In recent years, with the rapid development of society and the increasing demand for a better life, the demand of people living in Tibet is more diversified for meat products, especially for pork of three-way crossbred commercial pigs. To resolve this contradiction, the breeding of three-way crossbred commercial pigs was rapidly implemented under the unified leadership of the local government. According to statistics of the local government, in recent years, the scale of breeding for three-way crossbred commercial pigs has been more than 300,000 in Tibet, and the scale of breeding is steadily increasing.

At present, high-altitude hypoxic environment has been shown to increase the development of inflammation throughout the body or multiple-organ dysfunction possibly (Pham et al., 2021). For example, high-altitude hypoxic environment induces acute mountain sickness, high altitude pulmonary edema, and high altitude cerebral edema (Purushothaman et al., 2011), and can even also affect the growth and intestinal health (Jensen and Moore, 1997; Xu et al., 2014). In addition, it has also been shown that inflammatory signaling in response to high altitude hypoxic environment exist an adaptive mechanism (Pham et al., 2021). Tibetan pigs, also known as ginseng pigs, are the indigenous breed native to the Qinghai-Tibet Plateau. In order to adapt to high-altitude environments, Tibetan pigs have developed unique physiological mechanisms during the long process of evolution and have acquired stable structural and functional characteristics (Yang et al., 2021). In the last few years, the commercial pigs had also been raised in Qinghai-Tibet Plateau because of its higher growth rate, higher feed efficiency, and superior meat yield (Zhang et al., 2018). However, the three-way crossbred commercial pigs were purchased from plain areas of China soon after weaning. They may face immense living challenges from high altitude hypoxic environment due to environmental changes (Lanikova et al., 2017).

As is well known, the gut is an important physiological function organ (Brestoff and Artis, 2013). The microbiota located in the gut exerts important functions for gut homeostasis and host health. Studies have proven that intestinal microbiota is strongly linked to intestinal barrier function and systemic inflammation (Pickard et al., 2017). For example, enteric pathogen C. rodentium is a major hallmark of diarrheal disease for mice, its expansion can result in dramatic colonic mucosal hyperplasia and a local Th1 inflammatory response. Clostridium difficile infection can result in the occurrence of inflammatory bowel disease (Sokol et al., 2018). In contrast, the abundances of intestinal beneficial bacteria are positively related to the intestinal health, such as Lactobacillus and Bifidobacterium maybe improve intestinal barrier function and reduce intestinal inflammation (Wu et al., 2020). Meanwhile, gut microbiome is also influenced by many factors, such as genetic, foods and environmental factors (Goodrich et al., 2017), and it has been the widely validated. In addition to above factors, high-altitude environments affect the composition and function of the gut microbiota (Suzuki et al., 2019). At high altitude, there is a decrease in the barometric pressure and a consequent reduction in the oxygen partial pressure, which will lead to the increased abundances of the obligate anaerobes (Moreno-Indias et al., 2015). At present, a few studies have explored the microbial community composition of mammals living at different altitudes, including ruminants (Zhang et al., 2016), humans (Lan et al., 2017), and pikas (Li et al., 2016). However, the studies about that the effects of high-altitude hypoxic environment on colonic inflammation, intestinal barrier and gut microbiota in three-way crossbred commercial pigs have so far hardly been explored. Therefore, we selected Tibetan pigs and three-way crossbred commercial pigs living at 3500–3700 m as a positive control group and treatment group in the present trial, respectively. Moreover, three-way crossbred commercial pigs living at 800–1000 m were also selected as negative control group. We used 16S rRNA gene sequencing to analyze the microbiota between Tibetan pigs and three-way crossbred commercial pigs, analyzing the relationship between gut microbiota and intestinal parameters such as intestinal inflammation and intestinal barrier. This study thus sought to explore the effects of high-altitude hypoxic environment on gut microbiota and gut health of pigs.

Intestinal barrier, including ecological barrier, mechanical barrier, and immunity barrier, plays an integral role in maintaining gut homeostasis. Among them, the intestinal mechanical barrier is essentially a defensive layer composed of intestinal mucosal epithelial cells and tight junctions between cells and the bacterial membrane, and can effectively prevent intestinal injury (Odenwald and Turner, 2017). Break of mechanical barrier often show obvious mucosal ulceration, loss of crypt, goblet cells and epithelial damage, neutrophil infiltration and causes inflammation (Turner, 2009; Li et al., 2019a). In this study, the occurrence of local mild inflammatory cell infiltration was observed in the colon of CP-H group. The level of D-Lac, LPS and the activity of DAO in serum of CP-H group were higher compared with CP-L group. Moreover, we also found that relative mRNA expression levels of IL-1β, IL-6 and p65 in colonic mucosa of CP-H group were higher relative to CP-L group. These data evidenced that the colon of CP living in plateau were in the inflamed state, and the intestinal barrier was disrupted to the certain extent. This situation may be related to the altitude at which the CP were located (Yi et al., 2021). It has been reported that upon exposure to high altitude, oxygen deficit and oxidative stress can occur (Földes-Papp et al., 2005; Pichler Hefti et al., 2016). Subsequently, inflammatory factors are released in response to hypoxia and oxidative stress (Himadri et al., 2010; Querido et al., 2012). For example, Himadri et al. (2010) found that high-altitude hypoxic environment leads to activation of NFκB in brain of rats. The activated NFκB further upregulates the proinflammatory cytokines, such as IL-1, IL-6, and TNF-α. On the other hand, the development of the inflammatory reactions might also be involved in as well as acclimation to high altitudes, while there is a genetic basis to the adaptation of high altitude (Simonson et al., 2010; Yi et al., 2021). In one study, comparing the levels of systemic inflammatory factors in the serum of 36 healthy subjects who had been living on the Qinghai-Tibetan plateau for a short time and 24 subjects who prolonged living on the Qinghai-Tibetan plateau, the authors found that the 36 healthy subjects’ levels of IL-2, IL-3, MCP-1, IL-1β in serum were higher than those in the 24 subjects (Yi et al., 2021). Simonson et al. (2010). found that high-altitude adaptation in Tibetans has resulted from local positive selection on the genes of EGLN1 and PPARA. CP is not a local pig breed of Qinghai-Tibetan plateau. In this experiment, the CP were purchased from the plain area of China after weaning. Therefore, the occurrence of intestinal inflammation was observed in the colon of CP-H group, the reason may be that the change of genetic material is also very difficult to achieve in short life of CP in order to adapt to their environment.

Altitude is also an important factor that influence the gut microbiota composition. Most the studies demonstrated that significant differences of gut microbiota were observed among different altitudes (Lan et al., 2017). Researches compared the differences in intestinal microbiota between plateau pika and the low-altitude dauricus. Scleroderma was the most abundant genus in the plateau pika, and Prevotella, Oscillospira, Ruminococcus, and yrc22 were abundant in the plateau pika. While Proteobacteria, Actinobacteria, and Verrucomicrobia were abundant in low-altitude dauricus. The Shannon index of the plateau pika were significantly higher than those of the low-altitude dauricus, and eight genera, including Streptococcus and Pseudomonas, increased with the altitude (Li et al., 2018, 2019b). Additionally, Zeng et al. (2020). found that Acinetobacter, Pseudomonas, and Sphingobacterium were the top three abundant bacterial genera in high altitude fecal samples in both humans and pigs. In this study, a large variation had also been observed in microbiota composition between CP-H group and CP-L group, including the within community diversity (alpha diversity), the between community diversity (beta diversity) and the microbial composition in the different levels. Therein, the Terrisporobacter and UCG-002 were enriched in the CP-H compared with CP-L. The most likely explanation for this phenotype was that at high altitude, there was a decrease in the barometric pressure and a consequent reduction in the oxygen partial pressure, which lead to the abundances of these obligate anaerobes increased (Moreno-Indias et al., 2015).

Under normal conditions, intact intestinal barrier can inhibit pathogenic bacterial growth (Fukuda et al., 2011). However, some conditional pathogenic bacterial have the opportunity to grow quickly when the barrier is damaged. These pathogenic bacterial destroy the intestinal microecological barrier, then accelerate intestinal damage and result in bacterial antigen translocation from the intestinal cavity to the circulatory system and finally lead to systemic inflammation (Figueredo et al., 2018; Yin et al., 2019). In this study, the abundance of Terrisporobacter in CP-H group was significantly higher than CP-L group, and it was positive significant correlation between Terrisporobacter and relative mRNA expression levels of IL-1β, the LPS content and the activity of DAO. Very little is currently known about Terrisporobacter, but there were also indicated that it has been linked to oxidative stress and inflammation in preterm infants (Cai et al., 2019; Ho et al., 2019). The Terrisporobacter could produce the urinary toxin, such as trimethylamine-N-oxide (Cai et al., 2019). Trimethylamine-N-oxide may be involved in the pathogenesis of IBD by impacting ATG16L1-induced autophagy and activating NLRP3 inflammasome (Yue et al., 2017; Li et al., 2022). In addition, our data indicate that Clostridium_ sensu_stricto_1 and Streptococcus had higher abundance in colonic chyme of CP-H compared to TP. Meanwhile, Streptococcus was significantly positively correlated with D-Lac. In previous studies, Clostridium_ sensu_stricto_1 and Streptococcus are deemed to be opportunistic pathogens associated with colitis (Zou et al., 2020; Wen et al., 2021). Therefore, they may be another important factor to aggravate intestinal inflammation of CP-H in this study. Interestingly, we found that the Lactobacillus_johnson and Lactobacillus_reuteri were enriched in gut of TP. The Lactobacillus_johnson and Lactobacillus_reuteri is a well-known gut probiotic (Avall-Jääskeläinen and Palva, 2005; Sung et al., 2018). At present, they have been used as feed additive in swine industry, with very good results (Bampidis et al., 2021). Thus, it may be a good therapeutic candidate for intestinal diseases treatment with the addition of Lactobacillus_johnson and Lactobacillus_reuteri in the CP-H feed.

Short-chain fatty acids (SCFAs) are produced by anaerobic gut bacteria through saccharolytic fermentation of complex resistant carbohydrates (Muralitharan et al., 2020). The SCFAs predominantly include acetate, propionate, and butyrate which account for approximately 80% of all SCFAs. Therein, butyrate are important substrates for maintaining the colonic epithelium, regulating tight junction proteins and enhancing intestinal barrier function through increasing expression of claudin-1 and ZO-1 (Wang et al., 2012). Studies have reported that the majority of butyrate are produced by a small number of gut bacteria, including Faecalibacterium prausnitzii, Eubacterium rectale, Eubacterium hallii and Ruminococcus bromii (Louis et al., 2010), of which is dominated by Ruminococcus bromii (Sun et al., 2017). In this study, no major strains related to butyric acid production were found in the top 15 differential bacterial genera in abundance. Thus, this may be the main reason for the no significant differences found about the concentrations of butyric acid. However, it is worth noting that the concentrations of valerate in CP-H was significantly higher than CP-L and TP. Previous studies found that valerate, which is a SCFAs mainly converted from proteins or amino acids (Gao et al., 2022), was demonstrated that could enhance intestinal barrier functions at physiological concentration (McDonald et al., 2018). However, valerate exhibited the concentration-dependent paradoxical effects on intestinal barrier function. It can exert the opposing effects on normal colonocytes at high concentrations (Burgess, 2012). Interestingly, through the correlation analysis, we found that the concentrations of valerate is significantly positively correlated with Streptococcus and Clostridium_sensu_stricto_1 in this study. Based on our data, we could speculate that the most of the valerate in CP-H group was likely arose from Streptococcus and Clostridium_sensu_stricto_1. However, the mechanism underlying the production of valerate in gut is not fully understood and further investigation is needed.

In summary, high-altitude hypoxic environment changed compositions of gut microbiota, promoted the occurrence of colonic inflammation, and disrupted intestinal barrier of the three-way crossbred commercial pigs.

The data presented in the study are deposited in the NCBI SRA database with accession number PRJNA 872015.

The animal study was reviewed and approved by the Experimental Animal Welfare and Ethical Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences.

CL and GS designed the experiment. CL, GS, JD, and HH carried out the experiment. CL and GS wrote the manuscript. RZ, LC, BW, YZ, HZ and ZW revised the manuscript. All authors contributed to the article and approved the submitted version.

This research was supported by the Tibet Science and Technology Projects (XZ-2019-NK-NS-003) and Agricultural Science and Technology Innovation Program (CAAS-ZDRW202006-02, ASTIPIAS07).

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.

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