1. Introduction

Domestic egg-laying chickens have been continuously selected for high egg production with a high feed efficiency to meet the constant increase in human nutrition demand for eggs due to both population growth and rising individual consumption [1,2]. However, extreme selection is often at the expense of the animal’s health and welfare [3,4]; i.e., selecting one trait (such as production) could affect other traits, causing negative impacts on the animals [5]. Based on the traditional selection theory, an animal’s productivity is correlated with its competitive ability [6,7]. As unexpected results, the effects of selection for increased production reportedly resulted in increased interspecific competition and aggression [8,9,10]. In previous studies, egg production increased significantly in former commercial Dekalb XL hens through more than 20 years of selection, while mortality associated with aggression and related injurious behaviors (aggressive pecking, severe feather pecking (SFP), and cannibalism) in non-beak trimmed hens also increased about 10-fold [11]. Increased injurious behaviors could be related to selection unequally affecting the animals’ adaptability to their rearing environments and management practices. Within a socioecological environment, not all animal individuals have an equal ability to modify their physiological and behavioral characteristics (such as personality traits for boldness, activity, and aggressiveness) in response to practice-associated stressors (inter-individual differences in adaption) [12,13,14,15]. Based on a dominance hierarchy or a ranking order, subordinates that are in direct contention with a dominant individual within a social group (i.e., the interactions between dominant higher-ranking (alpha) animals and submissive lower-ranking (beta) animals) exhibit fear, reducing their adaptation to the rearing environments and related management practices. Consequently, the subordinates enter a ‘pre-pathological state’ or ‘pathological state’ with physiological and metabolic disturbances [16,17,18]. Dominant chickens then exhibit an increased frequency of aggression and related injurious behaviors via the brain award systems and reinforced learning pathways, which could be similar to the brain systems reported in humans [19].

Aggression in chickens, as in most other species of social animals, is a highly complex social behavior. From an evolutionary viewpoint, aggression, as a natural part of an animal’s life, is essential for the animal to establish and maintain social status, to protect valuable resources (food and territory), and to reproduce successfully (survival, growth, breeding, and rearing offspring) [20,21,22,23]. However, some forms of aggression in chickens, such as excessive aggression-related injurious behaviors, can be harmful, leading to devastating consequences with increased social stress, feather and body damage, and injury (leading to cannibalism) [24,25,26]. In addition, numerous studies focusing on the function of gut microbiota in behavioral development have indicated that the dysregulation of the microbiota–gut–brain (MGB) axis has been implicated in abnormal behaviors (aggressive pecking, feather pecking (FP), and cannibalism) in laying hens [27,28]. Feather pecking may not be associated with dominance status; however, recent studies suggested that FP is related to social-stress-associated fearfulness [29]. Injurious behaviors, as a socially transmitted learning behavior, can be spread among flocks [30]. It has been previously reported that FP could affect up to 80% of birds in current housing environments [25].

Those injurious behaviors may be reduced through genetic selection [31,32,33,34]. However, there is “no sign that breeders will be able to guarantee the ‘non-peck’ layers in time” for hens to be housed in cage-free systems [35,36]. Egg production facilities are transferring from the conventional (battery) cage system to cage-free systems in the United States. Approximately 230 corporate customers, such as McDonald’s, Walmart, Subway, and Kroger, have pledged to only buy cage-free eggs by or before 2025. In addition, recent studies showed that selection for low-FP chickens failed to eliminate FP completely in flocks [37], which suggests that genetic selection should be paired with other management strategies [38]. Currently, beak trimming (BT), a routine procedure practiced in the United States egg industry, is the most effective method for reducing social stress by preventing and/or inhibiting injurious behaviors. However, BT has been criticized for causing tissue damage and pain (acute, chronic, or both) [39,40], negatively impacting the welfare of billions of chickens annually [41,42]. In addition, the chicken beak is a multipurpose organ playing a vital role in a variety of functions, from eating to defense against predators and parasites [43]. Beak trimming damages these beak functions, leading to abnormal behaviors and frustration [44]. Considerable concerns from the public have led to a growing global movement against the procedures causing pain and suffering in farm animals. However, recent studies have reported that FP and cannibalism still occur in beak-trimmed, floor-reared, and cage-free flocks [45]. Based on the outcomes, several studies have advocated that “solutions have to be found before thinking about banning BT” [45,46]. In addition, recent studies have indicated that FP and foraging are uncorrelated, which is inconsistent with the original hypothesis that FP is redirected food-related foraging pecks [47]. Feather pecking can lead to cannibalistic pecking, consequently eating and removing flesh from the victims by further reinforcing the behavior via the gut–brain reward systems (the central serotonergic and dopaminergic systems) [48,49]. In addition, injurious-behavior-associated social stress can disturb intestinal bacterial balance, resulting in physiological and behavioral disorders via the MGB axis [50,51].

The gut microbiota plays a critical role in early programming and later activity of the central stress systems, i.e., the hypothalamic–pituitary–adrenal (HPA) and the sympathetic–adrenal–medullary (SAM) axes [52,53,54]. Like an endocrine organ, the gut microbiota is sensitive and reactive to various exogenous stimuli, functioning as an environmental sensor linked to the pathogenesis of stress-related illnesses through the bidirectional communication of the MGB axis [55,56,57,58,59] and the microbiota–gut–immune (MGI) axis [60,61] in various animals including chickens [62,63,64]. Maintaining gut microbiota balance and health is essential for animals (including chickens) to maintain their optimal physiological and behavioral functions of growth, reproduction, and welfare. In humans, various psychological (emotional and mental overstimulation) and/or physical (environmental conditions) stressors alter gut microbiota diversity, composition, or both and increase the inability to maintain a healthy gut microbial profile, leading to neuropsychiatric disorders [65,66,67,68,69,70,71]. Targeting the intestinal microbiota with the goal of restoring its balance has been recognized as a novel therapeutic option for patients with neuropsychiatric disorders [72,73,74]. Several probiotics, as psychobiotics, such as Bifidobacterium and Lactobacillus, which can benefit mental health, have been used for preventing and treating patients with behavioral impairment, such as anxiety, depression, and impulsively and compulsively disrupted social behavior, via regulating the MGB, MGI, or both axes [75,76,77,78,79,80,81,82,83,84]. However, the use of purified probiotics benefits has shown mixed results, with several weaknesses including transient beneficial effects, requiring continuous administration over time due to the host’s resident microbial populations and “colonization resistance” [85]. Thus, it has been proposed that using live commensals coming directly from a healthy gut may be more effective than probiotics [86,87]. However, this hypothesis has not been well investigated in chickens.

The avian cecum, as a multipurpose organ, has a greater biological role than the cecum in most mammals [88,89,90,91]. In addition, chicken lines’ differences in the cecal microbiota composition in response to environmental stressors (such as ambient stress) [92] and experimental challenge models [93] have been reported. For example, laying hens showing high or low FP have different gut microbial populations [94,95] and intestinal and peripheral metabolite profiles [96,97]. However, a recent study reported that these differences may not be associated with FP and antagonistic behavior, due to limited effects on microbiota composition between the divergently selected lines for high and low FP [98]. It is still unclear how the gut microbiota is involved in injurious behaviors. In addition, the effects of early-life microbiota transplantation on gut microbiota composition and its function have not been well established [99].

2. Cecal Microbiota Transplantation, Social Stress, and Injurious Behavior in Chickens

2.1. Stress and Gut Microbiota

Stress is a natural biological (physical and mental) response to internal and external challenges in living organisms, including chickens. Normally, it prompts chickens’ ability to adapt to their rearing environments, while abnormally, an overload of stress challenge (too much exposure to a stressor or combined stressors causing a long-term activation of the stress response systems) reduces gut microbiota diversity, composition, or both [131]. The gut microbiota is functionally like an endocrine organ, releasing numerous bioactive factors to activate the HPA and SAM stress systems in response to stimulations, consequently affecting host physiological and behavioral homeostasis via the bidirectional communication of the MGB and MGI axes [63,64]. Healthy intestinal microbial communities and functions are essential for animals to fit their living environments [132,133]. The intestinal microbial community has been named the “social or behavioral immune system” linked to the microbiota–gut–brain–immune axis [134] based on the two reciprocal themes: (1) gut microbiota influences host social behavior and (2) social behavior and social structure shape the composition of the gut microbiota across individuals [135]. Based on these theories, environmental factors causing changes in the gut microbiome are linked to stress-induced neurobehavioral disorders including aggression and related damaging behaviors [136,137]. In addition, the differences in gut microbiota composition and/or diversity are related to personality traits [15,138], temperament [139,140], and sociability [87,141] in humans and various social animals, including chickens.

Numerous psychological (an emotion and/or mental overstimulation) and/or physical (environmental conditions) stressors reduce gut microbiota diversity and/or alter microbiome composition by (1) disrupting the community stability of commensal bacterial populations, often accompanied by reduced beneficial bacteria and increased pathogens (causing a chronic low-grade inflammation); (2) increasing the survival translocation of pathogens and releasing virulence factors; (3) disrupting absorption of nutrients and minerals (metabolic disorders); (4) disrupting microbial neuroendocrine functions (alterations in synthesis of several signaling molecules and neurochemicals including serotonin (5-HT) in the gastrointestinal tract (GIT); (5) disrupting the gut epithelial barrier, thereby increasing intestinal permeability and releasing certain bacteria, bacterial antigens, and metabolites (leaky gut), resulting in both intestinal and systemic immune reactions; and (6) damaging epithelial cells, producing free radicals and reducing antioxidant capacity (oxidative stress) [142,143,144]. These changes in the gut microbiota with a chronic low-grade inflammation profoundly influence host health and behavioral homeostasis via the MGB and MGI axes [58,145]. Treatments aimed at restoring normal gut microbiota composition and homeostasis have become effective methods to prevent and/or reduce various stress-induced neuropsychiatric disorders [146,147].

2.2. Possible Pathophysiological Mechanisms Underlying Injurious Behaviors in Chickens

In mammals, chronic stress is a major risk factor in neuropsychiatric disorders [148]. Social stress induces numerous microbiota-derived neurochemicals (neuromodulators) to enter the blood stream and influence brain function, especially the functions of both the HPA and SAM axes [149,150], which affects the development and balance of emotional and mental behaviors. Alterations in neuroendocrine homeostasis, i.e., corticosterone (CORT) and catecholamines (such as epinephrine (EP) and norepinephrine (NE)) levels, have been identified as the final common pathways in controlling animal behavior and pathophysiological status [151]. Animals raised in a germ-free (GF) environment expressing an exaggerated HPA response to psychological stressors could be normalized with certain bacterial probiotic species, such as Bifidobacterium infantis [152,153] and Bacillus licheniformis [154]. Animals treated with probiotics had a blunted HPA response [155]. Similarly, FP in chickens is influenced by dysregulation of the gut microbiome, which consequently affects neurotransmitter and immune homeostasis [27,94,95]. Current studies have evidenced that changing prenatal and early postnatal brain developments are involved in the development of injurious behaviors in laying hens [156] and other farm animals [157]. Current studies have evidenced that early-life cecal microbiota transplantation (CMT) induced different levels of aggressive behavior in the male recipients, which is corrected with each donor line’s behaviors. The results indicate that transferred donors’ cecal microbiota uniquely modifies the serotonergic activity, stress response, innate immunity, and cecal microbiota populations in recipients through the MGB and MGI axes. The underlying mechanisms, such as the responsible individual bacterium (or bacteria), the released neuromodulators and/or metabolites, as well as the involved pathways, will be examined in upcoming studies.

2.3. Physiological Mechanisms of Modulation of Intestinal Microbiota to Regulate Social Stress and Related Abnormal Behaviors

A healthy intestinal microbial community plays a critical role in regulating stress responses of the HPA and SAM axes to maintain host behavioral and physiological functions to fit their living environments [132]. Accumulating studies from various animal models in gut microbiota investigations, such as GF (complete absence of microbial exposure) animals, specific pathogen-free (SPF) animals, antibiotic-treated (broad-spectrum antibiotic cocktails) animals, and animals exposed to pathogenic bacterial infections, suggest that the gut microbiota plays an important role in the regulation of anxiety, mood, and cognition, indicating the possibility of using probiotics to modify the gut microbiota to control impulsive and compulsive behaviors in patients with neuropsychiatric disorders [158,159,160,161]. Like mammals, the gut microbiome plays a critical role in poultry health and welfare [119,162,163]. Laying hens showing high or low FP have different gut microbial populations [27,94,95,164] and metabolite profiles [96,97]. Therefore, the gut microbiome represents a novel therapeutic target for stress-induced mental and mood disorders in humans and injurious behaviors in chickens.

Probiotics are commensal bacteria that offer potential health benefits to the host, including the allostatic load (cumulative chronic stress in the body), when administered in adequate amounts. Generally, probiotics may aid animals in adapting to their ambient environments and protect against pathogens by (1) altering the microbiota profile in favor of beneficial bacteria to prevent the growth of pathogens and compete with enteric pathogens for the limited availability of nutrient and attachment sites; (2) producing bacteriocins (including bacteriostatic and bactericidal substances) and short-chain fatty acids against pathogens to regulate the activity of intestinal digestive enzymes and energy homeostasis and increase mineral solubility; (3) modulating host immune and inflammatory responses to reduce oxidative stress, inflammation, and cell injury; (4) restoring/strengthening the intestinal barrier integrity, which prevents pathogens and toxic substances from crossing the mucosal epithelium; (5) stimulating the neuroendocrine system and attenuating stress-induced disorders of the HPA and/or SAM axes via the MGB and MGI axes; and/or (6) inducing epithelial heat shock proteins to protect cells from oxidative damage [165,166,167,168,169,170]. Both human and rodent studies indicated that probiotics reduce chronic-psychological-stress-induced abnormal brain activity and related cognitive dysfunctions by lowering plasma CORT and adrenocorticotropic hormone levels, restoring hippocampal 5-HT and NE levels, and normalizing immunity with low plasma levels of Tumor necrosis factor alpha but high levels of Interleukin-10, an anti-inflammatory cytokine [171,172,173]. Several probiotics, as psychobiotics, for example, Bifidobacterium and Lactobacillus, deliver mental health benefits with neurobehavioral effects, which have been used in humans for improving cognitive function and for preventing and treating patients with behavioral impairment in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, and diseases with neuropsychiatric disorders, such as anxiety, depression, and impulsively and compulsively disrupted social behavior [75,77,78,79,83,84]. Based on these findings, targeting the gut microbiota has been recognized as a novel therapeutic option for patients with neuropsychiatric disorders [63,73,74]. Current studies have evidenced that the influence of the gut microbiota on the host behavior as seen in mammals is shared in chickens [173]. For example, dietary supplements of probiotics-based Bacillus amyloliquefaciens reduce distress calls and aggressive behavior in turkey poults [174], and Lactobacillus rhamnosus [175,176] and Bacillus subtilis [177] decrease stress-induced FP in adult hens by restoring the gut microbiota and 5-HT metabolism [70]. However, the evidence for probiotic benefits is mixed, proposing that the use of live commensals coming directly from a healthy gut may be more effective than probiotics.

Fecal microbiota transplantation has recently become a novel method for modulating the gut microbiota in gastrointestinal disorders such as inflammatory bowel syndrome and CDI [178,179], and non-gastrointestinal diseases including neuropsychiatric disorders [180,181]. Fecal microbiota transplantation (FMT) is a method of directly restoring healthy gut bacteria by transferring stool from a healthy donor. Stool contains thousands of microorganisms and a vast number of metabolites and has been recognized as a rapid and effective method to reshape the intestinal microbiota and metabolic profiles in humans and animals [182,183]. For example, the gut microbiota of recipients from stressed donors mimics the effects of stress on control animals, which could be reversed by transferring microbiota from unstressed animals [184,185]. Studies in Clostridium difficile infection patients revealed that the diversity of gut microbiota is increased following FMT, which is critical for defense against pathogens via colonization resistance. Clinically, a single dose can have long-lasting effects [186,187,188]. However, recent studies indicate that a fecal sample is not reliable in mapping the complete cecal microbiome and cannot be used to monitor the shifts and changes in cecal content in chickens [189,190,191].

Taken together, in humans and rodents, microbial colonization impacts brain development in early life, with long-lasting effects on adult behavior. Fecal microbiota transplantation and probiotics repair social-stress-induced disturbance of microbial functions and attenuate stress-induced responses of the HPA and/or SAM axes by protecting neuronal plasticity at the hypothalamic level as well as promoting neurogenesis in the hippocampus. Fecal microbiota transplantation restores the negative feedback of the stress systems to regulate animal health and behavior, providing novel insights into understanding how the gut microbiota community prevents abnormal behavior in patients with psychological disorders. Researchers hypothesized that similar cellular mechanisms may be manifested in CMT recipient chickens, because chickens and mammals share a similarity in the interactions between the microbiome and the neuroendocrine systems, generally named microbial endocrinology [192,193,194]. This hypothesis has been tested and evidenced in recent studies.

2.4. Cecal Microbiota Transplantation and Injurious Behavior in Chickens

Early life (immediate post-hatch) in chickens is a critical window of time causing enduring effects on the development of the intestinal microbiome and related brain functions and behavior in later life. Although microbial complexity considerably increases in the cecum with age [157], modulation of the structure and function of the cecal microbiome during early life alters neurophysiology in adolescence [195]. In chickens, the avian cecum plays a vitally important role in maintaining pathophysiological homeostasis, especially during periods of stress [196,197,198,199]. With up to 10¹¹ cells per gram of content, the cecum has the greatest bacterial biodiversity (bacterial diversity, richness, and species composition) along the chicken GIT [200,201,202]. As a multi-purpose organ, it has a complex motility, pushing contents in two directions (a two-component system): the cloaca (excreting as cecal drop) and the ileum (providing bacteria (for bacterial proliferation and colonization)) involved in the bird’s biological homeostasis [197,203,204,205]. The cecum with its high level of diversity maintains intestinal microbial stability in responding to various stressors [206] and determines colonization resistance against invading pathogens [207]. As the bird’s primary fermentative organ, the cecum possesses higher levels of DNA replicative viability than feces [208]. A balanced cecal microbiota diversity and composition has been used as an indicator of growth and health in poultry [209,210,211]. However, unlike mammals, in a commercial production setting, microbial contact is completely interrupted between domesticated parents and chicks. Various technologies have been developed for the modification of gut microbiota diversity and composition and related functions, including CMT, in chickens.

The effects of early-life CMT on the development of the gut microbiota in recipient chickens with long-lasting effects have been previously investigated. Franco et al. reported that broiler chicks (recipients) that received cecal contents from organic hens or industry-raised broilers (donors) by oral application on day 1 had distinctly colonized bacterial microbiota profiles, which was similar to the cecal microbiota profiles of the donors, respectively [212]. The differences between the recipient broilers had been maintained from day 7 to day 42 (the end of this study). The results indicate that transferred microbiota can persistently colonize the newly hatched broilers. In addition, early intervention with cecal fermentation broth from donor broilers (180 days old) regulates the colonization and development of gut microbial function in newly hatched broiler chicks (recipients), increasing beneficial bacteria and the concentration of short-chain fatty acids (SCFAs), while reducing the abundance of pathogenic bacteria [213]. In another study, cecal contents collected from ISA Brown chickens or hens (donors) at 1, 3, 16, 28, and 42 weeks of age were orally applied to newly hatched broiler chicks (recipients) [214]. Results showed that the cecal proteome of recipient chicks was correlated to the composition of the donors’ microbiome following a single inoculation on the day of hatch, with a long-lasting effect, up to 45 days of age (an entire broiler production period). Taken together, early inoculation with cecal microbiota represents a novel method for modulating the host microbiome to improve production and reduce susceptibility to infection in chickens.

In the current studies, CMT from the divergently selected inbred donor lines has been evidenced functionally to reduce or inhibit stress response and related aggression and damage pecking in recipient chickens of a commercial strain. These findings further support the theory that the exhibition of injurious behaviors is a stress-induced neuropsychological disorder in chickens, which is comparable to human psychopathological disorders [215,216]. Stress-associated gut dysbiosis and low-grade chronic inflammation are common traits of these disorders. For group-living chickens as well as other social animals, individuals share microbes and interact around environments and resources, by which the gut microbiota may have considerable consequences for host social interactions, such as the social ranking of individual animals [217,218]. For laying hens, like other social animals, the development of injurious behaviors may therefore be a phenotypic behavioral consequence of an imbalanced gut microbiota composition and related dysregulation of the communication between the gut and brain [204]. Birds with a higher propensity performing injurious behaviors have distinct microbiota profiles compared to their non-pecking counterparts [27,112]. Similarly, the microbiota differencesbetween the selected inbred lines (line 6₃ vs. line 7₂) exhibit distinct phenotypes [112], and CMT may be a method with the potential to control and replicate the role of the gut microbial community after a single passage of transplanted cecal content. This hypothesis will be tested in upcoming studies.

Major microbiota colonization of the intestine occurs in post-hatched chicks. CMT in early life (day-old chicks) may have great protective effects against stress-induced physiological and behavioral changes [219,220]. The current study showed that recipient chickens (6₃-CMT vs.7₂-CMT) had different levels of aggression and related damaging behaviors, which was correlated with the degree of injurious behaviors of donors [123]. The early postnatal period is a vital window for birds as well as mammals to be colonized with the microbiome [213], whereby early-life CMT profoundly influences brain development and intestinal microbiota composition and diversity [221] with a long-lasting impact on gut-brain neural circuit development and its responses to stressful episodes [222,223]. However, inconsistent results of CMT-induced intestinal microbiota modulation have been reported across studies. Early-life homologous (within line) microbiota transplantation (a pooled donor’s ileum, ceca, and colon contents) increases activation in both selected high- and low-FP recipients, with limited effects on their microbiota composition, stress response, and FP [28]. It is still unclear how FP arises as a consequence of dysregulated communication between the gut and the brain. A recent study also reported that gut microbial composition (from the digesta and mucosa of the ileum and cecum) and predicted functions were not associated with FP and antagonistic behavior in laying hens [33]. Therefore, given the inconsistent results, there is a critical need to further identify the biofunctions of cecal microbiota in controlling injurious behaviors in laying hens via CMT from the divergently selected non-aggressive and aggressive lines. Taken together, the obtained results may potentially influence the common procedures used in controlling aggression and related injurious behaviors in chickens as well as other species of farm animals, such as the dehorning of calves in beef and dairy operations [224,225] and teeth clipping or tail docking in swine operations [226,227]. The work may also have implications for human medicine, providing information for developing next-generation psychobiotics [228,229] and impacting human mental health; currently, 1 in 6 U.S. youth aged 6-17 and 1 in 5 U.S. adults experience mental health disorders each year [230].

This entry is adapted from the peer-reviewed paper 10.3390/microorganisms12030471