**4. Chicken as an animal model for social stress and related aggression**

Human emotional susceptibility in an inter-group is associated with individual differences in the functions of the hormonal and neurochemical systems in response to internal and external stimulations [182, 183]. Various social and biological factors are associated with the development and expression of aggressive behaviors, including environmental, genetic, cognitive, hormonal, and neurotransmitter circumstances [184]. Aggression and associated mental illness are an emerging public health problem [29]. Animal models are critical for investigating the potential biological processes involved in human aggression and mental disorders.

Although there are dissimilarities between humans and chickens, as indicated, the neural circuitry for aggression and social behavior appear to be evolutionarily conserved across the vertebrates [185]; chickens have been used as an animal model in various clinical and psychopharmacological studies, such as anxiety, depression, and aggression [186, 187]. A commercial hen can have more than 300 offspring with similar genetic characteristics during her lifetime, and chicks can be hatched without maternal condition effects. Previous studies have reported that birds' brain possess a core "social behavioral network" which is humongous to the social behavioral network of mammals [188]. There are evidences that the central nuclei involved in moodiness in avian, at least in part, are morphofunctional homologous to the mammalian nuclei [189], such as the hypothalamus [190], nucleus taeniae (homolog to the amygdala of mammals, [191, 192]), and Raphe nucleus [193]. These nuclei exert similar cognitive abilities and consciousness [194] with capability of plasticity in response to environmental stimulations [195]. In addition, there are similar distributions of neurotransmitter receptors, including serotonergic receptors

*Oral Health by Using Probiotic Products*

robehavioral functions [128].

**2.2 Microbiota, stress-associated aggression**

various animals [149, 150] including chickens [24, 151–154].

Serotonin, as an ancient chemical, is a key neurotransmitter. It plays a critical role in shaping social responses by regulating both basic (proactive) behaviors

**3. Serotonin and aggression**

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

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

**34**


#### **Table 1.**

*Behavioral ethogram.*

between birds and mammals [196, 197]. Particularly, it has been evidenced that the development and function of avian HPA axis in response to stimuli are analogous to those of mammals [198, 199] and there are similar distributions of cortisol-like molecules in the same organs from both birds and mammals [200]. Birds' CORT biosynthesis and metabolism can be stimulated in the same way as that of mammals [201–203]. During breeding, functional integrations among behavior, physiology, morphology, and environment in chickens may create suites of various traits that are simultaneously acted upon each selection program [204]. For example, selection based on individual production capability (phenotypic characteristics) increases animals' competition and aggression [205, 206] such as potentially injurious feather pecking and cannibalism. For instance, through more than 20 years of selection, egg production had been increased significantly in a former commercial Dekalb XL strain, whereas mortality due to aggression and cannibalism in non-beak trimmed hens had also increased about 10-fold [132, 152].

Results from previous studies conducted in our laboratory have found that chicken strain differs in social reactions through a diversely genetic selection program. The DXL hens have distinct stress levels in attempting to adapt to their social environments [207, 208] and exhibit different levels of aggressiveness [168]. In addition, neurotransmitters, 5-HT and dopamine (DA), and the hypothalamicpituitary-adrenal (HPA) axis are regulated differently between chicken strains [168, 207]. There results suggest that injurious behaviors and related social sensitivity of chickens, similar to that in mammals, are regulated via the serotonergic system and the HPA axis [123, 209], and mechanisms underlying aggression in laying hens may be analogous to those of humans and rodents [210]. Similarly in humans and rodents, stress-induced destruction of intestinal bacteria disturbs the bilateral connection of the microbiota-gut-brain axis in chickens, affecting physiological homeostasis and behavioral exhibition [211]. In one of our studies, the role of probiotic, *Bacillus subtilis*, on aggression in DXL line was examined. In the study, one-day-old female chicks were reared in single-bird cages up to 24 weeks. At 24 weeks of age, the hens with similar body weight were paired for the first aggression test (pre-treatment, day 0) in a novel floor pen [212]. Behaviors were video-taped for 2 h immediately after releasing the two hens simultaneously into the floor pen to determine the dominant individual per pair. Following the behavioral test, the subordinate hens were continuously fed the regular diet, while the dominant hens were fed the diet mixed with 250 ppm probiotic (1.0 × 106 cfu/g of feed) for 2 weeks. The probiotic contains three proprietary strains of *B. subtilis.* After the treatment (day 14), the second aggression test was conducted within the same pair of hens. The video recording was analyzed for frequency of feather pecking, threat, aggressive pecking, and threat kick by the routine lab procedures (**Table 1**) [154, 213].

**37**

*Gut-Brain Axis: Probiotic,* Bacillus subtilis*, Prevents Aggression via the Modification…*

for detecting social stress-induced aggression and violence [219, 220].

As social animals, chickens show fear, depression, and/or anxiety in novel environments and show aggression toward ones for establishing social dominance rank in unfamiliar social groups [214, 215], which is similar to rodents used in human psychopharmacological studies [216, 217]. The paired social ranking-associated behavioral test used in this study has been routinely performed in chicken behavioral analysis [154, 213, 218]. The rationale and cellular mechanisms of the test is similar to the resident-intruder test which is a standardized method used in rodents

A healthy intestinal microbiota community is important for maintaining physiological and behavioral homeostasis as that the gut microbiota regulates appetite, local and systemic immunity, stress responses of the HPA and sympatheticmedullary-adrenal (SMA) axes, and circadian rhythms [5, 221]. The new strategies of psychotherapy aimed at restoring the normal gut microbiota and intestinal homeostasis have been developed for the prevention and/or reduction of stress-

Probiotics are commensal bacteria ("direct-fed microbials", DFM) that offer potential health beneficial bio-physiological effects to the host's stress response (acute, chronic, or both). Probiotics aid animals in adapting to their environments and protect against pathogens by: (1) altering the microbiota profile with beneficial bacteria to prevent the growth of pathogens and to compete with enteric pathogens for the limited availability of nutrient and attachment sites; (2) producing bacteriocins (such as bacteriostatic and bactericidal substances) and short-chain fatty acids against pathogens to regulate the activity of intestinal digestive enzymes and energy homeostasis and to increase mineral solubility; (3) reducing oxidative stress, inflammation, and acinar cell injury; (4) modulating host immune and inflammatory responses and restoring the intestinal barrier integrity which prevents pathogens from crossing the mucosal epithelium; (5) stimulating the endocrine system and attenuating stress-induced disorders of the HPA and/or SMA axes via the gut-brain axis; and (6) inducing epithelial heat shock proteins to protect cells from oxidative damage; and (7) synthesis and secretion of neurotransmitter such as 5-HT and tryptophan [16, 17, 222–226]. In both human and rodent studies, probiotics reduce cognitive dysfunction, decrease the stress response and related oxidative damage by lowing plasma CORT and ACTH levels, restore hippocampal 5-HT levels, and normalize immunity with low plasma levels of TNF-α (tumor necrosis factor-α), a proinflammatory cytokine, but high levels of IL-10 (Interleukin-10), an anti-inflammatory cytokine [67, 227–230]. It has been stated in nonhuman primate models that the composition of the gut microbiota has potential effects on hosts' aggressive behaviors and anxiety symptoms [127], which is similar to the findings reported in humans [137, 231–233]. In rodent studies, GF animals with exaggerated HPA responses to social stress can be normalized by certain probiotics [147]. In addition, probiotics have successfully attenuated anxiety and depressive behaviors

in rat offspring separated from their mother [234–236] and the obsessivecompulsive-like behaviors in house mice [28, 237]. These results support the psychobiotics theory [238] and provide a new insight into the possible use of probiotics

A proposed strategy for improving human health is dietary supplement with probiotic microorganisms including Bacillus species [52, 244]. *Bacillus subtilis* is spore-forming bacteria. They have heat stability and low pH-resistance (the

to improve a host's cognitive function in humans [9, 40, 41, 239–243].

*DOI: http://dx.doi.org/10.5772/intechopen.86775*

**5.1 Probiotics and** *Bacillus* **spp.**

**5. Probiotic,** *Bacillus subtilis***, and aggression**

induced abnormal behaviors and mental disorders.
