Obesity has been becoming one of the major health problems for humans, which is characterized by excessive fat accumulation, and imbalanced energy intake and expenditure, and accompanies with low grade of systemic and chronic inflammation. It is associated with a wide range of pathological disturbances in metabolic organs and then predisposes toward type 2 diabetes mellitus (T2DM) (Hanning and Diaz-Sanchez, 2015) and cardiovascular disease (Kahn et al., 2006; Canfora et al., 2015). Further, obesity is also related to certain types of cancer, osteoarthritis, and asthma. In pigs, fatness has been regarded as a typically complex and economic trait in pig production. It brings low feed conversion rate and unfavorable fat mass. Dissection of the mechanism for the fatness in pigs not only benefits the pig industry but also provides important information for understanding human obesity, because pigs possess greater similarity with humans in nutritional and metabolic physiology compared to other animal models (Swindle et al., 2012). Fatness is affected by many factors, such as genetics, nutrition, and lifestyle as well as gut microbiome. More and more studies in humans have shown that obesity is related to the gut microbiota (Ley et al., 2006; Turnbaugh et al., 2009; Munoz-Garach et al., 2016).

Mammalian gastrointestinal tract is resided by a complex, diverse, and dynamic community of symbiotic microbes that continuously interact with the host (Hooper et al., 2002; Backhed et al., 2005). Gut microbiota has demonstrated the great significance to animals by providing a large amount of functions that host lacks, such as fermenting undigested energy substrates, stimulating the host immune system development, participating in the metabolic processes, preventing growth of harmful and pathogenic bacteria and so on (Guarner and Malagelada, 2003). The relationship between human obesity and gut microbiota composition has been established for several decades with the hallmark study by Backhed et al. (2004) which demonstrated that the gut microbiota as an environmental factor modulates fat storage. Since then, the role of the gut microbiota in the pathogenesis of obesity has become a vigorous research area. The further studies showed that obesity is associated with the changes of two dominant phylum-level bacteria of Bacteroidetes and Firmicutes in the gut (Mariat et al., 2009; Gerritsen et al., 2011). A reduction of bacterial diversity and the altered metabolic pathways were demonstrated to associate obesity from a comparison study in obese and lean twins (Turnbaugh et al., 2009). An endotoxin-producing “obese microbe” was isolated from an obese human and was confirmed to cause the obesity in the germfree mice (Fei and Zhao, 2013). In pigs, the studies reported by Guo et al. (2008a,b), Wall et al. (2009), and Luo et al. (2012) inferred that the gut microbiota might participate in the process of fat storage and should be correlated with the porcine adiposity formation. In addition, Yang et al. (2016) identified tens of fatness-associated bacteria including Escherichia spp. which showed a higher relative abundance in high fatness pigs. Overall, accumulating evidences suggest that the endotoxin-induced inflammation, dysbiosis of gut microbiota composition and Firmicutes/Bacteroidetes ratio are involved in the development of obesity. But the mechanism remains controversial (Turnbaugh et al., 2006, 2009; Cani et al., 2007; Wen et al., 2008).

Bamaxiang is a Chinese indigenous mini pig breed, and Erhualian is another Chinese indigenous pig breed which is famous for its high prolificacy. Both breeds show a higher fatness than Western pig breeds (Ai et al., 2013). In this study, we evaluated the association of the gut microbiome with porcine fatness in both faces and cecum luminal samples.

For recent years, more and more researches have concentrated upon the microbiota inhabiting the host gastrointestinal tract since gut microbiome has been reported to associate obesity and metabolic dysfunctional diseases in both humans and mice. Exploring the effect of the gut microbiome on obesity and insulin resistance has gained insight into the role of the microbiota in the development of diabetes mellitus and cardiovascular disease. Fatness is an importantly economic trait in pig production. While no such studies have been reported in pigs. To our knowledge, for the first time, we evaluated the association of the gut microbiome with fatness in swine, especially with the samples from cecum. Moreover, we adopted a novel and powerful two-part model association analysis to interpret effects of both presence/absence and relative abundance of gut microbiota on porcine fatness.

As we expected, although all experimental pigs were raised in the same farm house and fed the similar formula diet, we observed distinct phylogenetic composition of gut microbiome among samples. This should be explained by (1) different sampling sites (cecum vs. feces). Microbiota in stool are a mix mucosally associated microbes, most from the colon and lumenal microbes (Eckburg et al., 2005); (2) different genetic background between two pig cohorts.

We noticed that most of the associations were trait-specific. This heterogeneity may be caused by distinct mechanism of fat deposition for different types of adipose involving different microbes among abdominal adipose, leaf fat and subcutaneous adipose. This condition was similar to that in genetic dissection of porcine fatness traits, in which different genomic loci were identified for each fatness trait (Qiao et al., 2015). To the best of our knowledge, this study first evaluated the fatness-associated microbes in the cecum samples. Compare with the fecal samples, significantly higher number of the fatness-associated OTUs was identified in the cecum samples. As we have well known, cecum has the great diversity and complex of microbiota (Looft et al., 2014). And fermentation of dietary indigestible fiber and polysaccharides occurs at the cecum. We suggested that the samples from the cecum would be better for studying the association between microbiota and fatness than stool samples.

Interestingly, many fatness-associated microbiota identified in this study have potential functions related to metabolisms. At the taxonomic level, some of the fatness-associated bacterial taxa have been reported to participate in the process of the utilization of undigested carbohydrates from the diets or the host polysaccharide. For examples, R. gnavus was positively associated with fatness traits in both fecal and cecum samples. R. gnavus plays a pivotal role in UDCA formation in the colon, which regarded as a supplement of the bile acid (Lee et al., 2013). A recent study found that the α-galactosidase 1 (Aga1) and α-galactosidase 2 (Aga2) which are two kinds of the glycoside hydrolase (GH) family from R. gnavus played an indispensable role in the degradation of dietary oligosaccharides and exerted a tremendous fascination on designing of galacto-oligosaccharide (GOS) prebiotics (Cervera-Tison et al., 2012). The studies in human and rat found that R. gnavus was enriched in the obese rats and humans (Petriz et al., 2014; Andoh et al., 2016). Furthermore, both R. gnavus and Coprobacillus identified in this study could ferment the indigested polysaccharide into the SCFAs from the food in gastrointestinal tract, and then the SCFAs were absorbed by the host and could regulate host body energy homeostasis (Layden et al., 2013). Both Anaerovibrio and Clostridium butyricum were negatively associated with LeafFatWt. A previous study indicated that the Anaerovibrio lipolytica from Anaerovibrio can produce lipase in hydrolysis of triglyceride (Henderson, 1971). Previous studies have summarized that, as a kind of probiotics, C. butyricum can produce butyrate that provides the majority energy to the gut epithelial and repairs the intestinal mucosa damaged by virus (Araki et al., 2002, 2004; Zhang et al., 2011). The M. schaedleri was negatively associated with AbdomenFatWt in fecal samples. Interestingly, in the diet induced obesity (DIO) mice, the abundance of Mucispirillum was decreased (Clarke et al., 2013). Actinobacteria which was negatively associated with both average and waist backfat, has been recognized as the producer of many bioactive metabolites including antibacterials and antivirals for humans (Mahajan and Balachandran, 2011), and growth promoting substances for plants and animals (Atta and Ahmad, 2009).

At the OTU level, the fatness-associated OTUs were mainly annotated to YS2 (Cyanobacteria), Lachnospiraceae, Ruminococcaceae, Prevotella, Treponema, and Bacteroidales (Figure 3). Lachnospiraceae is abundant in the digestive tract of many mammals and relatively rare elsewhere. Members of this family have been linked to obesity in humans (Cho et al., 2012), mainly due to the association of many species within the group with the production of butyric acid (Duncan et al., 2002). Ruminococcaceae has been reported to play the role of biohydrogenation, and is capable of producing butyrate via fermenting various substrates, which may exert potential physiological functions in host health (Huws et al., 2011; Onrust et al., 2015). Kim et al. (2012) observed that Ruminococcaceae was enriched in mice fed high fat diet. Geurts et al. (2011) also observed a higher abundance of Ruminococcaceae in the db/db mice compared to lean mice. Prevotella and Treponema belong to xylan-degrading bacteria, which contain a set of bacterial genes for cellulose and xylan hydrolysis, and act as a host mutualistic component to help to degradate dietary fiber which could produce significantly more short-fatty acids (Warnecke et al., 2007; Flint et al., 2008). De Filippo et al. (2010) proposed that the degrading polysaccharide-rich diet allowed gut microbiota to maximize energy intake from fibers diets, but also protect them from inflammation and noninfectious colonic diseases. Besides Prevotella and Treponema, Bacteroides was also annotated to the fatness-associated OTUs. These microbes ferment polysaccharides to short-chain fatty acids, such as propionate by Bacteroides spp. from succinate pathway and acetate by Prevotella spp. from pyruvate via acetyl-CoA. Maslowski et al. (2009) and Smith et al. (2013) showed that the interactions of acetate and propionate with GPR43 have an important role in anti-inflammatory effects via the modulation of cT_Reg cell. In addition, the SCFAs can influence host homeostasis, inhibit the accumulation of fat mass development in adipose tissue and promote leptin level. In diet-induced obese mice, Bacteroides-prevotella also showed a negative correction with fat mass development and inflammation (Neyrinck et al., 2011; Ridaura et al., 2013; Ivarsson et al., 2014). In the cecum samples, Prevotella, Bacteroides, and Treponema were negatively associated with fatness traits.

In addition, Blautia, Coprococcus, Ruminococcus, and Clostridiales were also annotated to the fatness-associated OTUs in the cecum samples. These bacteria were identified in a phylo-functional core of gut microbiota in healthy young Chinese cohorts (Zhang J. et al., 2015). The members of Blautia and Ruminococcus have been reported to produce acetate via acetyl-CoA from pyruvate and Wood–Ljungdahl pathway by fermenting glucose and indigestible diet fiber (Miller and Wolin, 1996; Barcenilla et al., 2000; Pryde et al., 2002; Liu et al., 2008; Kovatcheva-Datchary et al., 2009; Crost et al., 2013). Perry et al. (2016) reported that the increased acetate could promote hyperphagia and increase energy storage as fat. In mice, ingestion of high fat diet was associated with the higher abundance of Clostridiales compared to the low fat diet regardless of propensity for obesity (de La Serre et al., 2010; Delzenne and Cani, 2011).

We estimated that gut microbiome could explain 1.55–2.73% of the variation in the six fatness-associated traits. This effect size was similar to that of most quantitative trait loci (QTL) previously reported for porcine fatness traits (Ai et al., 2012). However, in humans, gut microbiota has been implicated as a pivotal contributing factor in diet-related obesity (Zhang C. et al., 2015). This different contribution size should be due to the reason that all experimental pigs were raised in the same farm house and provided the same diet. The environmental factors, such as diets displayed less effects on gut microbiome and the subsequent fat deposition. Although the effect size of gut microbiome on fatness is small, we have established that gut microbiome should be a risk factor for porcine fatness.

A recent study reported by Ross et al. (2013) described an efficient methodology for predicting complex traits from quantitative microbiome profiles, and demonstrated that microbiome profiles can be used to predict human inflammatory bowel disease (IBD) status and BMI, and methane production in cattle with high accuracy. And based on the phylotypes of gut microbiota, Zhang et al. (2010) used partial least square discriminate analysis (PLS-DA) to predict host genotypes, diets or obesity phenotypes. In the futural study, the construction of prediction methodology based on the gut microbiome profiles for porcine fatness traits would greatly promote the pig production by reducing fat mass and improving the feed efficiency. Furthermore, the results from this study gave important cues for isolation of the causative microbes for porcine fatness that would provide basic information for regulating the gut microbiome to reduce fat deposition in pigs.

We identified a number of taxa and OTUs that showed significant associations with porcine fatness traits in the cecum luminal samples and feces. The fatness-associated microbiota were mainly involved in fermenting dietary indigestible fiber and polysaccharides to produce short-fatty acids. The short-fatty acids have been reported to inhibit fat mass development and inflammation. Significantly higher number of fatness-associated OTUs were identified in the cecum suggesting that cecum luminal samples would be better used for investigation of fatness-associated microbes than stool samples. Although the effect size of gut microbiome on porcine fatness is not very large in this study, we established that gut microbiome should be a risk factor for porcine fatness. These results help us to better understand the structure and functional potential of swine gastrointestinal microbiota, and provide a new insight into the role of gut microbes in affecting the porcine fatness traits.

All samples were collected according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. Animal Care and Use Committee (IACUC) in Jiangxi Agricultural University specifically approved this study. All the experimental pigs used in this study were raised in the farm house affiliated to State Key Laboratory for Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University. The pig owners consented to this study. No vulnerable populations were involved in this study.

CC conceived and designed the experiments, analyzed the data, wrote, and revised the manuscript; MH performed the experiments, analyzed the data and wrote the manuscript; SF, XH, HY, ZL, and JG performed the experiments; YZ and SK collected the samples; LH conceived and designed the experiments, and revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.