In the present study, dietary OAE supplementation increased body weight, average daily gain and decreased feed conversion ratio in broilers and in a dose-dependent manner. Consistent with our findings, several recent studies have indicated that oregano extract improved the growth performance [18, 19, 20]. Aromatic substances of phytogenic feed additives were also reported to stimulate the intestinal secretion of digestive enzymes in broilers, increase absorption area and contribute to the stabilization of the gut microbiota, and growth performance improvement [21]. In addition, in this study, the reduction of intestinal crypt depth, improved immune homeostasis and altered microorganisms structure all suggested the role of OAE in enhancement of digestion absorption function and maintaining healthy intestinal condition as well as benefiting feed utilization in broiler.
Inflammatory reactions result in the production of numerous cytokine and inflammatory mediators, causing tissue and intestinal epithelial cell damage, which decreases intestinal barrier functions [22, 23]. In this study, the mRNA expression levels of IL-4 and IL-10 were decreased in the OAE group at D42. This result might be associated with flavonoids in OAE (including apigenin and toxifolin), flavonoids were reported to inhibit Th2-type cytokine production [24, 25, 26]. Berry polyphenol components such as flavonoids, proanthocyanidins, and anthocyanins have developed functions in suppressing the secretion of cytokines such as IL-4 [27]. In addition, the decreased expression of IL-4 and IL-10 might be due to the inhibition of intestinal pathogens by OAE and the reduction of the intestinal inflammatory response, so there is no need to secrete excessive anti-inflammatory factors. Furthermore, it has been demonstrated that SIgA acts as the first-line defense barrier in protecting the intestinal epithelium. In the present study, dietary OAE addition significantly increased SIgA secretion. Studies have found that SIgA could enhance the immune function of the intestinal mucosa [28]. SIgA was dysregulated and led to the change of microbial communities [29]. And the relationship between beneficial bacteria and IgA was bilateral, which contribute to improving the intestinal barrier. Thus, the reduced inflammatory response and improved intestinal barrier in response to OAE treatment would be beneficial for the maintenance of intestinal health and growth performance.
To better understand the positive effects of OAE, further analysis was conducted on gut microbiota. There are hundreds of millions of microorganisms in the intestine. The gut microbiota interactions play an important role in preventing pathogen colonization, maintaining immune homeostasis and nutrient metabolism. The active ingredient of oregano (such as thymol and carvatol) has strong lipid solubility, which can quickly penetrate the cell membrane of pathogenic microorganisms, change their permeability and cause the loss of contents. Moreover, it could effectively prevent the oxidative energy supply process of mitochondria, damaging pathogenic microorganisms due to lack of energy [30, 31]. Apigenin and toxifolin have been reported to significantly increased the abundance of Lactobacillus and inhibited the reproduction of E. coli [32, 33, 34]. In this study, OAE treatment increased the abundance of some beneficial bacteria such as Lactobacillus and Firmicutes, which were helpful for the maintenance of the overall microbial structure. Lactobacillus is recognized as beneficial bacteria which can promote the growth of animals, regulate the normal flora of the gastrointestinal tract and improve the body’s immunity. Intestinal colonization resistance means the inhibition of resident bacteria overgrowth within the intestinal tract. Lactobacillus can maintain intestinal colonization resistance and resist invasion [35, 36]. Thus, these results indicated that OAE effectively inhibits the colonization of pathogenic bacteria, which may be related to apigenin and toxifolin. Invasion of pathogens can cause secretion of pro-inflammatory factors such as IL-1β and TNF-α [37], while beneficial bacteria could resist the invasion of pathogens and inhibit the increase of pro-inflammatory factors. As reported, Firmicutes is the dominant species in poultry and most of them are beneficial bacteria. The ratio of Firmicutes/Bacteroidetes was usually used to represent the distribution of beneficial and harmful bacteria. These beneficial bacteria, such as all members of the Lactobacillus family, maintained intestinal health by modulating cytokine and chemokine gene expression [38, 39]. In this study, gut microbiota structure alteration might conduce to the enhanced intestinal barrier function and alleviate inflammation, thereby improving intestinal health.
Beneficial bacteria in the gut could ferment carbohydrates to produce short-chain fatty acids, which also inhibit the growth of harmful bacteria [40, 41]. Short-chain fatty acids provide energy for epithelial cell to meet the requirements of epithelial barrier function and cell division. SCFAs are regarded as mediators in the communication between the gut microbiota and the immune system [42, 43, 44]. Especially, Acetate could regulate intestinal inflammation by stimulation of GPR43 [45], helping to maintain intestinal epithelial barrier function [46]. It was reported that the main components of short-chain fatty acids were acetic acid, propionic acid and butyric acid, accounting for more than 95%, among which acetic acid content was the highest [47], which was consistent with our experimental results. It has been reported that dietary acetic acid could increase appetite and regulate metabolism [48, 49]. Therefore, the increase in growth performance might be positively correlated with acetic acid content. Butyric acid as the main substitute for Firmicutes could also develop function in anti-inflammatory and regulating gut microbiota [50, 51]. In the current study, OAE increased the abundance of Lactobacillus and Firmicutes, then increased the contents of acetic acid and butyric acid, thereby maintaining intestinal health.
OAE contains not only alcohol-soluble substances but also water-soluble substances, which contain the complete complex of the plant [52]. Moreover, the contents of total phenols and flavonoids in water extract are high [53]. Quercetin, apigenin and other flavonoids have low bioavailability and need to be metabolized by hindgut microbiota [54, 55]. This suggests that OAE had the potential to improve the growth performance in birds through modulating gut microbiota. Subsequently, cecal microorganisms were added to the basal medium with OAE, and the enriched microorganisms and their metabolites were orally administered to broilers to study the direct effect of microorganisms on intestinal health. The results showed that Lactobacillus was increased and Unclassified_Enterobacter was decreased in the OAE group. Enterobacter is the most common cause of gram-negative bacterial infection. It mainly includes Yersinella, Escherichia coli, Klebsiella and so on. E. coli produced toxins that disrupted the intestinal barrier, causing disorders of the gut microbiota and metabolic diseases [56]. Gut homeostasis is mediated by the preponderance of obligate anaerobic members of Firmicutes and Bifidobacteriaceae, whereas the increase in facultative anaerobic Enterobacteriaceae is a common marker of gut dysbiosis [57]. However, the gut microbiota imbalance appeared in the absence of OAE. These results indicated that the OAE can directly affect the microorganisms, and has good antibacterial activity in vitro, which was consistent with the results in vivo in the study. In addition, OAE increased SCFAs contents, then propionic and butyric acids might be converted to acetic acid in the absence of carbon sources, resulting in a decrease in their concentrations after 24 hours.
Subsequently, we evaluated the direct regulation of the gut microbiota and the role of SCFAs driven by the microbiota on intestinal health. The results showed that the mRNA expression levels of IL-4 and IL-10 increased in microorganism group to a certain extent, while the mRNA expression levels of MUC2 and secretion of SIgA were increased in supernatant group. Lactobacillus Plantarum increased the mRNA expression of IFN-γ and IL-4 in the jejunum of broilers [58, 59]. The results implied that enriched Lactobacillus could regulate the expression of cytokines to improve intestinal health. In addition, it was reported that acetic acid could maintain the integrity of intestinal epithelium [60]. Paassen et al. [61] found that acetic acid, propionic acid and butyric acid could all improve the expression of MUC2 gene and protein in LS174T cells. SCFAs were involved in the activation of B cells, thereby promoting the secretion of SIgA. The excellent effects in supernatant group may be mainly caused by acetic acid. Therefore, OAE could promote the growth of Lactobacillus, and inhibit the growth of harmful bacteria, consequently improving mucosal immunity. While the special microbiota could drive the production of acetic acid, which could enhance the protection of mucin and SIgA in the intestine.