Effects of oor- and net-reared systems on the intestinal growth and microbial diversity in the cecum of ducks

Rearing systems can affect livestock production directly, but the effects of oor-reared systems (FRSs) and net-reared systems (NRSs) on the intestinal growth states and microbial diversity in the cecum of ducks are largely unclear. Methods The ducklings in this study were randomly divided into FRS and NRS groups, weighed at 4, 8 and 13 weeks, respectively, then the duodenum, jejunum, ileum and cecum were sampled and measured, and the content of cecum were analyzed by 16S RNA. The values of relative weight (RW), relative length (RL) and RW/ RL of four intestinal segments in FRS were signicantly higher than that in the NRS during week 4, 8 and 13 (p < 0.05). A total of 157 genus were identied from ducks under the two systems,the dominant microorganisms in both groups were Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria at phyla level. The distribution of microorganisms in cecum of two groups showed signicant separation in three time periods, and the value of Simpson index in FRS was signicantly higher than NRS at 13 weeks (p < 0.05).Five differential microorganisms and 25 differential metabolic pathways were found in the cecum at week 4, 7 differential microorganisms and 25 differential metabolic pathways were found in the cecum at week 8, and 4 differential microorganisms and 2 differential metabolic pathways were found in the cecum at week 13. There were differences in intestinal growth and microorganism between FRS and NRS ducks.


Introduction
Digestion and nutrient absorption are the basic functions of the intestine and mainly occur in the small intestine, which is also the longest part of the digestive tract. The mucosa is a crucial component of the small intestine wall with many nger-like villi extending from the mucosal layer to the lumen, increasing the surface area of the small intestine 600 times compared to that of the whole intestinal cavity, and the nutrients in intestinal content can be easily absorbed because the villi have tight blood capillaries with thin vascular walls [1][2][3]. Premature mice with poor weight have lower villus height, crypt depth, cell proliferation, goblet cells and Pan's cells than normal mice [4]. Thus, increasing the length and weight of the intestinal tract helps to expand the digestive area of food and promote the digestion and absorption of nutrients.
Intestinal microorganisms are known as the "second genome" of the host, and approximately 35% of microbial enzymes in the intestine can be utilized by the host. Intestinal microorganisms play an important role in body growth and health by impacting intestinal villus and crypt morphology, nutrient metabolism regulation, mucosal immune activation, energy-rich short-chain fatty acid production, host behavior regulation, intestinal epithelial cell repair and pathogenic microorganism resistance [5][6][7][8][9]. Food rapidly passes through the front of the intestinal tract but stays for several hours in the tail end of the tract [10]. The cecum, as the main site of intestinal microbial colonization and the main area of microbial anaerobic fermentation with the highest content of short-chain fatty acids, has a higher fermentation ability than the small intestine [11], is considered to be of highest importance in poultry health and is a major pathogen reservoir [12][13][14]. The abundance and diversity of cecum microorganisms are in uenced by many factors [15][16][17], and rearing systems are an important factor. The microbial diversity in the cecum of wild red-crowned cranes is lower than that of captive and arti cial cranes, and the microorganisms composition is also signi cantly variable [18], which is consistent with the results of Kakapo parrots, Antarctic seals and wild captured rodents [19,20]. However, different from the above consequences, the microbial abundance of the cecum of outdoor Dagu chickens is higher than that of cage chickens [21].
After China's accession to the WTO, the export share of duck's main and by-products, such as duck meat, duck egg and down, has been greatly increased. According to the statistics of FAO, the number of ducks raised in China and the number of ducks in stock have ranked rst in the world in recent years. The oorreared system (FRS) and net-reared system (NRS) are the two main systems of intensive duck production; the FRS is bene cial for muscle growth and product quality [22], while the NRS can allow excreta to be removed through metal nets, thus keeping the environment clean. In this study, we aim to perform a comprehensive assessment of the intestinal growth and microorganisms in the cecum of ducks in an FRS and NRS. The results of this study will help to offer useful information for selecting an appropriate and healthy rearing system for ducks and provide a theoretical and practical reference for the further study of ducks in rearing systems.

Laboratory Animals
The Nonghua sheldrake ducks used in this experiment were provided by the poultry raising experimental farm of Sichuan Agricultural University. A total of 180 healthy ducks were randomly divided into an FRS and NRS equally after brooding with a feeding density of 4-5/m 2 , and the intake of feed and water for each duck were ensured ad libitum during the experiment to meet the National Research Council requirements. Table 1 shows the nutritional standards of the different stages of the duck. Thirty ducks in each group were randomly selected at 4, 8 and 13 weeks, respectively, and euthanized by cervical dislocation after fasting for 12 h. All animal handling procedures were reviewed and approved by the Animal Ethics Committee of Sichuan Agricultural University (Ya'an, China).

Measurement of Intestinal Growth
The intestine of FRS and NRS was taken out and tissues such as pancreas and fat attached to the intestine were removed; one end of the intestine was xed on a glass plate wetted with distilled water and gently straightened to measure the length of the duodenum, jejunum, ileum and cecum when the intestine no longer retracted. The contents of the intestine were removed, and the aforementioned four intestinal segments were weighed with an electronic balance. RW/RLrelative length (RL), relative density (RW) and RW/RL were calculated. RL = intestinal length (cm)/live weight (kg) RW = intestinal weight (g)/live weight (kg)

Determination of Cecum Microorganisms
The intestinal contents of 5 ducks in each reraring system were randomly selected at 4, 8 and 13 weeks respectively. Place the obtained contents of intestines in the marked EP tubes and put them into liquid nitrogen immediately. The 16S rRNA gene library was constructed by extracting total microbial DNA from intestinal contents and amplifyed 16S rRNA sequences of all microorganisms. PCR products were puri ed using Agencourt Ampure XP beads (Beckman, USA) accounting to the instructions of manufacturer nd quanti ed using the PicoGreen dsDNA Assay kit (Invitogen, USA), equally combined and followed by gel puri cation using a QIAquick Gel Extraction Kit (Qiagen, USA), and then requanti ed by PicoGreen. The prepared DNA library was then sequenced using the MiSeq platform (Illumina, USA). An operational taxonomic units (OTUs) table was generated using the Uparse clustering method (97 % cutoff), and all samples were rare ed to the same sequencing depth by resampling OTUs prior to downstream analysis.

Data Statistic Analysis
Mothur software (version v.1.30) and QIIME software (version 1.8) were used to evaluate the alpha diversity and beta diversity of the samples. The beta diversity distance matrix was calculated by Qiime (version 2), and the differences were re ected on the two-dimensional coordinate map for NMDS analysis. In LEfSe analysis (version 1.5.3), the nonparametric factor Kruskal Wallis rank sum test and Wilcoxon rank sum test were used to detect the difference between the two groups, and the biomarker was obtained based on LDA>4. The gures were drawn using the R language tool (version 3.6.0). SPSS 21.0 software (IBM, USA) was used to analyze the data, and a t-test was used to analyze the signi cance of the sample data. The data are expressed as the mean ± S.D. Statistically, p<0.05 represents a signi cant difference, and p<0.01 is an extremely signi cant difference.

Results
The Effects of Rearing Systems on the Growth of the Small Intestine The values of relative weight (RW)/ relative length (RL) of the duodenum, jejnum, and ileum and the value of jejunal RW in FRS were signi cantly higher than that in the NRS at 4 weeks (p < 0.05), and cecal RW and RW/RL in the FRS were great signi cantly higher (p < 0.01). The RW/ RW of cecum in FRS was signi cantly higher than that in the NRS at 8 weeks (p < 0.05), and all other intestinal growth related indexes, including RL, RW and RW/RW, in FRS were great signi cantly higher (p < 0.01). The values of RW/RL and RW of the duck small intestine and the RL of ileum in the FRS were still great signi cantly higher than those in the NRS at 13 weeks (p < 0.01), and cecal RW/RL in NRS was also signi cantly lower than that in FRS (p < 0.05). In addition, the body weight of ducks in the two systems was also statistically analyzed and it was found that the value in FRS was signi cantly higher than NRS at 4 weeks (p < 0.05), while signi cantly lower at 8 weeks (p < 0.05). However, there was no signi cant difference in body weight between the two systems at week 13 (p > 0.05) ( Table 2). The Effects Of The Rearing System On Cecum Microorganisms A total of 4,612,553 clean tags were generated from 104 samples of duck cecal contents after splicing and ltering for quality, and each sample produced at least 25,925 clean tags. The rarefaction curve with the number of OTUs based on sequencing tended to reach a saturation plateau, suggesting that the 104 samples were adequate to estimate the phenotype richness and microbial community diversity of cecum microorganisms at a 97% similarity threshold, and broadly, the microbial abundance in the FRS was higher than that in the NRS (Fig. 1A). To investigate the microbial community of the cecum in the FRS and NRS, pairwise comparisons of microbial similarity among the two systems were performed, and analyses of the common and unique OTUs were conducted. A total of 157 genus were identi ed from ducks under the two systems. However, no speci c microorganism was found in each intestinal segment at 4,8 and 13 weeks (Fig. 1B). The bacterial phyla of the top 10 most abundant microorganisms of the cecum were determined, and the dominant microorganisms in both groups were Firmicutes(43.87%4 9.61% vs 41.58%~ 57.40%), Bacteroidetes( 20.54%~ 28.06% vs 14.14%~ 18.51%), Actinobacteria( 9.79%~ 22.67% vs 12.34%~ 33.89%) and Proteobacteria( 5.93%~ 6.41% vs 3.66%~ 6.58%); the abundance of Bacteroidetes at 13 weeks was higher than that of Actinobacteria in the FRS( 27.56% vs 9.79%), which was the exact opposite of the NRS (14.14% vs 23.69%) (Fig. 1C).
Simpson index had no signi cant difference between FRS and NRS at 4 and 8 weeks by analyzing the microbial diversity of cecal contents (p > 0.05), while the value of FRS was signi cantly higher than NRS at 13 weeks (p < 0.05) ( Fig. 2A). Besides, the distribution of cecal microorganisms in the two rearing systems were obviously separated in the three time periods of the experiment (stress1 = 0.1314, stress1 = 0.1226, stress1 = 0.1441) (Fig. 2B). LEfSe analysis was carried out to determine the speci c microorganisms at species level responsible for microorganism diversity. The abundances of Ruminococcaceae-uncultured-bacterium, Ruminococcaceae-UCG-014 and Desulfovibrio were higher in FRS than in NRS at 4 weeks, while Brachybacterium and Lactobacillus genera hold higher abundances. The genera Brevibacterium, Brachybacterium and Bacteroides were enriched in the FRS at 8 weeks, while Subdoligranulum, Akkermansia, Blautia and Collinsella were enriched in the NRS. The abundances of the genera Bacteroides and Ruminococcaceae-uncultured-bacterium were higher in the FRS at 13 weeks, while Subdoligranulum and Brachyspira were more abundant in the NRS (Table 3).  (Fig. 3A). There were 25 signi cantly different pathways between the two systems at 4 weeks, and 9 pathways were more abundant in the FRS, including drug resistance, environmental adaptability, energy metabolism and cell motility pathways (Fig. 3B). Similar to the results at 4 weeks, 25 different pathways, including 11 functional pathways, such as immune diseases, cofactors and vitamin metabolism, endocrine system and amino acid metabolism, were more abundant in the FRS at 8 weeks (Fig. 3C). However, only cofactors and vitamin metabolism were more abundant in the FRS at 13 weeks, while the abundances of the translation pathway were higher in the NRS (Fig. 3D).

Discussion
The host and environment both in uence the microorganisms of the cecum, and environmental factors are more signi cant than the host [23,24]. The FRS and NRS are two main methods of intensive farming of ducks. The FRS is the most primitive method of duck farming in China due to its low cost and high meat quality. However, FRS require a particular rearing area and frequent replacement of cushions, which are still challenges, and diseases occur easily in FRS due to direct contact with feces. Currently, most farmers build grid structures approximately 60 cm above the ground and lay metal nets to remove excreta, but the cost is relatively high, and the cleaning and disinfection of the nets are inconvenient. In this study, we reared ducks with the same density, intake and drink under uni ed management in both an FRS and NRS to determine the differences in the intestinal growth and microorganisms of the cecum.
The growth of the small intestine in the outdoor environment, such as grazing and arti cial grassland, was signi cantly higher than that in the cage [21,25], and a similar situation appeared in this experiment. Both intestinal relative length, which can re ect the intestinal capacity, and the ratio of relative weight to relative length, which could re ects intestinal motility, were higher in FRS, suggesting that ducks reared in oor system have stronger intestinal peristalsis ability and larger food digestion areas, and it may result from increased activity in a swimming pool. Many reports have shown that the length and weight of poultry intestines are affected by the level of ber, for example, dietary sun ower hulls could increase the length of the small intestine of broilers [26]. Considering the speci c situation of this experiment, it may be because in addition to arti cial feeding, ducks in the FRS also consume mattresses on the ground and algae in ponds, which increase their ber levels. Therefore, it can be concluded that FRSs is more conducive to duck intestinal growth than NRSs.
The comprehensive characterization of duck intestinal microbial communities is a critical precondition to understand and predict how rearing systems alter these communities. In order to further explore the differences between the FRS and NRS, the sequences of the cecum content were detected. Although there are no speci c microorganisms in cecum of ducks in both systems at genus level, the diversity showed a signi cant difference at 13 weeks. In general, the diversity of intestinal microbial composition of poultry gradually increases with the increase of age after birth. All kinds of microorganisms rise and fall one after another, and then tend to a relatively stable state in youth [15]. Therefore, the difference in diversity of duck intestinal microorganism in this study is convincing. Intestinal microorganisms were affected by many factors, including age, gender and environment [27][28][29]. Different rearing systems provided different growth environment for ducks, which made the diversity in ground-based ducks higher, and the result is consistent with that of Dagu chicken [21]. Considering that long-term stress could reduce the diversity of intestinal microbiota [30], this result may be because the ducks were raised on a net and unable to contact the natural environment. Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria were dominant in the cecum in both systems, and this result coincides with wild turkeys, captive broilers, caged Beijing ducks and oor-raised Landes geese [31][32][33][34].
The environmental adaptability pathways of the microorganisms in the cecum of ducks in the FRS at 4 weeks were higher, and the metabolic-related pathways were lower, including xenobiotic biodegradation and metabolism, amino acid metabolism and lipid metabolism, suggesting that the change in the living environment after the brooding period caused stress. The abundance of most pathways related to diseases, including cardiovascular disease pathways, substance dependence and viral infectious diseases, in the NRS was higher than that in the FRS at 4, 8 or 13 weeks, and these diseases cause serious harm to the body, which is consistent with previous studies [35,36].. Of the 8 different metabolic pathways at 8 weeks, the abundance of 5 of them were greater in the FRS than in the NRS, suggesting that ducks in the FRS had adapted to the environment and needed more substance, improving the meat quality. Studies have shown that the meat of outdoor chickens is darker and has a better water-holding capacity [37,38]. In addition, of the 25 functional pathways at 8 weeks, 16 pathways appeared at 4 weeks, and their abundances changed between 4 weeks and 8 weeks. Only the cofactors and vitamin metabolic pathways in the FRS were more abundant due to the higher abundance of Bacteroides and Ruminococcaceae-uncultured bacteria, while the abundance of the translation pathway in the NRS was higher at 13 weeks due to the presence of Subdoligranulum and Brachyspira, implying that the differences in the functional pathways of the microorganisms in the cecum of ducks in the FRS and NRS gradually decreased with time. As discussed before, the colonization of intestinal micro ora is a process that changes with age [39], thus the differential metabolic pathways and microorganisms are also a gradually stable process. When ducks enter the youth period and the intestinal environment is relatively stable, the in uence of FRS and NRS on duck intestinal development and microorganisms can be revealed.

Conclusion
There were differences in intestinal development and microorganism between oor-reared systems (FRS) and net-reared systems (NRS) ducks. The values of intestinal relative length, relative weight and relative weight / relative length in FRS were higher than NRS. The cecum microorganisms of ducks were obviously separated under the two rearing systems, and the diversity of cecal microorganisms was higher in FRS at13 weeks. The differential metabolic pathways of cecal microorganisms decreased with the increase of age, and the abundances of the translation pathway were higher in the NRS at week 13, while cofactors and vitamin metabolism were more abundant in FRS.
Abbreviations was the supervisor of the project. XC was a major contributor in writing the manuscript. All authors read and approved the nal manuscript. shown. In A, B and C, FRS represents oor-reared systems, and NRS represents net-reared systems. 4W, 8W and 13W represent 4 weeks of age, 8 weeks of age and 13 weeks of age.  Differential function pathways at 8 weeks. D: Differential function pathways at 13 weeks. In A, B, C and D, FRS represents oor-reared systems, and NRS represents net-reared systems. 4W, 8W and 13W represent 4 weeks of age, 8 weeks of age and 13 weeks of age.