**2.1 Social stress and the hypothalamic-pituitary-adrenal axis**

The social world is filled with different types of interactions, such that social challenges (fight-or-flight) and social environmental changes (group instability), as stressors, can cause highly potent stress [101–103]. Some management practices used in the intensive farm animal production systems, for example, may cause stress in animals. Farm animals are often housed in large groups at high densities, and during the animals' lifetimes, they are repeatedly transported to new environments and mixed with unfamiliar individuals. Based on the social network theory, animal group disruption can lead to social stress and related aggression [104] when they are unable to cope with these management practices and/or to reset their dominance rank (a form of animal social structure hierarchy) [105, 106].

Aggression has been commonly defined as feeling of anger or antipathy during social interactions, leading to hostile or destructive behaviors or attitudes, provoking physical or verbal attack or confront toward another individual [1]. Aggressive encounters among conspecifics for obtaining or maintaining a socially dominant position or rank cause a negative or unstable social environment to activate the hypothalamic-pituitary-adrenal (HPA) axis which is known as the key stress response system [107–109]. Cortisol (or corticosterone, CORT, in rodents and birds), as one of the final hormones released from the adrenal cortex, has multifunctional functions in both normal and abnormal states, regulating behavioral styles, metabolic patterns, and endocrine and immune functions, and ensuring an adequate coping strategy and well-being [110–112]. In mammals, hypercortisolism in response to novel environment exposures has been evidenced to be of adrenal origin [113, 114]. The value of circulating cortisol (or CORT) has been used as a criterion of stress response in humans and various animals [115–117]. Corticotrophinreleasing factor facilitates aggression [118, 119], and related elevation of CORT (or cortisol) also affects neuroendocrine functions through binding to their receptors [120, 121], causing neuron loss in the hippocampus [122], dysfunction of the serotonergic system [123], and inhibition of immunity (increased pro-inflammatory cytokines as neuroinflammatory response) within stress-sensitive brain regions [124]. These changes finally lead to the development of psychological disorders such as aggression [109, 125]. Similar to that the HPA hyperactivity generates aggressive behavior, exogenous glucocorticoid treatments increase exacerbated aggressiveness in both humans and experimental animals [123, 126]. Taken together, emerging

#### **Figure 1.**

*The microbiota-host interaction occurs at the level of the gastrointestinal mucosa via local neural, endocrine, and immune activities, influencing brain neurotransmitter expression, physiological homeostasis, and immunity (modified from [79]).*

*Oral Health by Using Probiotic Products*

humans and experimental animals [23–26].

reduces aggressive behaviors following social challenge.

**2. Gut microbiota and the gut-brain axis**

axis [2–10]. Intestinal bacteria, as well as *Bacillus subtilis*, used as probiotics are involved in tryptophan metabolism [11–14]. Tryptophan metabolites have roles in protecting intestinal mucosa from inflammation and regulating gut immune homeostasis [15–18]. Tryptophan, a precursor of 5-HT, directly affects brain 5-HT synthesis as that tryptophan can pass the brain-blood barrier [19, 20]. The brain serotonergic system plays a critical role in regulating behaviors, especially aggression [21, 22]. Concentrations of 5-HT and its metabolites, as well as the density of 5-HT receptors, have been used as major indicators of aggressive behaviors in

Probiotics (also called psychobiotics or bio-friendly agents), defined "as a source of live (viable) naturally occurring microorganisms (direct-fed microbials, DFMs)", have been used as dietary supplements to target gut microbiota (microbiome) for a novel promising therapeutic approach of various diseases including social stressinduced mental disorders in humans and various experimental animals [27–36]. Different probiotic strains, for example, have been investigated as functional food or therapeutic treatment of various diseases, including *Bifidobacterium bifidum* [37–39]; *Bifidobacterium bifidum* (BGN4) and *Bifidobacterium longum* (BORI) [40, 41]; *Bifidobacterium pseudocatenulatum* [42]; *Lactobacillus helveticus* [31, 32]; *Lactobacillus plantarum* [43]; *Lactobacillus paracasei* (KW3110, [44]); *Lactobacillus rhamnosus* [45]; and *Clostridium butyricum* [46]. The results collected from these studies indicate that the effects of probiotics on physiological homeostasis, immunity, stress resistance, and related health status are affected by multiple factors, including the probiotic species, its concentration, and duration as well as the host's age and health status [47, 48]. *Bacillus subtilis*, as one of the three most common species of probiotic products in the United States [49], has been widely used as functional feed supplements, such as in a number of dairy and non-dairy fermented foods, for improving human health and well-being [50–52]. Similarly, *Bacillus subtilis*, as growth promoters, has been demonstrated to improve chickens' growth performance [53–55]; regulate intestinal microstructure [56] and digestive enzymes [57, 58]; synthesize and release antimicrobial and antibiotic compounds [15, 59–63]; increase immunity [57, 64] and neurochemical activities including serotonin [8, 65, 66] as well as affect animal behavior [67, 68] following various stressors. In addition, *Bacillus subtilis* can overproduce l-tryptophan [11, 69], consequently increase 5-HT in the hypothalamus [70], function as an antidepressant and anti-anxiety agent [71, 72], and eliminate nervous tension in mice [73]. In the current study, chickens were used as an animal model to assess if dietary supplementation of probiotic, *Bacillus subtilis,*

Gut microbiota is a collective name of ten of trillions of microorganisms living in our intestine, including more than 35,000 different species of known bacteria [74]. Microbiome refers to the collective genomes of more than 3 million genes of the microorganisms in a particular environment. Gut microbiota, like a virtual endocrine organ, reacts to various internal and external stimuli [5, 75–78]. Consequently, gut microbiota influences and regulates hosts' health and mood statue including aggression by integrating metabolic, immune, endocrine, and neural reactions through the bidirectional communication of the gut-brain axis (**Figure 1**) [7, 33, 79–83]. Gut microbiota in hosts, for example, regulates brain neurotransmitters [84–87] such as serotonin (5-HT) through releasing its precursor, tryptophan, an essential amino acid [75, 88–90]. Serotonin is a key neurotransmitter within the brain, contributing to the development of the central nervous system (CNS)

**32**

evidence have indicated that the changes of the programming of HPA stress reactivity [127] cause long-term effects on the host physiological homeostasis and neurobehavioral functions [128].

#### **2.2 Microbiota, stress-associated aggression**

Aggression within a group is to establish a dominance hierarchy when the animals are first brought together in a common environment [129–131]. From an evolutionary perspective, aggression is adaptive behaviors that are related to an individual's survival, growth, and reproductive success within a group [132, 133]. However, aggression with long-term impact is often destructive and maladaptive in today's society, affecting millions of people's health and well-being around the world annually, resulting in significant social destruction and economic costs [134–136].

Intestinal microbiota plays an important role in regulation of social behavior, emotional expression, and mental health within the animal kingdom [3, 137, 138]. They are essential players in stabilizing homeostasis of the GI tract in response to both acute and chronic stress via the microbiota-gut-brain axis [16, 139]. Normally, intestinal microbiota provides protection for animals by competing for attachment sites and nutrients with pathogens as well as production of antimicrobial peptides and neuroactive compounds [140, 141]. The gut microbiota is also a key pathway to modulate brain processing the integrated information received from the peripheral nerve systems (the vagus nerve, enteric nerve, and autonomic nervous system), hormone signaling, the immune system, and microbial metabolites (short-chain fatty acids) [5, 137]. Under social challenges, stress and related oxidative damage cause anatomical and functional disorders of the GI tract by: (1) disrupting the commensal bacterial populations and colonization, thus reducing beneficial bacteria and increasing pathogens; (2) increasing pathogen survivability and innovating capability; (3) disrupting absorption of nutrients and minerals, including calcium; (4) disrupting microbial neuroendocrine functions; (5) disrupting the gut epithelial barrier, thereby increasing intestinal permeability causing the gut to leak certain bacteria (leaky gut), resulting in metabolic disorder; (6) damaging epithelial cells, thus producing free radicals and reducing antioxidant efficacy; and (7) interrupting intestinal integrity, thereby leading to intestinal inflammation [142–144]. These changes in gut microenvironment affect brain functions, resulting in exacerbated HPA axis activity, increased chronic inflammation, and/or disrupted neurotransmitter balance, leading to emotional damage [139, 145] and mental disorders [137, 138]. Sudo et al. [146] reported a correlation between the changes of gut microbiota and the function of the HPA axis. Germ-free (GF, antibiotic-treated microbiota-deficient or raised without any exposure to microorganisms) rats show exaggerated HPA responses to psychological stress [75, 147] with significantly higher levels of both ACTH and CORT in response to restraint stress compared to control rats [146]. In GF mice, gut microbiota also modulates the 5-HT synthesis and release at both the brain and peripheral levels directly and indirectly via the microbiota-host interactions [148]. Reduced 5-HT activity (a 5-HT deficiency) has been associated with personality traits (interspecific social behavior), such as impulsivity and aggression, and deteriorated stress coping capability (increased stress response) in humans and various animals [149, 150] including chickens [24, 151–154].
