Introduction
The routine use of antimicrobials in animal production has been identified as a potential factor in the development of antimicrobial resistance (1) and societal concern is driving increased stewardship, using the 5Rs model of responsibility, reduction, refinement, replacement, and review (2), especially in the use of medically-important antimicrobials. In the United States, increased stewardship has led to a 33% reduction in the domestic sale and distribution of all medically-important antimicrobials intended for use in food-producing animals, between 2016 and 2017 (3). However, certain swine production practices continue to impose increased risk of stress-induced disease susceptibility, and hence the use of antimicrobials is perceived to confer production advantages (4), although removal (5) or replacement with an alternative (6) may not be detrimental.
Stress can be defined as “an environmental effect on an individual which overtaxes its control systems and results in adverse consequences, eventually reduced fitness” (7). One of the most stressful events for pigs during production is that of weaning (8). At this time, they are subject to removal from the sow, an abrupt change in diet from milk to solid food and mixing into groups with unfamiliar pen-mates. In some countries, they may also be subjected to an additional stressor of transportation (9). These combined stressors often result in post-weaning diarrhea and compromised welfare post-weaning (10). The implication of various bacteria in post-weaning diarrhea has resulted in routine use of antimicrobials at this time-point, which impacts intestinal microbiota (11) but is seen to improve welfare, at least in the short term. The longer-term impact may be opposite. Reduced microbiome diversity and richness can reduce adaptability should the animal be challenged by dietary disturbances (12) or pathogens (13). This study used a combination of chlortetracycline, a medically-important antimicrobial aimed at prevention and treatment of gastrointestinal diseases, and tiamulin, a non-medically important antimicrobial aimed at prevention and treatment of respiratory diseases. This combination is the most commonly-prescribed antimicrobial mix used for 14d in post-weaning diets in the U.S., and has no adverse effects used at the concentrations prescribed. Alternatives to antimicrobials are being sought that will have the same health and welfare benefits, without the implications for antimicrobial resistance. These include phages, bacteriocins, peptides, probiotics, synthetic compounds, and nutraceuticals (14, 15). One potential nutraceutical is L-Glutamine, a conditionally-essential amino acid and immunomodulator that inhibits pro-inflammatory cytokines (16, 17). Dietary L-Glutamine supplementation can improve overall health and growth of newly weaned pigs (16–20) through a variety of mechanisms including: increased intestinal health and immune function, enhanced oxidative-defense capacity, prevention of intestinal atrophy, improved antibacterial activity, and greater nutrient absorption. It is well-known that L-Glutamine supplementation can improve piglet health and increase productivity (16–20), but little has been reported regarding its effects on swine welfare. In a study on simulated transport, weaned piglets fed diet with added L-Glutamine showed decreased intestinal damage, increased feed intake and growth, and decreased behaviors associated with illness compared to piglets provided diet without antibiotics (6). However, effects on other behaviors, and indicators of welfare and emotional state are not known.
Whereas, the potential effects of diet on behavior in pigs has been described (21), it is only in the last decade that the link between the gastrointestinal microbial population and brain function has begun to be elucidated (22, 23). We now know that the microbiome and the brain communicate bi-directionally via multiple suggested mechanisms, known collectively as the microbiome-gut-brain axis (24, 25). The existence of microbiome-gut-brain axis, and its relationship with behavior, affective state, cognition and stress response, is reported for numerous animal species, primarily rodents and humans. From human and other animal literature, the microbiome has been shown to play a role in anxiety and depression (26), fearfulness (27) and aggression (28) with shifts in microbiome linked to changes in these behaviors or affective states. As a route by which health and welfare of pigs can be manipulated, it is only recently receiving attention and is so far poorly understood (29, 30) but, theoretically, modification of the pig's gut microbiota offers a potential method to improve the pig's response to weaning and other lifetime stressors, and to improve overall health and welfare.
Since we know of the links between gut microbiome and fearfulness/anxiety and aggression, measures of interest will be behavioral responses during novel object tests carried out in the home pen so as to remove the confound of novel environment (31) and skin lesions post-mixing —a validated proxy measure of aggression (32). Tear staining will also be recorded, as the secretion of the Hardarian gland has been demonstrated as a potential tool for assessment of welfare in pigs on farm (33) and in a laboratory setting (34).
The objectives of the study were (1) To evaluate whether the administered diet can impact behavioral and welfare indicators during the administration period; (2) To determine if any diet effects can still be observed once the administration period is over; (3) To correlate behavioral indicators with specific microbiota composition.
Discussion
Specific feeding strategies for pigs to improve their ability to cope with stress or to alter behavior is a relatively unexplored area. In most studies dealing with feeding and stress and behavior in pigs, dietary changes have aimed to affect the time spent eating and the feeling of satiety to reduce aggressions, stress (43) and aberrant behaviors (44). However, there are a few studies that have emerged over the past 20 years that have revealed that dietary components per se can influence behavior.
Aggressiveness can be modulated via mineral intake, such as magnesium (45–47) and tryptophan supplementation (48–51). Besides preventing agonistic interactions from happening, an optimal ratio of fat, cholesterol and carbohydrate can even promote positive non-agonistic social interactions (52). Stress levels and fearful emotions can also be decreased with a large range of feed supplementation: vitamin E (46, 53), magnesium (47), tryptophan (48, 54–56), aromatic plant extracts (53, 57), chitosan (58), and the ratios of fat, cholesterol, carbohydrate (52), and linoleic acid in the diet (59). Finally, tryptophan has also demonstrated consistent effects in terms of reduction of aberrant behaviors, such as tail biting (49, 60), as well as changes in exploration in behavioral tests (61, 62). Exploration has also been increased by high linoleic acid ratio (59) or dietary cholesterol supplementation (63, 64). In the present study, pigs received three different diets: a diet including supplementary L-glutamine (GLN), a diet including an antibiotic treatment composed of chlortetracycline and tiamulin (A), and a diet without any prophylactic antibiotics or feed supplements (NA). The antibiotics and the L-glutamine were provided in feed. To the best of our knowledge, only one study recorded the behavior (excluding feeding or drinking) of pigs raised with either no supplementation in their feed, or orally supplemented with a common commercial prophylactic antibiotic or with L-glutamine (6). The authors demonstrated an increase in lying behavior and a decrease in standing behavior for NA pigs for the 48-h following a combined weaning and transport day. Those changes in behavior were correlated with reduced growth performance compared to GLN and A groups. They did not find any differences between the A and GLN groups. They suggested that these changes in behaviors for the NA group could reflect a higher degree of illness. Three welfare indicators—two of which are behavioral—have been recorded in the current study: tear staining, as a non-invasive indicator of stress (65); number of lesions, as an indirect indicator of aggressive behavior (32); and behavior during a novel object test, as an indicator or fear or anxiety (31). Similar to the study of Johnson and Lay (6), the NA treatment was the only treatment to differ from the other treatments. Oral antibiotics or L-glutamine provided similar beneficial effects regarding stress as minerals, amino-acids or plants extracts mentioned above. The NA piglets also had a higher number of lesions located in the front part of their body, as well as total lesions 48-h after mixing, representing more overall aggression in this treatment, with pigs likely more engaged in fighting (66). Results about aggression were only visible 48-h after mixing coinciding with the establishment of the hierarchy (67). Both A and GLN diets conferred similar positive effects in terms of aggression, as magnesium (45–47) and tryptophan supplementations (48–51). Finally, piglets supplemented with both A or GLN showed more withdrawals to a novel object dropped in their home pen at d16, but interacted longer with a novel object at d85 and interacted sooner with a novel object at d111. The three behaviors mentioned demonstrated a higher interest of the A and GLN treatment pigs for a novel object no matter their age and the time gap after the end of the feed supplementation. The higher number of withdrawals, in this particular situation, could also be interpreted as an increase in interest for the novel object as withdrawal appeared because of direct touching of the object that made it move. NA pigs adopted a strategy of flight in a corner of the pen. A better measure would have been the duration of cowering.
Feed supplements can be powerful tools as some studies have demonstrated effects on behavioral and welfare indicators only a few hours (62) or a few days after the beginning of the supplementation (46, 48–51, 54, 55). However, the majority of the studies have used a longer inclusion time of a few weeks (47, 52, 53, 56–61, 63, 64). Unfortunately, all the data collection is usually done only during the supplementation period or sometimes for a few days after. There is a critical knowledge gap about the longer-term effects of specific feed supplements over time. In the present study, the effects of short-term dietary treatments on stress and fear indicators were still significant up to 97 d after the end of the supplementation period. Inclusion of A or GLN treatments for 14 d induced similar short and long term effects on stress and behavioral indicators (tear staining, lesions and reactions during a novel object test). Those effects may come from changes in the microbiota composition. Indeed, GLN is a key regulator of microbiota composition as it participates in the nitrogen balance in the gut, which regulates the metabolism of bacteria (68). GLN promoted microbial richness and diversity in comparison to the A and NA treatments. Reduction of gut microbiota richness and diversity was associated with depression and anxiety-like behaviors in rodents (69, 70). In a study with obese humans, supplementation in 14 d with GLN induced a shift for the phyla Actinobacteria and Firmicutes, especially for the genera Dialister, Dorea, Pseudobutyrivibrio, and Veillonella (71). Shifts in the Firmicutes and Bacteroidetes phyla have also been observed in mice supplemented for 14 d with 1% L-glutamine (72). In the present study, pigs fed with the two dietary treatments A and GLN differed in terms of gut microbiota composition, while pigs from the NA group did not differ from the two other groups. Firmicutes was also the phylum mainly discriminating GLN and A groups after the 14-day dietary supplementation, followed by the phyla Actinobacteria, Bacteroidetes, Proteobacteria, and Spirochaetes. Shifts in bacterial populations were visible across all gut locations, ileum, caecum and colon, and in both gut content samples and mucus samples. Shifts over time and along the entire gut tract, observed in our study, is consistently reported in swine microbiota studies (73). The microbial composition is following a dynamic process around weaning, mainly depending on changes from liquid to solid feed (74, 75).
Antibiotics are known to strongly affect the microbiota composition by the depopulation of sensitive bacterial (58, 76). This reduction in diversity and bacterial abundances of the gut microbiota disrupts the gut ecosystem and weakens host immunity to pathogens (77, 78). In the current study, A treatment pigs had a richness and diversity comparable to NA pigs and significantly lower than GLN pigs. The lower richness and diversity of the gut microbiota in NA pigs could indicate intestinal disruptions caused by a high pathogenic load. Overall, α-diversity reported in the present study, matched results demonstrated in comparable pig studies. There was an ascending gradient of α-diversity, richness and evenness from the upper part (ileum) to the lower part (caecum and colon) of the gut tract (79–85). In term of microbial composition, ileum samples were drastically different from caecum and colonic samples which were highly similar (81, 83, 84, 86, 87). Moreover, in the present study we also confirmed that mucosa samples have higher richness than lumen samples regardless of the location in the gut tract (82, 85, 88). The three most abundant phyla determined in this study: Firmicutes, Bacteroidetes, and Proteobacteria also matched what is always reported for pigs at that age (73, 79, 82, 86, 87, 89). In terms of behavioral traits, GLN and A groups behaved similarly and were different from NA pigs, while in terms of microbiota composition the two groups were contrasted. L-glutamine conferred similar effects to antibiotics in terms of welfare and behavior, and should therefore be considered as a serious alternative to antibiotics, which may cause a decrease in microbiota diversity, select for antibiotic resistance, as well as increase the risk of colonization by pathogenic bacteria especially because of the creation of free ecological niches (90).
On d34, i.e., 20 d after the end of the dietary treatment, the differentiation of microbiota composition between dietary groups observed on d14 was not noticeable anymore. It could derive from two main causes: a cross contamination between groups, due to the fact that pigs in the neighboring pens had limited physical contact, leading to a convergence in gut microbiota composition; or an inevitable reversion to the commensal microbiota composition because of a powerful gut microbiota homeostasis. The first hypothesis is possible in the present study as pigs were penned separately by dietary treatment groups but were all located in the same room, which made cross contamination possible. Despite this loss of difference in microbiota composition, behavioral effects were still visible up to 97 d after the end of the dietary treatment. One possible explanation is related to the effect of microbiota on epigenetics. Indeed, it has already been demonstrated that microbial metabolism of diet can affect host gene expression via epigenetics pathways (91), including genes involved in the regulation of locomotor activity and anxiety-like behaviors, such as time spent in light in a light/dark box test compartment or open arms in an elevated plus maze test (92). During the supplementation period, substrates available for methylation can, for example, be altered or the activity of enzymes can be modulated by the presence of unusual compounds. Those alterations will then modify persistently the gene expression, as it will be transferred from one cell division to the next one enabling long term heritable changes. To promote long term gut microbiota differentiation, supplementation should also preferentially be given during the perinatal period, when the vulnerability of the entire organism is enhanced (92).
The gut microbiota plays a crucial role in the regulation of brain development, as well as in the emotional state of individuals (92–95). Relationships have been demonstrated between pathological emotional behaviors, such as anxiety and depression, and gastrointestinal disorders like irritable bowel syndrome (23). Challenges of rodents with pathogenic bacteria have provoked changes in behaviors within a few hours, resulting in an increase in anxiety and a decrease in exploratory behaviors (96, 97) via a direct action on the vagus nerve (98–100). However, Bercik et al. (101) also showed an effect of microbiota composition on emotional behavior of mice with a sectioned vagus nerve, revealing the existence of other modes of communication between the microbiota and the HPA axis (23, 102). Several studies using the rodent model demonstrated the importance of the microbiota on the emotional state of individuals, especially anxiety (97, 101–106). In humans, changes in gut microbiota through the supplementation of a probiotic reduced cortisol concentration, and therefore acting as an anxiolytic (107). Changes in gut microbiota composition affect the production of cytokines and tryptophan in the host, which are the main two pathways involved in the behaviors of depression and anxiety (108). Nowadays, the influence of gut microbiota on behaviors have been demonstrated in rodents (23). However, studies in livestock remain uncommon and detailed effects of specific bacterial populations on behavioral traits are unknown. Even if studies on livestock are still uncommon, they have all implicated the gut microbiota in host behavior (109–113). In hens, the aggressive damaging behavior of feather pecking was associated higher relative abundances of Clostridiales and a lower relative abundance of Lactobacillus in the luminal microbiota composition of ileum, caecum and colon sections combined (114, 115). In humans, Parashar and Udayabanu (94) have reviewed the relationships between specific bacteria populations and both stress and anxiety. They showed that a rise in stress coincides mostly with an increase in Bacteroidetes, a decrease in Actinobacteria, Firmicutes, and Proteobacteria, whereas a rise in anxiety coincides with either an increase or a decrease in Bacteroidetes and Firmicutes, an increase in Actinobacteria or a decrease in Proteobacteria. In the current study, we also revealed that some indicators of aggression, responses to stress and exploration have medium to strong correlations with specific bacterial populations. The phyla identified were Actinobacteria, Bacteroidetes, Fibrobacteres, Firmicutes, Proteobacteria, and Spirochaetes. That means that any feed supplements that can promote their development may affect the behavioral response of its individuals. Indeed, the brain is shaped both directly and indirectly by the gut microbiota through several pathways involving metabolites and neurochemicals' secretions. These molecules reach the brain via the blood stream or the nervous system and affect its development, neural and pain processes, and modulation of the HPA axis, therefore modulating the behaviors of individuals (116).