 36.74 ± 3.75b 35.74 ± 7.22b CF (%) 38.93 ± 2.21a 26.63 ± 1.56b 25.29 ± 2.72c 22.56 ± 2.38b,c NDF (%) 33.25 ± 0.62a 30.05 ± 1.08b 27.99 ± 0.61b 18.34 ± 0.59c ADF (%) 26.07 ± 0.67a 14.34 ± 0.71d 19.58 ± 0.64b 17.58 ± 0.45c a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing diets with the same fiber content, the effects of fiber source on digestibility were as follows: digestibility of NDF in the LJ group was significantly higher than in the LM group, and the digestibility of CP, CF, NDF, and ADF in the HJ group was significantly higher than in the HM group. Other values were not significantly different (Table 3). Effect of Dietary Fiber on Immune Organ Indices The bursa, thymus, and spleen indices in the 8% fiber groups (HJ and HM) were significantly higher than those of the 5% fiber groups (LJ and LM). In particular the thymus index of the HJ group was 2.76 times that of the LJ group (Table 4). Table 4. Immune organ indices for geese fed diets of contrasting fiber level and source. HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 4. Immune organ indices for geese fed diets of contrasting fiber level and source. HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b HJ LJ HM LM Thymus index (%) 0.47 ± 0.01a 0.17 ± 0.00d 0.33 ± 0.00b 0.20 ± 0.00c Bursa index (%) 0.07 ± 0.00a 0.04 ± 0.00b 0.09 ± 0.00a 0.05 ± 0.00b Spleen index (%) 0.09 ± 0.00a 0.06 ± 0.00b 0.10 ± 0.00a 0.06 ± 0.00b a–dIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing groups being fed the same amounts of fiber, the thymus index of the LM group was significantly higher than that of LJ group, but that of HM group was significantly lower than that of HJ group (Table 4). Effect of Dietary Fiber on Cellulase Activity As shown in Table 5, in the corn straw groups higher dietary fiber levels were associated with a trend towards greater cellulase activities in the duodenum, ileum, and cecum. In the alfalfa groups, fiber level affected cellulase activity differently; compared with the LM group, it was significantly lower in the jejunum and higher in the ileum and cecum of the HM group. Table 5. Cellulase activity in intestine for geese fed diets of contrasting fiber level and source. Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b a,bIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Table 5. Cellulase activity in intestine for geese fed diets of contrasting fiber level and source. Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b Intestinal section HJ LJ HM LM Duodenum 0.99 ± 0.03a 0.93 ± 0.04a 0.86 ± 0.02a 1.29 ± 0.05a Jejunum 0.93 ± 0.04a 0.93 ± 0.03a 0.80 ± 0.07b 1.07 ± 0.05a Ileum 1.16 ± 0.10b 1.06 ± 0.06b 2.31 ± 0.31a 1.12 ± 0.04b Cecum 3.27 ± 0.21a 3.14 ± 0.12a,b 3.40 ± 0.22a 3.09 ± 0.23b a,bIn the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. View Large Comparing groups fed the same amount of fiber, fiber source also had an effect on cellulase activity. There was no significant difference in cellulase activity between groups LM and LJ, but cellulase activity in the jejunum of the HM group was significantly lower than that of HJ group, whereas those of the cecum of the HM group were significantly higher than that of the HJ group. Analysis of Microflora at the Genus Level To compare the differences in composition of the intestinal microflora at the genus level, bacterial genera with relative abundances of >0.1% were counted, and then the dominant bacteria (relative abundance ≥ 1%) were analyzed by variance analysis. Analysis of Duodenal Microflora In the duodenum of the HJ group, there were 20 phyla and 298 genera, of which 34 genera had a relative abundance of >0.1%; in the LJ group, there were 21 phyla and 312 genera, of which 32 genera had a relative abundance of >0.1%; in the HM group, there were 18 phyla and 287 genera, of which 31 genera had a relative abundance of >0.1%; and in the LM group, there were 17 phyla and 312 genera, of which 33 genera had a relative abundance of >0.1%. The differences in the dominant flora among the groups are shown in Table 6. Table 6. Analysis of duodenal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 6. Analysis of duodenal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 28.80 ± 14.1a 19.58 ± 8.35b 36.96 ± 20.11a 20.16 ± 9.69b Helicobacter 8.27 ± 7.03a 5.84 ± 1.96a 7.44 ± 5.28a 12.36 ± 10.64a Pediococcus 6.24 ± 2.41a 4.71 ± 1.78b 7.89 ± 3.83a 4.88 ± 1.93b Pseudomonas 1.67 ± 0.58b 2.44 ± 1.19a 1.24 ± 0.61b 2.83 ± 0.39a Roseburia 0.11 ± 0.06a 0.17 ± 0.19a 0.07 ± 0.05a 2.67 ± 1.75a Oscillospira 1.73 ± 0.09a 1.70 ± 0.74a 2.25 ± 1.13a 1.97 ± 0.15a Desulfovibrio 1.50 ± 0.36a 1.61 ± 0.37a 3.37 ± 2.01a 1.92 ± 0.68a Escherichia 2.16 ± 0.52b 5.26 ± 3.03a 0.71 ± 0.02c 1.85 ± 0.28b Bacteroides 1.45 ± 0.44a 1.35 ± 0.92a 2.48 ± 1.63a 1.67 ± 0.81a Phyllobacterium 0.07 ± 0.03b 1.20 ± 0.58a 0.01 ± 0.00b 1.34 ± 0.58a Tolumonas 1.13 ± 0.66a 1.14 ± 0.69a 0.57 ± 0.30a 1.11 ± 0.86a Megamonas 4.31 ± 2.95a 1.49 ± 0.41a 1.23 ± 0.65a 0.80 ± 0.28a Proteus 0.12 ± 0.05b 7.77 ± 3.88a 0.11 ± 0.02b 0.66 ± 0.33c Ruminococcus 1.39 ± 0.60a 0.34 ± 0.16b 0.81 ± 0.45a 0.63 ± 0.22b Clostridium 1.06 ± 0.23a 0.42 ± 0.23b 0.48 ± 0.27b 0.28 ± 0.07b Prevotella 0.22 ± 0.04b 0.40 ± 0.32a,b 1.21 ± 0.46a 0.28 ± 0.06b Corynebacterium 1.50 ± 0.51a 0.05 ± 0.01b 0.08 ± 0.06b 0.04 ± 0.00b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large When a higher-fiber diet was fed, the abundance of the Lactobacillus, Pediococcus, and Ruminococcus genera were significantly higher, and that of the Pseudomonas, Escherichia, Phyllobacterium, and Proteus genera were significantly lower. In addition, the abundance of the Escherichia, Clostridium, Corynebacterium, and Megasphaera genera were significantly higher in the HJ group than in the HM group. Analysis of Jejunal Microflora In the jejunum of the HJ group, there were 25 phyla and 308 genera, of which 34 genera had a relative abundance of >0.1%; in the LJ group, there were 21 phyla and 287 genera, of which 30 genera had a relative abundance of >0.1%; in the HM group, there were 21 phyla and 266 genera, of which 23 genera had a relative abundance of >0.1%; and in the LM group, there were 20 phyla and 289 genera, of which 24 genera had a relative abundance of >0.1%. The differences in the dominant flora and in some cellulolytic bacteria among the treatment groups are shown in Table 7. Table 7. Analysis of jejunal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 7. Analysis of jejunal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 25.66 ± 13.32a 24.16 ± 1.04a 18.09 ± 8.42a 22.21 ± 5.88a Helicobacter 4.98 ± 2.15b 7.13 ± 4.52a 9.35 ± 4.14a 2.55 ± 0.83b Pediococcus 4.62 ± 3.15a 5.53 ± 0.95a 3.53 ± 1.97a 5.25 ± 0.94a Pseudomonas 2.84 ± 1.18a 3.31 ± 1.25a 2.27 ± 2.02a 1.89 ± 0.81a Escherichia 1.54 ± 0.69a 1.80 ± 0.35a 1.80 ± 0.39a 1.42 ± 0.35a Oscillospira 1.53 ± 0.40a 1.93 ± 0.62a 1.26 ± 0.51a 1.46 ± 0.71a Desulfovibrio 1.49 ± 0.65a 1.71 ± 0.96a 1.34 ± 0.60a 1.22 ± 0.33a Tolumonas 1.22 ± 0.78b 1.09 ± 0.19b 3.76 ± 2.15a 2.71 ± 1.30a Bacteroides 0.76 ± 0.21a 1.03 ± 0.46a 0.66 ± 0.18a 0.85 ± 0.26a Ruminococcus 0.73 ± 0.18a 0.87 ± 0.33a 0.58 ± 0.24a 0.62 ± 0.28a Prevotella 0.68 ± 0.44b 1.77 ± 0.98a 0.17 ± 0.07d 0.27 ± 0.06c Clostridium 0.39 ± 0.18a 0.35 ± 0.12a 0.21 ± 0.07a 0.19 ± 0.01a Brevundimonas 0.20 ± 0.08b 1.78 ± 0.51a 0.01 ± 0.01c 0.20 ± 0.074b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a–cIn the same row, different letters represent significant differences, with P < 0.05. View Large Feeding a larger amount of dietary fiber led to a decrease in abundance of the genus Brevundimonas; this genus was most abundant in the LJ group. Ruminococcus showed a similar trend, but the difference was not significant. In groups fed corn stover, the abundance of the genus Helicobacter was significantly lower when high fiber levels were fed, but the opposite association was shown in the alfalfa groups. In addition, the abundance of the genus Tolumonas was significantly higher than in the corn stover groups, but there were no significant differences among the other genera. Analysis of Ileal Microflora In the ileum of the HJ group there were 25 phyla and 348 genera, of which 36 genera had a relative abundance of >0.1%; in the LJ group, there were 22 phyla and 295 genera, of which 24 genera had a relative abundance of >0.1%; in the HM group, there were 19 phyla and 309 genera, of which 29 genera had a relative abundance of >0.1%; and in the LM group, there were 24 phyla and 303 genera, of which 27 genera had a relative abundance of >0.1%. The differences in the dominant flora and some cellulolytic bacteria among the treatment groups are shown in Table 8. Table 8. Analysis of ileal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 8. Analysis of ileal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Genus HJ (%) LJ (%) HM (%) LM (%) Lactobacillus 12.90 ± 5.87a 5.33 ± 1.53a 18.09 ± 11.31a 14.08 ± 9.56a Pediococcus 3.41 ± 1.47a 3.08 ± 2.57a 4.45 ± 2.78a 3.16 ± 1.32a Pseudomonas 3.30 ± 1.51a 2.22 ± 1.28a 4.56 ± 3.34a 2.52 ± 1.02a Clostridium 2.76 ± 1.09a 0.40 ± 0.16b 2.72 ± 1.43a 0.27 ± 0.14b Oscillospira 2.25 ± 0.61a 1.89 ± 0.37a 2.58 ± 0.80a 1.89 ± 0.66a Escherichia 1.92 ± 0.71a 1.5656 ± 1.05a 2.5544 ± 2.10a 1.59 ± 0.89a Turicibacter 1.72 ± 0.40a 2.98 ± 1.75a 2.46 ± 2.20a 1.43 ± 0.58a Helicobacter 1.62 ± 0.77a 1.11 ± 0.24a 4.55 ± 2.22a 1.06 ± 0.40a Desulfovibrio 1.60 ± 0.55a 1.66 ± 0.49a 2.10 ± 0.39a 1.68 ± 0.66a Bacteroides 1.29 ± 0.56a 1.31 ± 0.30a 1.19 ± 0.36a 1.39 ± 0.60a Ruminococcus 0.95 ± 0.30a 0.75 ± 0.03b 1.17 ± 0.31a 0.83 ± 0.32b Tolumonas 0.40 ± 0.24a 2.08 ± 1.51a 0.54 ± 0.18a 0.83 ± 0.80a Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large The abundance of the genera Clostridium and Ruminococcus was significantly higher when dietary fiber content was higher, but there were no significant differences among the other genera. Analysis of Cecal Microflora In the cecum of the HJ group there were 20 phyla and 225 genera, of which 25 genera had a relative abundance of >0.1%; in the LJ group, there were 18 phyla and 212 genera, of which 24 genera had a relative abundance of >0.1%; in the HM group, there were 17 phyla and 199 genera, of which 22 genera had a relative abundance of >0.1%; and in the LM group, there were 19 phyla and 226 genera, of which 24 genera had a relative abundance of >0.1%. The differences in the dominant flora and some cellulolytic bacteria among the treatment groups are shown in Table 9. Table 9. Analysis of cecal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large Table 9. Analysis of cecal microflora at the genus level. Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Genus HJ (%) LJ (%) HM (%) LM (%) Desulfovibrio 14.93 ± 6.43b 27.13 ± 7.77a 22.02 ± 6.63a 17.80 ± 7.00b Bacteroides 14.83 ± 4.58b 12.95 ± 2.92b 11.24 ± 5.71b 17.83 ± 2.16a Oscillospira 6.37 ± 1.05a 5.49 ± 0.48a 4.87 ± 0.55a 5.41 ± 1.09a Prevotella 4.49 ± 2.75a 1.74 ± 0.51b 7.48 ± 6.32a 2.56 ± 0.86b Ruminococcus 2.47 ± 0.55a 2.13 ± 0.09a 2.11 ± 0.69a 1.97 ± 0.50a Lactobacillus 1.89 ± 0.53a 1.63 ± 0.47a 1.79 ± 0.33a 2.26 ± 0.73a Megamonas 1.81 ± 0.72a 3.00 ± 0.42a 3.19 ± 1.75a 2.41 ± 0.66a Faecalibacterium 1.25 ± 0.11a 1.17 ± 0.33a 1.62 ± 0.57a 1.07 ± 0.31a Helicobacter 1.19 ± 0.40a 0.63 ± 0.24b 1.24 ± 0.84a 0.57 ± 0.15b Akkermansia 0.25 ± 0.09b 1.61 ± 1.06a 1.29 ± 0.62a 0.46 ± 0.06b Note: In the same row, different letters represent significant differences, with P < 0.05. Data were obtained from 2 geese per pen and 6 geese per treatment. HJ group: geese were fed a diet containing 8% corn stover CF; LJ group: geese fed a diet containing 5% corn stover CF; HM group: geese fed a diet containing 8% alfalfa CF; LM group: geese fed a diet containing 5% alfalfa CF. a,bIn the same row, different letters represent significant differences, with P < 0.05. View Large The abundance of the genera Desulfovibrio and Akkermansia was affected differently by fiber level between the corn stover and alfalfa groups. The abundance of the genus Bacteroides in the LM group was significantly higher than in the other 3 groups, but the differences between the other 3 groups were not significant. When dietary fiber content was higher, the abundance of the genera Prevotella and Helicobacter was significantly greater. Screening for Cellulose Degrading Bacteria Using the CMC-Na plate and the Congo red staining methods, 8 strains with cellulolytic ability were isolated from the intestinal mucosa of the geese, comprising M4, M5, and M8 from the cecum, H2, H4, and H6 from the ileum, and S2 and S6 from the duodenum. Morphological and Biochemical Identification of 8 Strains As shown in Table 10, all 8 strains could use D-glucose and sucrose, and liquefy gelatin. Strains H2, H4, M5, and S6 were positive on the methyl red test, and only strain S6 was negative for the Voges-Proskauer (V-P) and citrate utilization tests. Strains S2 and S6 were negative on the indole test, whereas the others were positive. Strains M8 and S6 could not hydrolyze starch and only strain S2 was negative on the catalase test. Strains H6, M5, and M8 were positive on the hydrogen sulfide test, whereas the others were negative. Only strain M8 was negative on the urease test, strains M5 and S2 were negative for nitrate reduction, and strains M8 and S6 were negative for gram staining, whereas the others were positive. By reference to Microbial Taxonomy and the Berger Bacterial Identification Manual, strains H2, H4, M4, and M5 were preliminarily identified as Bacillus, strain H6 as Clostridium, strain M8 as Enterococcus, strain S2 as Staphylococcus, and strain S6 as Pseudomonas. Table 10. Physical and biochemical properties of functional strain. H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − Note: “+”represents positive, “−”represents negative. View Large Table 10. Physical and biochemical properties of functional strain. H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − H2 H4 H6 M4 M5 M8 S2 S6 D-Glucose + + + + + + + + Sucrose + + + + + + + + Methyl red − − + + - + + − V-P test + + + + + + + − Indole test + + + + + + − − Starch hydrolysis + + + + + − + − Catalase + + + + + + − + H2S − − + − + + − − Urease + + + + + − + + Gelatin liquefaction + + + + + + + + Citrate + + + + + + + − Nitrate reduction + + + + − + − + Gram staining + + + + + − + − Note: “+”represents positive, “−”represents negative. View Large Molecular Identification of the 8 Strains The genomic DNA of these 8 strains was extracted and used as templates for PCR. The sequencing results for each strain were analyzed using BLAST, which showed that the H2, H4, H6, M4, M5, M8, S2, and S6 sequences were most homologous with Paenibacillus cookii (99%), Bacillus subtilis strain CJ2 (100%), Clostridium lentocellum DSM 5427 (99%), Bacillus licheniformis strain C1-5-8 (98%), Bacillus cereus strain P14 (99%), Clostridium cellulolyticum H10 (99%), Staphylococcus haemolyticus strain CIFRI P-TSB-72 (99%), and Pseudomonas aeruginosa strain F1 (99%), respectively. When combined with bacterial morphology, strain H2 was identified as Paeniacillus mucilaginosus, H4 as Bacillus subtilis, H6 as Clostridium, M4 as Bacillus licheniformis, M5 as Bacillus cereus, M8 as Clostridium cellulolyticum, S2 as hemolytic Staphylococcus, and S6 as Pseudomonas aeruginosa. Orthogonal Combination Analysis of Strains for Cellulase Production Because strains H2 and S2 were antagonistic to most other strains, the other 6 strains were isolated and tested for cellulase production. As shown in Table 11, the enzyme activity of strain S6 was the highest, at 7.52 ± 0.24 U/mL. The combined enzyme activity of 2 strains was mostly additive, but some strains also showed synergistic effects. The combined enzyme activity of strains M5 and S6 was 9.86 ± 0.22 U/mL, and this was therefore considered to be the optimal combination. Table 11. Orthogonal combination analysis of cellulase activity (U/mL). H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 View Large Table 11. Orthogonal combination analysis of cellulase activity (U/mL). H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 H4 H6 M4 M5 M8 S6 H4 3.44 ± 0.12 3.56 ± 0.09 4.33 ± 0.11 6.53 ± 0.08 4.22 ± 0.12 7.34 ± 0.25 H6 3.56 ± 0.09 4.31 ± 0.13 3.21 ± 0.19 6.43 ± 0.14 4.22 ± 0.16 7.12 ± 0.21 M4 4.33 ± 0.11 3.21 ± 0.19 4.49 ± 0.12 6.77 ± 0.15 4.46 ± 0.14 7.77 ± 0.19 M5 6.53 ± 0.08 6.43 ± 0.14 6.77 ± 0.15 6.55 ± 0.09 6.17 ± 0.18 9.86 ± 0.22 M8 4.22 ± 0.12 4.22 ± 0.16 4.46 ± 0.14 6.17 ± 0.18 4.23 ± 0.18 6.12 ± 0.14 S6 7.34 ± 0.25 7.12 ± 0.21 7.77 ± 0.19 9.86 ± 0.22 6.12 ± 0.14 7.52 ± 0.24 View Large DISCUSSION In this study, we aimed to investigate the effect of dietary fiber quantity and source on the microflora of goose intestines. We first studied the influence of dietary fiber on digestibility and immune organ indices, then analyzed the effects on the flora in the various intestinal segments by high-throughput sequencing. Finally, we isolated and identified 8 strains of bacteria with cellulose-degrading ability from the geese. Dietary fiber plays a vital role in the nutrition and physiology of animals. It can promote intestinal movement, increase the emptying rate of chyme (Franklin et al., 2002; Girard et al., 1995), improve gastrointestinal function, promote the digestion, absorption, and utilization of nutrients, and enhance immune function. In this study, a greater quantity of fiber in the diet increased feed intake, the digestibility of NDF, ADF, and CF, and immune organ indices, findings that are consistent with those of Gao et al. (2003) and Wang et al. (2008). However, there were some limitations to this study. In particular, the digestibility measured here was total tract digestibility, which is less specific than ileal digestibility, because it is altered by microbial activity in the hindgut. The intestinal flora is a very complicated system that plays a significant role in the immunity, nutrition, and metabolism of geese (Hooper and Gordon, 2001; Manco, 2012; Cryan and Dinan, 2012; Cryan and O’Mahony, 2011). The function of the microbial flora depends on its composition. Geese are capable of utilizing cellulose because microorganisms in the intestinal tract secrete digestive enzymes, especially cellulase. Yu et al. (1998) found that the addition of fiber to the diet, especially alfalfa powder, could increase the activity of cellulase in the cecum, and we have generated consistent data. In addition, cellulase activity varied between the intestinal segments, and was highest in the cecum (Huang et al., 2010). Dietary composition has a major influence on the composition of the intestinal microflora (Guo et al., 2011; Malenovic et al., 2009), and a dynamic balance in the intestinal microflora is essential for efficient absorption and metabolism of nutrients (Belanche et al., 2012). Variation in intestinal nutrition, metabolism, and immunity leads to contrasting bacterial composition in the various intestinal segments (Finkmann et al., 2000). Awati et al. (2005) showed that the intestinal microflora are affected by dietary composition, and dietary fiber is an important component of this. In our study, we found that the species present and the relative abundance of the dominant bacteria in the various intestinal segments were different, and were affected by the amount and source of dietary fiber. Bacteroides, Ruminococcus, Clostridium, and Pseudomonas spp. are important cellulolytic bacteria (Desvaux et al., 2000). In this study, the abundance of Bacteroides and Ruminococcus in the cecum was much greater than in other intestinal segments (Tables 6 to 9), implying that the cecum plays a significant role in fiber utilization by geese, consistent with previous studies. The Ruminococcus genus includes R. flavefaciens, R. albus, R. bromii, R. callidus, and R. gaavus, which directly participate in the decomposition of cellulose. A high-fiber diet significantly stimulated the growth of Clostridium in the duodenum and ileum, indicating that Clostridium likely has its effects in the duodenum or ileum. When diets containing different fiber sources or quantities were compared, the frequency of many bacterial genera were different, indicating that fiber absorption and utilization by geese are complex processes, and the result of synergism among the species comprising the intestinal microflora. Guo et al. (2010) screened a compound microbial system with the ability to degrade cellulose, which was composed of Clostridium, Bacteroides, Pseudomonas, and Alcaligenes. In our study, Clostridium, Bacteroides, and Pseudomonas existed in most intestinal segments, and they were abundant, implying that they might be an important part of the cellulose-degrading machinery in goose intestine. The homology comparison of 16S rDNA sequences obtained from the microflora is an important method for the identification of bacteria, and when this was combined with physiological and biochemical methods, we successfully identified 8 strains isolated from the gut of the geese, all of which had already been detected by high-throughput sequencing. The decomposition of dietary fiber by geese is a complex process involving multiple microbial interactions. 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Poultry ScienceOxford University Press

Published: Feb 14, 2018

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