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

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

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

*Oral Health by Using Probiotic Products*

**Behavior Description**

Feather pecking

Aggressive pecking

*Behavioral ethogram.*

**Table 1.**

hens had also increased about 10-fold [132, 152].

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

Threat One bird standing with its neck erect and hackle feathers raised in front of another bird

Forceful downward pecks directed at the head or neck of other birds

Threat kick One bird forcefully extending one or both legs such that the foot strikes another bird

which feathers are often pulled, broken, or removed)

One bird pecking at feathers of another bird, can be gentle (nibbling or gentle pecking in which feathers are not removed or pulled) or severe (vigorous pecking to feathers in

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

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

**36**

(**Table 1**) [154, 213].

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 stressinduced abnormal behaviors and mental disorders.

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

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

cfu/g

gastric barrier), and tolerate multiple environmental stressors [245, 246]. Several Bacillus spp. such as *B. coagulans* and *B. subtilis* have been used as probiotics in both humans [247–249] and animals [246] including poultry [65, 67, 250, 251]. Several mechanisms of action of *Bacillus* spp. have been proposed: the improvement of hosts' growth, survival, and health status via their anti-inflammatory functions through immunomodulation and cytoprotection [252, 253]. And, they have been used as antibiotic growth promoters alternatives with health-promoting benefits by naturally synthesizing proteins, enzymes, antimicrobial peptides, vitamins, gut flora modulation to promote beneficial microbiota along the GI tract and to correct and repair immunological and gut morphological alterations [244, 246, 250, 254, 255]. In addition, numerous studies have shown that probiotic-induced changes in the composition of gut microbiota lead to alterations of neuroendocrine functions. For example, in response stimulations, *B. subtilis* alleviate oxidative stress, provoke a specific biological response, and improve mood status of hosts via the gut-brain axis [52, 147, 256].

#### **5.2** *Bacillus subtilis***-based probiotic and social challenge-induced aggression**

Aggression, in nature, is associated with competition (natural selection) for survival and reproduction [22, 257, 258] (please also see above for the detailed description). However, in artificial production environments, such as in the poultry industry, aggression causes increased social stress and feather and body damage, in some instances these injuries leading ultimately to cannibalism. Cannibalism is a major concern related to non-beak trimmed bird deaths in current housing environments [259, 260]. Beak trimming (BT) is a routine procedure practiced in the US egg industry for reducing social stress by preventing and/or inhibiting feather pecking and cannibalism. However, BT is criticized for causing tissue damage and pain (acute, chronic, or both), negatively affecting the welfare of billions of chickens annually [261, 262]. Considerable concern from the public has led to a growing movement against procedures causing pain and suffering in farm animals. In response to growing pressures, housing environments of laying hens have been modified and/or various dietary supplementations have been provided in attempts to prevent social stress and stress-associated injurious behaviors. For instance, modifications include reducing light intensity, changing the nutritive value or taste of diets [263–265], providing straw or grain [266, 267] or pelleted diets [263], housing hens in floor-pens [268], and developing enriched cages [269, 270]. However, these methods have had limited success and provide no guarantee of preventing these injurious behaviors. Therefore, an obvious solution is to develop a welfare-friendly alternative to BT that minimizes social stress, thereby preventing feather pecking and cannibalism. The hypothesis was tested in this study: probiotics could be an alternative to beak trimming in chickens for reducing feather pecking and aggression via regulating the gut-brain axis.

Based on the 5-HT deficiency theory of aggression, social challenge-induced changes in 5-HT concentration were examined in this study. In the current study, the data showed that prior to the treatment (day 0), plasma 5-HT levels were higher (26% increase) in the dominant hens than that of subordinate hens but were not statistically significant (*P* = 0.24. Dominant5-HT = 17.46 ng/ml, Subordinate5-HT = 13.87 ng/ml). This finding is in agreement with the results reported previously [152, 168]. In those studies, higher plasma levels of 5-HT were detected in hens from mean bad bird (MBB) strain, a high aggressive strain selected for both low productivity and low longevity resulting from injurious pecking and cannibalism, compared to hens from kind gentle bird (KGB), a low aggressive strain selected for both high productivity and high longevity. In

**39**

**Figure 2.**

*differ (P < 0.05).*

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

addition, Bolhuis et al. [271] proposed that blood 5-HT activity is correlated with the development of severe feather pecking in laying hens. A similar correlation between blood 5-HT levels and aggressiveness has also been detected in humans and various other animals, that is, a lower blood 5-HT level was associated with less aggressive individuals in humans [180, 272] and canine [273], while an elevated blood 5-HT level was determined in patients with aggressive behaviors [274] and in

Post-treatment (day 14), plasma 5-HT levels were reduced toward the levels of controls (subordinates) in the probiotic fed dominant hens (*P* = 0.02) compared to their related levels prior to treatment (day 0) (**Figure 2**). There were no treatment effects on plasma 5-HT concentrations in subordinate hens fed a regular diet (*P* = 0.88). Although the reasons of the reduction of plasma 5-HT concentrations in probiotic fed hens are still unclear but could be similar to those proposed in probiotic-treated patients with intestinal inflammatory disorders such as irritable bowel syndrome (IBS) and IBS experimental animals. Probiotics reduce IBS-associated abdominal pain and abnormal bowel habits [276, 277] through regulation of both the central and peripheral serotonergic systems via the microbiota-gut-brain axis [278, 279] and gut epithelial enterochromaffin cells [19]. In the peripheral system, probiotics reduce or inhibit IBS-associated serotonergic system abnormalities, that is, great hypersensitivity and spontaneous release of 5-HT [280, 281]. Serotonin reuptake transporter (SERT or 5-HTT) has functions in inactivating 5-HT. Downregulation of SERT receptors has been found in the intestinal mucosa of IBS patient whose symptoms are similar to those found in the SERT knockout mice [282]. Serotonin 5-HT3 receptor antagonist also has shown efficacy in treating IBS patients [278, 283]. In addition, Wikoff et al. [284] reported that conventional mice had lower concentrations of 5-HT compared to GF mice. GF mice also had an exaggerated stress response [146] with anxiety-like behaviors [285]. These abnormal behaviors in GF mice can be inhibited or reduced by feeding probiotics [286] or transplanting fecal samples of conventional mice [287]. The current and previous

*Plasma serotonin (5-HT) levels at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed dominant hens and regular diet fed subordinate hens. Compared to subordinate hens, plasma 5-HT concentrations were higher in dominant hens at day 0 but without statistical difference (P = 0.24); the difference disappeared at day 14. Compared to the levels at day 0, blood concentrations of 5-HT were reduced in probiotic fed dominant hens at day 14 (P = 0.02) but were not in regular diet fed subordinate hens (P > 0.05). a,b between the concentrations at day 0 and day 14, least square means lacking common superscripts* 

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

aggressive teleost fish [275].

*Gut-Brain Axis: Probiotic,* Bacillus subtilis*, Prevents Aggression via the Modification… DOI: http://dx.doi.org/10.5772/intechopen.86775*

addition, Bolhuis et al. [271] proposed that blood 5-HT activity is correlated with the development of severe feather pecking in laying hens. A similar correlation between blood 5-HT levels and aggressiveness has also been detected in humans and various other animals, that is, a lower blood 5-HT level was associated with less aggressive individuals in humans [180, 272] and canine [273], while an elevated blood 5-HT level was determined in patients with aggressive behaviors [274] and in aggressive teleost fish [275].

Post-treatment (day 14), plasma 5-HT levels were reduced toward the levels of controls (subordinates) in the probiotic fed dominant hens (*P* = 0.02) compared to their related levels prior to treatment (day 0) (**Figure 2**). There were no treatment effects on plasma 5-HT concentrations in subordinate hens fed a regular diet (*P* = 0.88). Although the reasons of the reduction of plasma 5-HT concentrations in probiotic fed hens are still unclear but could be similar to those proposed in probiotic-treated patients with intestinal inflammatory disorders such as irritable bowel syndrome (IBS) and IBS experimental animals. Probiotics reduce IBS-associated abdominal pain and abnormal bowel habits [276, 277] through regulation of both the central and peripheral serotonergic systems via the microbiota-gut-brain axis [278, 279] and gut epithelial enterochromaffin cells [19]. In the peripheral system, probiotics reduce or inhibit IBS-associated serotonergic system abnormalities, that is, great hypersensitivity and spontaneous release of 5-HT [280, 281]. Serotonin reuptake transporter (SERT or 5-HTT) has functions in inactivating 5-HT. Downregulation of SERT receptors has been found in the intestinal mucosa of IBS patient whose symptoms are similar to those found in the SERT knockout mice [282]. Serotonin 5-HT3 receptor antagonist also has shown efficacy in treating IBS patients [278, 283]. In addition, Wikoff et al. [284] reported that conventional mice had lower concentrations of 5-HT compared to GF mice. GF mice also had an exaggerated stress response [146] with anxiety-like behaviors [285]. These abnormal behaviors in GF mice can be inhibited or reduced by feeding probiotics [286] or transplanting fecal samples of conventional mice [287]. The current and previous

#### **Figure 2.**

*Oral Health by Using Probiotic Products*

the gut-brain axis [52, 147, 256].

gastric barrier), and tolerate multiple environmental stressors [245, 246]. Several Bacillus spp. such as *B. coagulans* and *B. subtilis* have been used as probiotics in both humans [247–249] and animals [246] including poultry [65, 67, 250, 251]. Several mechanisms of action of *Bacillus* spp. have been proposed: the improvement of hosts' growth, survival, and health status via their anti-inflammatory functions through immunomodulation and cytoprotection [252, 253]. And, they have been used as antibiotic growth promoters alternatives with health-promoting benefits by naturally synthesizing proteins, enzymes, antimicrobial peptides, vitamins, gut flora modulation to promote beneficial microbiota along the GI tract and to correct and repair immunological and gut morphological alterations [244, 246, 250, 254, 255]. In addition, numerous studies have shown that probiotic-induced changes in the composition of gut microbiota lead to alterations of neuroendocrine functions. For example, in response stimulations, *B. subtilis* alleviate oxidative stress, provoke a specific biological response, and improve mood status of hosts via

**5.2** *Bacillus subtilis***-based probiotic and social challenge-induced aggression**

Aggression, in nature, is associated with competition (natural selection) for survival and reproduction [22, 257, 258] (please also see above for the detailed description). However, in artificial production environments, such as in the poultry industry, aggression causes increased social stress and feather and body damage, in some instances these injuries leading ultimately to cannibalism. Cannibalism is a major concern related to non-beak trimmed bird deaths in current housing environments [259, 260]. Beak trimming (BT) is a routine procedure practiced in the US egg industry for reducing social stress by preventing and/or inhibiting feather pecking and cannibalism. However, BT is criticized for causing tissue damage and pain (acute, chronic, or both), negatively affecting the welfare of billions of chickens annually [261, 262]. Considerable concern from the public has led to a growing movement against procedures causing pain and suffering in farm animals. In response to growing pressures, housing environments of laying hens have been modified and/or various dietary supplementations have been provided in attempts to prevent social stress and stress-associated injurious behaviors. For instance, modifications include reducing light intensity, changing the nutritive value or taste of diets [263–265], providing straw or grain [266, 267] or pelleted diets [263], housing hens in floor-pens [268], and developing enriched cages [269, 270]. However, these methods have had limited success and provide no guarantee of preventing these injurious behaviors. Therefore, an obvious solution is to develop a welfare-friendly alternative to BT that minimizes social stress, thereby preventing feather pecking and cannibalism. The hypothesis was tested in this study: probiotics could be an alternative to beak trimming in chickens for reducing feather pecking and aggression via regulating the gut-brain axis.

Based on the 5-HT deficiency theory of aggression, social challenge-induced changes in 5-HT concentration were examined in this study. In the current study, the data showed that prior to the treatment (day 0), plasma 5-HT levels were higher (26% increase) in the dominant hens than that of subordinate hens but were not statistically significant (*P* = 0.24. Dominant5-HT = 17.46 ng/ml, Subordinate5-HT = 13.87 ng/ml). This finding is in agreement with the results reported previously [152, 168]. In those studies, higher plasma levels of 5-HT were detected in hens from mean bad bird (MBB) strain, a high aggressive strain selected for both low productivity and low longevity resulting from injurious pecking and cannibalism, compared to hens from kind gentle bird (KGB), a low aggressive strain selected for both high productivity and high longevity. In

**38**

*Plasma serotonin (5-HT) levels at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed dominant hens and regular diet fed subordinate hens. Compared to subordinate hens, plasma 5-HT concentrations were higher in dominant hens at day 0 but without statistical difference (P = 0.24); the difference disappeared at day 14. Compared to the levels at day 0, blood concentrations of 5-HT were reduced in probiotic fed dominant hens at day 14 (P = 0.02) but were not in regular diet fed subordinate hens (P > 0.05). a,b between the concentrations at day 0 and day 14, least square means lacking common superscripts differ (P < 0.05).*

results indicate that normal health gut microbiota plays an important role in regulating social stress and stress-associated behaviors.

Whether the changes of blood 5-HT levels in probiotic fed dominant hens represent a similar change of 5-HT concentrations in the brain is unclear, as 5-HT cannot pass the brain-blood barrier and is regulated differently between brain neurons and peripheral tissues [288]. The plasma 5-HT is synthesized mainly by the EC cells of the gut and stored in the platelets [289]. However, it has been proposed that platelet 5-HT uptake is a peripheral marker of brain 5-HT [273]. Dietary probiotic, *Lactobacillus plantarum strain* PS128, increases the levels of 5-HT as well as dopamine in the striatum, which is correlated with the improvement of anxietylike behaviors in GF mice [290]. Similar results have been received from our current studies. In one study, chickens (broilers) were fed *Bacillus subtilis* from day one. At day 43, *Bacillus subtilis* fed chickens had higher levels of 5-HT in the raphe nuclei and lower levels of norepinephrine and DA in the hypothalamus than controls [291]. Probiotic fed chickens also had improved bone traits (bone mineral density, bone mineral content, and robusticity index). Under heat stress (32°C), *Bacillus subtilis* fed chickens had lower heat stress-related behaviors and inflammatory response and reduced IL-6 levels in the hypothalamus compared to controls [67]. Further studies, however, are needed to examine if there are correlations between the regulations of peripheral 5-HT and CNS 5-HT in probiotic fed hens.

The gut commensal microflora may have an indirect effect on 5-HT synthesis by regulating tryptophan metabolism. The degradation of tryptophan, a precursor of 5-HT, is mainly through the kynurenine pathway which regulates over 95% of tryptophan in the peripheral system and is functionally mediated by gut microbiota and probiotics [147, 292]. In the present study, the tryptophan level was not significantly affected by dietary probiotic (*P* = 0.35), but the initial levels of tryptophan in dominant hens were approximately 28% higher than the subdominant hens (*P* = 0.21) (**Figure 3**). The pattern of changes in blood concentrations of tryptophan in probiotic fed dominant hens was correlated with the changes of peripheral concentrations of 5-HT, indicating that probiotic may directly or indirectly regulate 5-HT synthesis in the peripheral system.

In this study, behavioral changes in dominant hens were correlated with the changes of blood 5-HT concentrations following fed probiotic. In the probiotic fed dominant hens, the levels of threat kick were reduced (**Figure 4a**, *P* = 0.04), and the frequency of aggressive pecking tended to be lower (**Figure 4b**, *P* = 0.053). The levels of feather pecking in dominant hens were reduced compared to their initial levels at day 0 but without statistic significant (**Figure 4c**, 58%, *P* > 0.05). There was no change in injurious behaviors in the regular diet fed subordinate hens between day 0 and day 14 (**Figure 4a–d**). The same or similar cellular mechanisms proposed in humans and rodents may be applied to the changes in the probiotic fed dominant hens. In humans and rodents, probiotics directly manipulate commensal bacteria releasing neuroactive factors, such as 5-HT and norepinephrine [8], and indirectly affect the neurotransmitter metabolisms, such as tryptophan as well as cytokines, through the microbiota-gut-brain axis [5, 40, 41, 279, 293–297]. In addition, the effects of probiotics, including *B. sublitis*, on behavioral exhibitions have been conducted on GF mice. Bercik et al. [287] reported that anxiety behaviors can be induced in less anxious phenotypic mice by colonization of the gut bacteria from anxiety-like phenotypic mice (FMT, fecal microbiota transplant). Probiotics also have therapeutic effects on neurodevelopmental disorders [28, 95, 298–301], for example, reduced anxiety-like behaviors by providing *L. helveticus*, *Mycobacterium vaccae*, and/or *Bifidobacterium* strains [231, 287, 302, 303], and alleviated autismrelated stereotypic behaviors by treating with *Bacteroides fragilis* [304] and behavioral dysfunction with *Lactobacillus reuteri* [301, 305].

**41**

**Figure 4.**

*difference (0.05* ≤ *P < 0.10).*

**Figure 3.**

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

*Plasma tryptophan levels at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed hens and regular diet fed hens. Prior to treatment, dominant hens had higher tryptophan concentrations compared to subordinates but the difference did not reach statistical difference (P = 0.21). There were no treatment effects on tryptophan concentrations in both probiotic fed hens and regular diet fed hens (P > 0.05, respectively).*

In the current study, body weight gain and hen-day egg production were not affected in the probiotic fed hens compared with the regular diet fed control hens (Control = 2.83%, Probiotics = 2.2%, *P* = 0.76; Control = 73.6%, Probiotics = 87.5%,

 *a trend* 

*Frequency of aggressive behaviors at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed hens and regular diet fed hens followed the paired social test. The exhibitions of aggressive behaviors in the regular diet fed subordinates were not affected by treatment (P > 0.05, respectively), while the frequency of threat kick (P = 0.04) was reduced, aggressive pecking (P = 0.053) was tend to be lower, and feather packing was declined (60%, P = 0.33) in probiotic fed dominates post-treatment. Notes: the treatment effects on the measured behaviors were reversed between dominants and subordinates during the 2nd social rank test. a,b between the frequency at day 0 and day 14, least square means lacking common superscripts differ (P < 0.05); and †*

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

*Gut-Brain Axis: Probiotic,* Bacillus subtilis*, Prevents Aggression via the Modification… DOI: http://dx.doi.org/10.5772/intechopen.86775*

#### **Figure 3.**

*Oral Health by Using Probiotic Products*

lating social stress and stress-associated behaviors.

5-HT synthesis in the peripheral system.

ioral dysfunction with *Lactobacillus reuteri* [301, 305].

results indicate that normal health gut microbiota plays an important role in regu-

Whether the changes of blood 5-HT levels in probiotic fed dominant hens represent a similar change of 5-HT concentrations in the brain is unclear, as 5-HT cannot pass the brain-blood barrier and is regulated differently between brain neurons and peripheral tissues [288]. The plasma 5-HT is synthesized mainly by the EC cells of the gut and stored in the platelets [289]. However, it has been proposed that platelet 5-HT uptake is a peripheral marker of brain 5-HT [273]. Dietary probiotic, *Lactobacillus plantarum strain* PS128, increases the levels of 5-HT as well as dopamine in the striatum, which is correlated with the improvement of anxietylike behaviors in GF mice [290]. Similar results have been received from our current studies. In one study, chickens (broilers) were fed *Bacillus subtilis* from day one. At day 43, *Bacillus subtilis* fed chickens had higher levels of 5-HT in the raphe nuclei and lower levels of norepinephrine and DA in the hypothalamus than controls [291]. Probiotic fed chickens also had improved bone traits (bone mineral density, bone mineral content, and robusticity index). Under heat stress (32°C), *Bacillus subtilis* fed chickens had lower heat stress-related behaviors and inflammatory response and reduced IL-6 levels in the hypothalamus compared to controls [67]. Further studies, however, are needed to examine if there are correlations between

the regulations of peripheral 5-HT and CNS 5-HT in probiotic fed hens.

The gut commensal microflora may have an indirect effect on 5-HT synthesis by regulating tryptophan metabolism. The degradation of tryptophan, a precursor of 5-HT, is mainly through the kynurenine pathway which regulates over 95% of tryptophan in the peripheral system and is functionally mediated by gut microbiota and probiotics [147, 292]. In the present study, the tryptophan level was not significantly affected by dietary probiotic (*P* = 0.35), but the initial levels of tryptophan in dominant hens were approximately 28% higher than the subdominant hens (*P* = 0.21) (**Figure 3**). The pattern of changes in blood concentrations of tryptophan in probiotic fed dominant hens was correlated with the changes of peripheral concentrations of 5-HT, indicating that probiotic may directly or indirectly regulate

In this study, behavioral changes in dominant hens were correlated with the changes of blood 5-HT concentrations following fed probiotic. In the probiotic fed dominant hens, the levels of threat kick were reduced (**Figure 4a**, *P* = 0.04), and the frequency of aggressive pecking tended to be lower (**Figure 4b**, *P* = 0.053). The levels of feather pecking in dominant hens were reduced compared to their initial levels at day 0 but without statistic significant (**Figure 4c**, 58%, *P* > 0.05). There was no change in injurious behaviors in the regular diet fed subordinate hens between day 0 and day 14 (**Figure 4a–d**). The same or similar cellular mechanisms proposed in humans and rodents may be applied to the changes in the probiotic fed dominant hens. In humans and rodents, probiotics directly manipulate commensal bacteria releasing neuroactive factors, such as 5-HT and norepinephrine [8], and indirectly affect the neurotransmitter metabolisms, such as tryptophan as well as cytokines, through the microbiota-gut-brain axis [5, 40, 41, 279, 293–297]. In addition, the effects of probiotics, including *B. sublitis*, on behavioral exhibitions have been conducted on GF mice. Bercik et al. [287] reported that anxiety behaviors can be induced in less anxious phenotypic mice by colonization of the gut bacteria from anxiety-like phenotypic mice (FMT, fecal microbiota transplant). Probiotics also have therapeutic effects on neurodevelopmental disorders [28, 95, 298–301], for example, reduced anxiety-like behaviors by providing *L. helveticus*, *Mycobacterium vaccae*, and/or *Bifidobacterium* strains [231, 287, 302, 303], and alleviated autismrelated stereotypic behaviors by treating with *Bacteroides fragilis* [304] and behav-

**40**

*Plasma tryptophan levels at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed hens and regular diet fed hens. Prior to treatment, dominant hens had higher tryptophan concentrations compared to subordinates but the difference did not reach statistical difference (P = 0.21). There were no treatment effects on tryptophan concentrations in both probiotic fed hens and regular diet fed hens (P > 0.05, respectively).*

#### **Figure 4.**

*Frequency of aggressive behaviors at day 0 (pre-treatment) and day 14 (post-treatment) in probiotic fed hens and regular diet fed hens followed the paired social test. The exhibitions of aggressive behaviors in the regular diet fed subordinates were not affected by treatment (P > 0.05, respectively), while the frequency of threat kick (P = 0.04) was reduced, aggressive pecking (P = 0.053) was tend to be lower, and feather packing was declined (60%, P = 0.33) in probiotic fed dominates post-treatment. Notes: the treatment effects on the measured behaviors were reversed between dominants and subordinates during the 2nd social rank test. a,b between the frequency at day 0 and day 14, least square means lacking common superscripts differ (P < 0.05); and † a trend difference (0.05* ≤ *P < 0.10).*

In the current study, body weight gain and hen-day egg production were not affected in the probiotic fed hens compared with the regular diet fed control hens (Control = 2.83%, Probiotics = 2.2%, *P* = 0.76; Control = 73.6%, Probiotics = 87.5%, *P* = 0.18, respectively). Previous studies have reported the beneficial effects of dietary supplementation of probiotics on daily weight gain, finished body weight, and feed conversion rate in broiler chickens [306, 307], turkeys [308], and swine [309]. Several studies also reported that probiotic diets improved egg production in hens [310]. The underlying mechanisms of these effects may be related to the beneficial bacterial growth in the gastrointestinal tract to facilitate the fermentation process which improves the digestion and utilization of nutrients in animals [311]. The beneficial effects on growth performance, however, are affected by the bacterial strains, preparation process, dosage, animal's age, and genetic type [312, 313]. In the current study, the probiotic was provided for 2 weeks only, which may not be sufficient to functionally improve both growth and production performance.
