Given the ban on in-feed antibiotic growth promoter usage in Europe and the global trend to reduce the utilization of antibiotics as growth promoters in livestock diets, determining alternatives to the utilization of antibiotic growth promoters has attracted increasing attention (Van et al., 2009). Probiotics, live cultures of bacteria provided to benefit the host are one of the alternative treatment strategies that can be used (Upadhaya et al., 2016). To date, a number of probiotics have been used in the poultry industry. As extensively used probiotics in the poultry industry, Lactobacillus strains can promote growth performance (Nakphaichit et al., 2011; Shen et al., 2014), improve meat quality (Keokamnerd et al., 2007; Lv et al., 2012), enhance immune response (Huang et al., 2004; Stringfellow et al., 2011), and prevent some avian diseases (Pascual et al., 1999; Yamazaki et al., 2012; Youn et al., 2012). However, it has proved difficult to demonstrate the mechanism whereby probiotics affect the gastrointestinal tract because probiotic strains exert their beneficial effects via different mechanisms in which other microbiota may be involved as well (Rubio et al., 2014; Serban, 2014; Patten and Laws, 2015). One probiotic strain may exhibit properties and clinical effects relatively different from another, even when the strains belong to the same bacterial species (Vieira et al., 2013). In addition, previous studies demonstrated the difficulty of reliably extrapolating the in vitro influences of probiotics to the in vivo situation (Jacobsen et al., 1999). Although numerous studies have focused on the positive effects of probiotics in animals, the mechanisms by which probiotics can successfully exert beneficial effects remain unclear. Therefore, the practical value in gaining further in vivo information about a useful probiotic strain should not be ignored. For example, it is important to find out whether or not live microorganisms are needed to induce the benefits.

Lactobacillus johnsonii BS15 (CCTCC M2013663) was isolated from homemade yogurt collected from Hongyuan Prairie, Aba Autonomous Prefecture, China. This probiotic strain can attenuate inflammation and mitochondrial injury, improve gut environment, and prevent non-alcoholic fatty liver disease in obese mice (Xin et al., 2014). In a previous study in broilers, L. johnsonii BS15 (LJ) supplementation improved growth performance and significantly enhanced the meat’s nutritional value, including the fatty acid composition, especially the polyunsaturated fatty acid content (Liu et al., 2016). However, the information on how LJ exerts its beneficial effects in broilers is limited. Thus, the present study aimed to assess the effects of LJ on growth performance, lipid metabolism, gut microbiota, intestinal development, and digestive ability. This study also aimed to examine whether the effects of LJ are related to the living state of microorganisms. The latter goal was achieved by comparing the results from the addition of live and disrupted bacterial cells.

A thorough understanding of the mechanisms underlying the direct and indirect effects of a probiotic strain on the host will certainly help improve the efficiency in applying probiotics in the broiler industry. Our recent study demonstrated the ability of L. johnsonii BS15 to improve meat quality from poultry by prolonging shelf life and increasing flavor and nutritional content (Liu et al., 2016). The effect of LJ BS15 was characterized by altering parameters related to lipid metabolism, including the fatty acid composition of meat and abdominal fat, which were markedly decreased with increased LJ concentration (Liu et al., 2016). Although lactobacilli exert numerous beneficial effects, such as improved nutrient absorption, vitamin synthesis, and reduced pain perception (Turpin et al., 2010), the exact in vivo mechanisms of action of these well-known beneficial bacteria are poorly understood. Hence, the present study aimed to prove the effect of LJ as a probiotic on growth performance and fat deposition. The present work also determined the changes in lipid metabolism, gut microbiota, intestinal development, and digestive ability upon LJ addition to elucidate the mechanisms underlying LJ effects. An appropriate LJ concentration was selected on the basis of a previous study (Liu et al., 2016). Moreover, the D-LJ group was added to determine whether or not the effects of LJ are related to live microorganisms.

Growth performance of the starter and finisher phases were measured in the present study. The enhanced ADG, slightly decreased FCR (P > 0.05), and unchanged ADFI at the starter phase suggested that the FCR improvement might be due to improved nutrient digestibility. These results are similar to the findings reported by Shen et al. (2014), who fed broilers with Lactobacillus plantarum. Moreover, the FCR was unchanged by LJ at days 22–42 and 1–42, although the ADG and ADFI were both increased. This result indicated that the enhanced ADG was strongly correlated with ADFI and may have contributed to the high body weight at day 22.

As a complicated process involving catalysis by abundant enzymes, lipid metabolism is modulated by numerous hormones. INS is an anabolic hormone that promotes glucose uptake by tissues (Huang et al., 2006). GH decreases lipogenic enzyme activities and adipocyte INS sensitivity. These effects result in reduced lipogenesis and decreased adipose tissue mass (Etherton, 2000). Serum LEP levels are highly related to fat deposition in pigs (Cameron et al., 2000). This marker serves as an indicator of fat content in the human body (Gill et al., 1997). As thyroid hormones, FT3 and FT4 are considered as the main hormones involved in the catabolic processes of lipids (Sheridan and Kao, 1998). The present research showed that LJ exerted no effect on the concentrations of the measured hormones in the sera of broilers. No sign of any kind of hepatotoxicity was observed, as demonstrated by the unaltered serum activities of AST and ALT after 21 and 42 days of LJ supplementation (Huang et al., 2015). The levels of serum lipids and lipoproteins can indicate the metabolic regulations in a steady state, especially the basal adjustment of fatty acid circulation between the liver and the adipose tissue (Mossab et al., 2002). HDLs form a class of lipoproteins with a range of sizes (8–11 nm in diameter). Fatty acids and cholesterol are carried by these lipoproteins from the body’s tissue to the liver (Zhang et al., 2013). Low concentrations of TG and LDL-C but high levels of HDL-C were observed in the LJ group. This observation suggested that LJ altered lipid metabolism. Similar effects were observed by Zhou et al. (2016), who studied Bacillus licheniformis. Barter et al. (2010) reported that a link may exist between the changes in HDL-C and TG, but they found no apparent relationship between LDL-C reduction and HDL-C increase. However, Zhang et al. (2011) and Zhao et al. (2013) observed that diet containing Clostridium butyricum and Enterococcus faecium exerted no effect on the concentrations of TG, TC, HDL-C, and LDL-C in broiler sera. Meanwhile, the serum INS levels were enhanced by C. butyricum. To date, most studies on lipid metabolism-associated hormones have been performed on mammals, which have different lipid metabolic processes in comparison to birds (Chung et al., 1983; Dunshea et al., 1992; Mossab et al., 2002). Information on the role and action of growth-associated hormones in broilers is also relatively limited. The discrepancy among these studies may be mainly attributed to the different animal and probiotic species investigated.

Consistent with our former study, the present result on abdominal fat percentage indicated that LJ can decrease fat deposition which is considered waste in the poultry industry (Farmani and Rostammiri, 2015; Liu et al., 2016). The result in fat deposition suggested that LJ can promote economic performance by decreasing extra costs. Likewise, a recent study reported the LJ-induced reversal of the trend of obesity in mice (Xin et al., 2014).

Unlike in mammals, LPL mRNA expression in growing chickens is not particularly reactive to aging and nutritional manipulation. This finding indicated the specificity of the physiological response of the broiler chicken LPL (Sato and Akiba, 2002). Chicken LPL is critical in fat accumulation in adipose tissue. The decrease of LPL in adipose tissue through nutritional means may be effective in retarding fatness in broiler chickens (Zimmermann et al., 2004). In the present study, LPL expression in LJ group was repressed at days 21 of age, which may have a relationship with the reduction in fat deposition. Although the ATGL and PPARγ mRNA expression levels in adipose tissue play a significant role in the regulation of fat deposit, these levels remained unchanged in the present study (Youssef and Badr, 2004).

A considerable portion of lipid metabolism occurs in the liver. Thus, the gene expression that alters enzyme capacity in relevant metabolic pathways has attracted significant attention. In this study, all the measured gene expression levels of lipid metabolism-related enzymes, except for ACC, were changed by LJ. This result indicated that LJ can influence both lipid synthesis and catabolism in the liver. SREBP-1c is linked to basic/helix-loop-helix/leucine zipper transcription. This protein can bind to the promoters of several lipogenic enzyme genes and induce their expression (Tabor et al., 1998). SREBP-1c also serves as a lipogenic nuclear transcriptional regulator that can directly affect the expression of ACC, FAS, and SCD1 (Richards et al., 2003). In our study, the expression of ACC exhibited no crucial response. Even so, LJ supplementation significantly inhibited the expression of SREBP-1c and its target genes, FAS and SCD1. FAS is a rate-limiting enzyme in the de novo synthesis of fatty acids (Wang and Tian, 2001). SCD1 is an endoplasmic reticulum enzyme that catalyzes the biosynthesis of monounsaturated fatty acids from saturated fatty acids (Cohen et al., 2002). These changes showed certain inhibitory effects caused by LJ on hepatic fatty acid de novo synthesis in broilers. Therefore, the reserved SCD1 expression in the present study may have caused low serum TGs and reduced abdominal fat deposition. Research on chickens reported that different diets can change the expression of PPARs in the liver of broilers (Maryam et al., 2011). As a nuclear receptor, PPARα is predominantly observed in the liver; the enzyme facilitates fatty acid catabolism by increasing CPT-1 and ACOX expression. This effect results in the stimulation of mitochondrial and peroxisomal fatty acid β-oxidation (Han et al., 2013). In the present research, LJ supplementation considerably upregulated the gene expression of hepatic PPARα, ACOX, and CPT-1 in the liver and enhanced the fatty acid β-oxidation.

As indicators of intestinal health or morphology, Vh, Cd, and Vh/Cd ratio may be related to intestinal health. Increased Vh may improve nutrient absorption and performance. The Vh/Cd ratio is critical in the evaluation of the developmental state of the intestine (Wu et al., 2013). As a key regulator, IGF-1 is an important regulator that controls long bone growth and energy metabolism. Hence, this growth factor facilitates the growth and function of almost every organ in the body (Giovannucci, 2001). IGF-1 could also increase the Vh in rats (Mantell et al., 1995). EGF, a heat- and acid-stable peptide, along with the EGF receptor, facilitates the proliferation and differentiation of epithelial cells. The two components could significantly enhance gut integrity and heal damaged mucosa or renew epithelial cells (Dvorak, 2010). EGF could also produce different kinds of biologic responses, most of which involve cell replication regulation, cell movement, and cell survival (Harris et al., 2003). In the present study, LJ exerted a positive effect on the jejunum and ileum; LJ increased the Vh and Vh/Cd ratio and the levels of IGF-1 and EGF. This result indicated that LJ could improve the development of the jejunum and ileum. Probiotics can also enhance the growth performance by improving the activities of different digestive enzymes (Timmerman et al., 2006; Fan et al., 2015). Similarly, the present study showed considerably higher activities of trypsin in the jejunum and lipase in both the jejunum and ileum relative to those of the control group.

Numerous animal studies have proved that ingestion of exogenous probiotics has a beneficial effect on growth performance through alteration of the gut microbiota. In chickens, Shen et al. (2014) found that Lactobacillus plantarum D22 could enhance the total count of anaerobes, as well as the individual counts of Lactobacillus spp. and Bifidobacterium spp. Samli et al. (2010) also reported that supplementing Enterococcus faecium (3.5 × 10⁸ cfu/kg) positively influenced ileal and cecal microbiota. In the present study, a significantly increased abundance of Bacteroidetes and a low Firmicutes/Bacteroidetes ratio were observed in the LJ group. This result is consistent with the effect of LJ in mice in preventing non-alcoholic fatty liver disease (Xin et al., 2014). Firmicutes and Bacteroidetes can also contribute to host metabolism through several mechanisms, including increased energy harvested from the diet, modulation of lipid metabolism, altered endocrine function, and increased inflammatory response (Greiner and Bäckhed, 2011). A low Firmicutes/Bacteroidetes ratio can be effective against obesity (Ley et al., 2006). Thus, the changes in this study suggested that the low abdominal fat percentage in the LJ group may be attributed to the low Firmicutes/Bacteroidetes ratio. Simultaneously, microbial flora plays a crucial role in inhibiting harmful bacteria and regulating immune responses. In this study, a potentially harmful family of microorganisms was suppressed by LJ BS15. The increased of Lactobacillus spp. level could also be indicative of the probable suppression of other non-beneficial bacterial groups (Munita et al., 2012).

Disrupted probiotics can enhance immune responses in broilers. This effect may be caused by a certain amount of bacterial antigens that could stimulate the gastrointestinal immune system (Huang et al., 2004). Some of the parameters in the D-LJ group were slightly improved relative to those in the control group in our study. Significant positive changes in FAS mRNA levels in the liver and Enterobacteriaceae 16S rRNA gene abundance in the ileum were also observed. Lactobacilli-produced exopolysaccharides exert various biological effects that could theoretically confer the host with a range of local and systemic health benefits (Patten and Laws, 2015). These exopolysaccharides may be partly associated with the results in the D-LJ group. Moreover, the enhanced Lactobacillus spp. level in the LJ group indicated that LJ may exert its beneficial effects by colonizing the intestinal tract and continuously producing certain metabolites with health benefits. However, whether other factors related to live microbes are involved remains unclear.

Notably, most of the positive influences caused by LJ, including those on growth performance; the mRNA expression levels in the liver and adipose tissue; and the contents of EGF, IGF-1, and digestive enzymes in the small intestine, were noted at day 21. Nevertheless, the serum biochemical parameters and abdominal fat percentage were altered by LJ only at day 42. Hence, altered lipid metabolism-related functional genes and digestive enzymes may result in the manifestation of its related phenotype. A commercial chick exits the egg while harboring a sterile intestinal tract that is fast colonized on the basis of the bacteria present in the environment and not by the bacteria from the hen (Barnes et al., 1980). As a result, the common succession of intestinal colonization is delayed, which enables the exogenous bacteria to be more effective to exert beneficial effects in the chick’s intestinal tract at the starter phase (van der Wielen et al., 2002). Therefore, LJ tended to confer benefits during the starter phase of production.

Feed supplementation with LJ BS15 may promote growth performance and lower fat deposition in broilers. These changes could be related to an improvement in lipid metabolism in the liver and adipose tissue. Inclusion of LJ BS15 may also enhance intestinal development and digestive ability, as well as help to balance the gut microflora in the small intestine. Based on the results shown in the two production phases and in the D-LJ group, we conclude that live LJ supplementation may provide health benefits by improving lipid metabolism, intestinal development, and gut microflora at the starter phase.

HW, DZ, and XN designed the experiments. LL, KP, XQ, and BJ performed the experiments. LL, GL, and ML analyzed the experimental data. HW and DZ wrote this paper. All authors read and approved the final 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.