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Effect of a novel nutraceutical supplement (Relaxigen Pet dog) on the fecal microbiome and stress-related behaviors in dogs: A pilot study

Simona Cannas, *, Barbara Tonini, Benedetta Belà, Roberta Di Prinzio, Giulia Pignataro, Daniele Di Simone, Alessandro Gramenzi

Abstract

Anxiety and stress can trigger functional gastrointestinal disorders, whereas gastrointestinal symptoms can significantly increase anxiety and depression levels. These patterns are associated with the connection of the intestine and the brain through the “gut-brain axis,” a bi-directional communication of the nervous, endocrine, and immune systems. This clinical trial sought to investigate the effects of a novel nutraceutical supplement (Relaxigen Pet dog) containing natural anti-inflammatory compounds (CLA, Krill), pre/probiotics, 5-HTP, and L-theanine on stress-related behavior and to assess the connection between these stress-related behaviors and the fecal microbiome in dogs. Forty dogs, ranging in ages from 1 to 10 years, took part in this double-blind, placebo-controlled clinical trial. Ten dogs were control dogs without overt clinical signs of anxiety. Thirty dogs (experimental dogs) with signs of stress and anxiety were randomly assigned to the treatment (N 1⁄4 20) and placebo (N 1⁄4 10) groups. The treatment sample group (20 dogs balanced for sex) was administered with Relaxigen Pet dog tablets and the placebo group (10 dogs balanced for sex) with placebo tablets by mouth once a day for 60 days. A basic history questionnaire that focused on all aspects of dog’s behavior, management, health issues, and behavioral signs of stress and anxiety, was collected at days 0, 30, and 60 for each experimental dog. A fecal sample was collected for all dogs at day 0 to compare the microbiome of the anxious and non- anxious dogs. The anxious dogs in the treatment and placebo dogs groups had a fecal sample collected again at 30 and 60 days. These samples were used to extract the DNA for microbiological analysis and to determine the leading bacterial group. ANOVA showed an influence of treatment  time and the group that received the treatment had a probability of improving greater than 10% (P 0.05). This study revealed a different structure of the intestinal microbiota between healthy dogs and those with stress-related behaviors at baseline. Supplementation with the Relaxigen seems to bring some changes in bacterial groups concentration in the treatment group of anxious dogs compared to the placebo one, but we must be very cautious in presenting these results due to the limitations of this pilot study (number of subjects, no dietary control, and no evaluation of microbiota over time for the control group dogs).

Introduction

The animal organism has an adaptive response to real or sup- posed dangers that includes two different mechanisms to alleviate a state of stress in adverse situations that threaten homeostasis. These responses consist of developing behavioral changes to annul or counteract the effects of the threat and physiological changes that are necessary to restore and maintain internal homeostasis (Casey, 2002). When an animal is unable to escape the stressor through an appropriate behavioral response, the stress response becomes chronic, which leads to adverse effects on the physical and emotional state of the individual (Casey, 2002). When the stress response is prolonged or when the stressor persists, the emotional response to ensure that the animal escapes the situation also con- tinues. In dogs, chronic stress likely underlies a wide range of behavioral problems such as anxiety, fear, and aggression (Casey, 2002; Ksenofontova et al., 2020).

These common behavioral conditions compromise biological functions and alter the well-being and quality of life (Cahill and McGaugh, 1995; Cahill and McGaugh, 1998; Casey, 2002; Ksenofontova et al., 2020). In protracted stressful events, the indi- vidual becomes unable to exploit an effective behavioral mecha- nism for reducing his physiological response while the cortisol produced in excess has adverse effects that contribute to at different levels to a series of concerns: hypertension, diabetes, infertility, growth inhibition, loss of libido, reduction of the level of attention, alteration of memory, inhibition of inflammatory re- sponses, and alteration of immune function (Cahill and McGaugh, 1995; Cahill and McGaugh, 1998; Casey, 2002).

In particular, protracted stressful events seem to determine behavioral and endocrine alterations and neuroinflammation (Fang et al., 2012; Carney and Gourkow, 2016).

Inflammation that acts as a protective function in controlling infections and promoting tissue repair is usually self-limited. However, a prolonged or repeated stimulus delays the release of pro-inflammatory mediators and neurotoxins that worsen tissue damage and negatively affect disease outcome leading to an anxiety-like behavior state (Wohleb et al., 2015). Although the biological mechanisms connecting neuroinflammation to mental health complications are not well understood, several studies in humans underline how inflammation and altered immune signaling significantly contribute to the etiology of many psychi- atric symptoms and disorders (Evans et al., 2005), depression (Walker et al., 2014), sickness behavior (Biesmans et al., 2013), anxiety (Pace and Heim, 2011), and cognitive decline also in dogs (Frank-Cannon et al., 2009; Smolek et al., 2016).

Furthermore, anxiety and stress appeared to trigger functional gastrointestinal disorders, as well as the appearance of gastroin- testinal symptoms, they can significantly increase anxiety and depression levels; the intestine and the brain are connected in the “gutebrain axis,” a bi-directional communication of the nervous, endocrine, and immune systems (Zhu et al., 2017)…

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