Working dogs, such as those used in search and rescue, law enforcement, military, and competitive sports, face significant physical and mental strain. These animals frequently experience stressful events, environmental stressors (heat, cold, and terrain variability), psychological stress related to task performance and training, and altered feeding and rest patterns, all of which differ substantially from the lifestyle of companion (pet) dogs.

Previous studies have demonstrated that these cumulative stressors are associated with sustained activation of stress-responsive neuroendocrine pathways. Such conditions have been shown to influence the oxidative stress markers, modulation of immune parameters, and alterations in gastrointestinal function and gut microbiota composition. Therefore, working dogs represent a biologically relevant and sensitive model for evaluating the effects of dietary interventions aimed at improving antioxidant defense, immune competence, and gut health, particularly under conditions of elevated physiological stress.

Nutritional approaches that can boost immunological function, increase physiological resilience, enhance performance, and recovery outcomes in these animals are therefore gaining attention. In this context, the use of nutraceuticals has attracted growing interest in canine health management, referring to nutrient-based foods or supplements that exert health-promoting effects beyond their fundamental nutritional roles.

Among these, the blue-green microalga Spirulina (Spirulina plantensis) has emerged as a promising functional ingredient due to its high content of bioactive chemicals. Spirulina platensis is a multicellular, filamentous, and photosynthetic cyanobacterium with a spiral shape. that has been marketed and consumed as a food for humans and animals. Spirulina possesses an exceptional nutritional profile, containing 60–70% high biological value crude protein. It is also rich in vitamins, minerals, essential fatty acids, and antioxidant pigments. These bioactive compounds are linked with broad-spectrum and numerous health benefits, including immunomodulatory, hypolipidemic, antioxidant, anti-inflammatory, hepatoprotective, nephroprotective, anti-viral, and anti-bacterial effects. Owing to these properties, Spirulina has been increasingly incorporated as a nutraceutical feed additive in animal nutrition, with growing interest in its potential to modulate metabolic, immunological, and gut-related molecular pathways under physiologically demanding conditions.

Nutrigenomics is an interdisciplinary field that integrates nutritional science with molecular biology to investigate how specific nutrients modulate gene expression and physiological responses within a given population. Understanding the nutrigenomic effects of nutraceuticals can reveal the mechanisms of their physiological and health-promoting actions, particularly in relation to gut integrity, immune regulation, and inflammatory pathways.

Emerging evidence in canine nutritional physiology indicates that dietary bioactive compounds can influence molecular pathways involved in inflammatory regulation, oxidative balance, and intestinal barrier function, particularly under conditions of increased physiological demand. Working dogs exposed to sustained stress may exhibit alterations in inflammatory signaling mediators and epithelial integrity markers, which are increasingly recognized as nutrigenomically responsive targets.

There are several studies on the incorporation of Spirulina plantensis as a nutraceutical in the diets of both humans and animals for its remarkable effects on general health, immune status, antioxidant activity, lipid profile, and gut immune health. Previous studies have evaluated Spirulina supplementation in dogs across a range of doses (0.04–0.19 g/kg BW/day) and these inclusion levels were reported to be well tolerated and palatable, with no significant adverse effects on gastrointestinal parameters, behavior, or general health status. More recently, Stefanutti et al. showed that Spirulina supplementation in overweight dogs over 12 weeks enhanced antioxidant status and exerted beneficial metabolic effects, including reductions in triglycerides and bilirubin concentrations. Importantly, no negative effects on health or weight management were observed.

Although Spirulina has been widely investigated for its health-promoting effects in various animal species, to date, limited in vivo studies have evaluated the effects of dietary supplementation of Spirulina platensis powder in working dogs. In particular, there is a lack of integrated studies evaluating gut-related nutrigenomic responses, fecal microbiota composition, metabolic as well as inflammatory and immunological biomarkers, and physiological performance. Moreover, dose-dependent responses of Spirulina platensis supplementation in working dogs have not been sufficiently characterized.

Based on this concept, we hypothesized that dietary supplementation of Spirulina platensis would improve the nutritional, biochemical, antioxidant, and immunological parameters in adult working dogs in a dose-dependent manner.

Accordingly, the present study aimed to evaluate and compare the effects of dietary supplementation of Spirulina platensis powder at two inclusion levels for adult working dogs. The objectives of the present study were to assess the potential dose-dependent responses in selected nutritional status parameters and fecal characteristics, protein profile and lipid metabolism, liver and kidney function, antioxidant biomarkers and fecal microbiota composition of adult working dogs. In addition, the study investigated some selected genes related to gut integrity (OCLN, FABP2) and systemic inflammatory and immunological (IL-6, TRAF6) response to determine the potential nutrigenomic impact of Spirulina platensis in dogs.