Metagenomic analysis of intestinal microbiota in geese from different farming systems in Gunungpati, Semarang

The diversity of intestinal bacteria in geese correlates with environmental conditions, rearing methods, and consumed feeds. The intestinal bacteria composition is useful for the absorption of nutrition, improving the metabolism, and may be related to the immune system. This study was conducted to examine the intestinal bacteria composition and the diversity of maintained goose in aviaries and barns. This research was an observational exploratory. Five geese were taken purposively from local breeders in Gunungpati District, Semarang City. A total of 5 g of intestinal contents from each sample was used for microbial genome isolation. Then, the genome was amplified to collect 16S rRNA gene region V3‐V4. The amplicons were then sequenced using the next generation sequencing (NGS) method (Illumina high‐throughput sequencing; paired‐end reads) and analyzed using QIIME2 to identify bacterial species. In addition, GC‐MSwas performed to identify and measure fatty acid contents in the intestinal. The results showed that both rearing and caged goose contained nine phyla of intestinal bacteria. The number of intestinal bacteria of barn geese (SU) reached 32,748 Operational Taxonomy Units (OTU); higher than aviary geese (SK), which was 11,646 OTU. The intestinal bacteria community in barn geese was approved by Phylum TM7 (Saccharibacteria candidate) (53.18%), followed by Firmicutes (32.51%) and Bacteriodetes (5.42%). Whereas on SK Firmicutes was compiled 49.34% of total OTU, TM7 (S. candidate) up to 21.17%, and Actinobacteria up to 15.99%. The abundance of TM7 may contribute to high 9,12‐octadecadienoic acid production, while Firmicutes was related to the high production of oleic acid. Based on these data, the reared geese had a more abundant diversity of bacteria than the caged one.


Introduction
Intestinal microorganisms are a bacterial community that settles inside the intestinal tract of living things, including geese. The intestinal bacteria composition is influenced by environmental conditions, feed and the host's internal body condition. It directly affects metabolism, absorption of nutrients, physiology, and immune regulation (Altizer et al. 2011). Goose is a waterfowl that can be an influenza virus reservoir in their digestive tract (Zhou et al. 2006; Harris et al. 2010; Kim 2018. However, it does not trigger to a severe condition of viralinfectiondiseases (Susanti et al. 2018). The ability to be a virus reservoir may be re lated to intestinal bacteria (Phuong et al. 2011; Mandl et al. 2015. In other case, an imbalance of microhabitat condi tions of intestinal bacterial or dysbiosis has been proven to increase the risk of diseases in the digestive system. It may actively contribute to food vulnerability in the future (Al tizer et al. 2011). It makes the geese play an essential role in preventing outbreaks of infectious disease pandemics. At present, the studies related to this case have not been focused on many researchers. Therefore, efforts to exam ine the role of the intestinal bacteria in geese as a reservoir of disease agents must be continuously investigated.
The rearing pattern is well known as one of the mech anisms for controlling intestinal microbiome conditions. Both method, aviary (caged geese) and barn (freeliving) cultivations, may contribute directly to the commensal and pathogenic bacteria in the intestinal (Leung and Ko privnikar 2016). Previous research from (Dominguez Bello et al. 2010) showed that the barn goose has more diverse and abundant intestinal bacteria comparing to the aviary. Freerange or barn geese also produced a high quality of meat and healthier (Yamak et al. 2016).
The metagenomic approach is the most appropriate technique for determining the composition of bacteria, both in the environment and the digestive tracts of geese. This technique is the basis for understanding the intesti nal bacterial taxonomy, which helps determine which one is the best cultivation method based on the condition of intestinal bacteria. By analyzing the metagenome condi tion, it will be easier to identify various bacteria species. Some uncultured bacteria in the intestinal tract are impos sible to identification using the conventional polymerase chain reaction (PCR) technique. Based on the previous research, it is necessary to study the microbiota of goose intestines to analyze the composition of intestinal bacte ria and the metabolism quality of aviary and barn farming systems related to host immunity.

Materials and Methods
This research was an exploratory observational study to analyze the composition, abundance of intestinal bacteria, and its correlations with feed and environmental condi tions. Aviary geese (SK) and barn geese (SU) samples were collected from the local community's goose hus bandry in Gunungpati Subdistrict, Semarang. Samples were obtained purposively with sampling criteria (inclu sions), i.e. aviary and barn geese, males or females aged at least three months, did not receive feed or drugs con taining antibiotics within two weeks. Samples obtained were excluded from the study sample if known to be lay ing eggs.
In this study, the aviary geese (the caged geese) were fed only concentrates, grains, and leftovers from house hold food waste. Then, barn geese farming was in the broader foraging area, and it may increase the intake of various types of food and nutrients from the environment, especially root and leave as plant fiber sources. However, despite having a different maintenance pattern, both the aviary and barn geese were always getting additional pel lets fed in, including mixed with rice bran, rice, and corn.

Sample preparation
Five femalegeese, which consisted of three geese from the aviary and two geese from the barn farming system, were collected. The geese were sacrificed by slaughtering ac cording to the farmer's standard procedure, then as much as 5 g intestinal contents were taken aseptically from each goose for two groups. The intestinal content of the water fowl sample was mixed per each group and homogenized using a vortex. After that, samples were collected in 3 mL microtubes and frozen at 20°C until further NGS and GCMS analysis.

DNA isolation and next generation sequencing (NGS) analysis
The microbial genome was extracted from intestinal con tents samples using the QIAamp DNA Stool Mini Kit (Qi agen, San Diego, California, US) according to the man ufacturer's protocol. The extracted DNA was stored in the 20°C freezer. Species identification was performed by amplifying 16S rRNA genome in the V3V4 region for accurate and precise results (Dennis et al. 2013; Yarza et al. 2014). The amplification process was run using Il lumina HiSeq 2500 platform for 20 cycles according to a procedure by Holm et al. (2019). The primers used were forwardprimer (5′ACTCCTRCGGGAGGCAGCAG3′) and reverseprimer (5′GGACTACHVGGGTWTCTAAT 3′) (Holm et al. 2019).

Bioinformatic analysis
The 16S sequence metagenomic analysis was performed using QIIME2 (Ver. 2019.4) (Caporaso et al. 2010). Pairedend files were demultiplexed using the demux plu gin. Then, quality control was performed on each sample using the Dada2 plugin (Callahan et al. 2016). Further more, the diversity index value was generated using six diversity indexes: Shannon (Shannon and Weaver 1949), Simpson (Simpson 1949

GC-MS analysis
Predicted bacteriasideproduct shortchain fatty acids (SCFAs) were analyzed using gas chromatographymass spectrometry (GCMS). A total of 20 g of intestinal con tent was dried at room temperature for three days to re duce water content, then 250 mg of sample was taken, and mashed using a mortarpestle and wrapped in filter paper and placed in the Soxhlet apparatus extraction tool. The sample was dissolved in 250 mL of nhexane to extract the fatty acid group components. The Soxhlet extraction procedure was conducted by modification steps from En eroth et al. (1968) and Batta et al. (2002), and repeated for 12 cycles for each sample. The extracted intestinal content samples that had been obtained were evaporated at 68°C for 6 h to get their constant weight before being injected into the GCMS tool. The GCMS process was performed using Shimadzu QP2010S with AGILENTTJ% W HP5 column (30 m long, ID 0.25 mm) and helium as the carrier gas. MS op erating conditions were as follows: ionization induced by electrons (EI) at 70 eV and ion source temperature up to 250°C. This compound was identified by conducting a li brary search using Shimadzu NIST / WILEY + mass spec tral database.

Result
Based on the analysis, both aviary (SK) and barn (SU) geese were maintained in a closedarea or did not mixed with other waterfowl or poultry species. However, the foraging area of barn goose was more extensive than the aviary. Therefore, it can be seen that aviary geese consume more starch than plant fibers. Intestinal bacteria can utilize the food substrate to modulate the goose's digestive and immune systems' de velopment and function. Instead, bacteria get habitat and nutrients for growth. The composition of intestinal bacte ria is influenced by feed intake. Interaction between vari ous bacteria in the intestinal can also increase growth and reduce the risk of enteric infection by pathogens.
The metagenomic analysis results showed differences in the diversity and abundance of intestinal bacteria be tween aviary and barn geese. There were at least nine phyla that have been identified from intestinal content. The abundance of reared goose intestinal microbiome reached 32,748 Operational Taxonomy Unite (OTU), more than the goose caged (11,646 OTU). The relative composition of each phylum is shown in Figure 1. In SU geese, intestinal bacteria were dominated by Phy lum TM7 (53.18%), followed by Firmicutes (32.51%) and Bacteriodetes (5.42%). Six other phyla (Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Synergis tetes, and Verrucomicrobia) were in the range of 0.69 3.11%. Whereas in SK, intestine colonies dominated by Phylum Firmicutes (49.34%), followed by TM7 of 21.17% and Actinobacteria 15.99%. Six other phylum (Bacte riodetes, Cyanobacteria, Planctomycetes, Proteobacteria, Synergistetes, and Verrucomicrobia) were in the range of 0.433.94%. Although the diversity of intestinal bacteria in SU individuals was abundant, most phylum (Cyanobac teria, Planctomycetes, Proteobacteria, and Verrucomicro bia) only have a composition <1.00% of total intestinal bacteria. While in SK, the composition of almost all bac terial phylum, except Synergistetes and Verrucomicrobia, was more than 1.00 At the lower taxonomic levels, aviary goose's bacteria were dominated by the F16 family (23.09%), followed by Ruminococcaceae (16.16%), Coriobacteriaceae (14.42%), Clostridiales (9.55%), Lactobacillaceae (7.25%), Lach  (Figure 2). Eight families were in the range of 13%, and nine other families were bellowing 1.00%. The intestinal bacteria in barn geese was dominated by Rs045 family (family groups of Saccharibacteria candidate) (55.04%), followed by Ruminococcaceae (15.62%), Clostridiales (7.50%), and Christensenellaceae (2.47%). Six families were in the range 12.46%, and 12 other families were in the range <1.00. The Paenibacillaceae family was only found in the aviary goose (2.07%) and was not found in the barn goose. Overall, the barn goose has higher bacte ria composition, which was 31,217 OTU; compared to the aviary goose, which was 9,693 OTU. The composition of intestinal bacteria in barn goose was more diverse than the aviary. It can be seen from the number of species that have not been identified. In aviary goose, approximately 4.78% of species were not identi fied, whereas, in barn goose, only around 3.02% of species were not identified. Of the total bacteria (Figure 1), sev eral OTUs were shared species owned by both aviary and barn goose.
The appearance of intestinal bacterial density shows that some OTUs identified from SK has a darker color than SU (Figure 3). It shows that some bacteria may be abundant in the barn but lower in aviary geese. The di versity shown by the diversity index shows that SU geese have higher variability (Table 1). Overall, there were no anomalies, which are deviations of one or more values to the other values, in the diversity index. It shows that all parameters of the diversity index state that barn goose has higher intestinal bacterial diversity than the aviary.
Intestinal bacteria indirectly play an essential role in regulating host metabolism through the production of vi tamin compounds, essential amino acids, and shortchain fatty acids (SCFAs). Therefore, the abundance of in testinal bacteria has an impact on the production of these SCFA. The results of GCMS analysis showed that the intestinal content was composed of SCFA, hexadecanoic acid, 9octadecenoic acid (Z), methyl ester, and oleic acid. While in barn goose, there were hexadecanoic acid and 9,12octadecadienoic acid (Z, Z) ( Table 2).

Discussion
In general, most people perform geese farming as a source of food and pets. Goose rearing patterns in the commu nities generally do not develop husbandry on an extensive and intensive (closed) scale, as does the community in Gu nungpati, Semarang. Goose has a smaller proportion of the digestive tract, so that the transit time for food to be digested is shorter than a mammal. However, based on the analysis of intestinal contents shows that most of the organic fiber has been completely digested (unpublished data). This research focuses on studies related to culti vation techniques, environment-moreover, diversity of goose intestinal bacteria that conducted in Gunungpati, Se marang.
Intestinal bacteria play an essential role in the metabolism of carbohydrates and fibers, proteins, and lipids. Cultivation patterns affecting livestock access in foraging activity. The root content of plants and leaves of grass is the dominant component found in the barn goose's gizzard. It indicates that the grass and the remaining or ganic material is the leading fiber supply of geese. High fiber is a component of nonstarch polysaccharide (NSP) known as the primary material digested or fermented by intestinal bacteria. The fermentation of NSP breakdown products produces shortchain volatile fatty acids (SCFAs) (Jamroz et al. 2002), absorbed by the mucosa and catabo lized.
In this study, the most dominant phyla is TM7 in barn goose and Firmicutes in the aviary, followed by Actinobacteria, Bacteroidetes, Planctomycetes, and Pro teobacteria, which is in line with a study by Yang et al. (2018). Wang et al. (2016) also showed that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in poultry feces. Furthermore, more microbiota phyla are found in the cecum than in other parts of the intestine.
The domestication process of the goose aims to in creases body mass by increasing carbohydratebasedfeed. It increases the Firmicutes colony (Grond 2017). Firmi cutes produces SCFA as a byproduct of the fermentation process, which can be directly absorbed by the host in testinal cell (den Besten et al. 2013). Several studies have shown a positive relationship between the excess quantity of Firmicutes and metabolic functions. Thus, prebiotic containing feed, such as Bacillus subtilis and Enterococ cus faecium, can increase nutrient uptakes and general metabolic efficiencies .
In contrast to the high Firmicutes density in the aviary goose group, barn goose bacteria is dominated by TM7 bacteria. The TM7 is known as a Saccharibacteria candi date that is predicted to be epibiotic parasites growing on the other bacteria surface (He et al. 2015). The increase in the density of Saccharibacteria candidates remains un clear. However, it may cause by maintenance patterns of goose, which the bacteria density increases in the dryland rearing (Zhao et al. 2019) and high cellulose consumption (He et al. 2015). Saccharibacteria candidates are predomi nantly found in the duodenum (Zhu et al. 2020), where the cellulose remains and not split into smaller components. The polysaccharides, especially the cell wall, mostly de graded by Bacteroidetes (Thomas et al. 2011). However, the abundance Bacteriodetes phyla in waterfowl, espe cially goose, is still not well understood. It is likely due to differences in diet, and the wide dietary range between wa terfowl species (further investigations of this relationship are needed). In this research, the Firmicutes are mostly composed of bacteria from the Clostridia family and may contain pathogenic bacteria Clostridium botulinum, well known as avian botulinum infection agent, but it is needed further analysis to identify the specific pathogenic species.
Bacteroides are an important bacteria related to fibrol ysis and or active fermentation of microbial ecosystems in the intestine (den Besten et al. 2013). Bacteroides are thought to be related to this part's specific role in the health and performance of poultry through fermentation prod ucts, SCFA, with host's genes (Pan and Yu 2014). Also, there are Actinobacteria bacteria that are more dominant in aviary goose than barn goose. The abundance of Acti nobacteria is positively correlated with the fiber intake (Dominianni et al. 2015).
The primary function of Proteobacteria in the digestive tract of poultry is not yet certainty known. However, an increase in the quantity and abundance of Proteobacteria groups increased in wild waterfowl species associated with the ocean ecosystem, 57.5%, compared to the terrestrial environment that only 45.5% (Grond 2017). Proteobacte ria are also known to be the most essential intestinal bac teria involved in the degradation of active acid herbicides (Liu et al. 2011), indicating the possible role of detoxifi cation in the digestive tract.
Geese do not have a complete metabolic cycle to me tabolize polysaccharides until they are ready to be ab sorbed. Based on research, as many as 20% of genes in poultry intestinal bacteria are the genes responsible for polysaccharides metabolism (Thomas et al. 2011; Singh et al. 2014, including major enzymes such as carbohy drate esterase, amylase, and glycoside hydrolase, which less on geese (Beckmann et al. 2006; Yeoman et al. 2012. Beside, intestinal bacteria provides unique compounds that can improve the quality and viability of duck poul try. During polysaccharide digestion, microbiota produces various kinds of SCFA, mostly acetate, propionate, buty late, valerate, isobutylene, and isovalerate, and vitamin K (Yeoman et al. 2012). Intestinal bacteria can utilize the food substrate to modulate the development and function of the digestive and immune systems in swan hosts. In stead, the host is a permissive habitat and nutrition for bac terial colonization and growth. Intestinal bacteria can be affected by feed, and usually, different feed interventions are used by feed production to increase goose growth and reduce the risk of enteric infection by pathogens.

Conclusions
The barn pattern provides more opportunities for the goose to expand the foraging areas, which may increase the in take of various types of organic fibers. Various feed di rectly contributes to the diversity of intestinal bacteria. It was proved from an abundance of intestinal bacteria in barn goose, which reaches 32,748 Operational Taxonomy Unite (OTU), while aviary goose is 11,646 OTU. It may also correlate with bacteria diversity, which showed that barn geese have a higher diversity score in overall indices. However, the highest bacteria composition in barn geese was Saccharibacteria candidate phylum, followed by Fir micutes, and the opposite in the aviary geese. Meanwhile, the abundance of Saccharibacteria may contribute to high 9,12octadecadienoic acid production, while Firmicutes is related to the high production of oleic acid. Nevertheless, further study is needed to help in understanding the roles of bacteria in terms of SCFA production.