PhD in Veterinary Sciences for Animal Health and Food Safety

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Curriculum vitae

Phd thesis

PhD Thesis Title

Improving Gastrointestinal Health and Wellbeing in Dogs under Breeding Conditions: Efficacy of a Natural Dietary Supplement

Summary

This three-year PhD project focused on the development and evaluation of a novel natural dietary supplement composed of bromelain, quercetin, and Lentinula edodes (shiitake mushroom) to improve gastrointestinal (GI) health and overall wellbeing in dogs kept under stressful breeding (kennel) conditions. The supplement was tested through a combination of in vitro and in vivo studies, assessing its antioxidant potential, effects on inflammatory markers, and influence on gut microbiota composition.

Background: Gastrointestinal (GI) disorders remain a significant health concern in dogs, particularly those exposed to chronic stressors of breeding or kennel environments. Under such conditions, characterized by repeated pregnancies, environmental instability, and social stress, dogs are especially vulnerable to chronic stress and gut disturbances that can lead to dysbiosis, gastroenteritis, diarrhea, leaky gut syndrome, and other inflammatory GI disorders. Oxidative stress has been identified as a key contributor to the pathogenesis of many of these conditions, exacerbating inflammation and compromising intestinal barrier function. Natural nutraceuticals such as bromelain, quercetin, and Lentinula edodes (Shiitake mushroom) have individually demonstrated promising antioxidant, immunomodulatory, and anti-inflammatory effects. However, their combined efficacy in improving GI health in dogs, particularly under breeding or kennel conditions, has not been explored. This study addresses this gap by evaluating the potential of a novel dietary supplement composed of these three natural ingredients to modulate gut health and maintain microbiome balance in kennel-housed dogs. Aim: This study aimed to evaluate the impact of a novel natural dietary supplement composed of bromelain, quercetin, and Lentinula edodes on the GI health in healthy adult dogs living under kennel conditions. Methods: The research was conducted in three phases: In vitro, in vivo, and microbiome analysis phases. The in vitro phase evaluated the antioxidant capabilities of five chosen nutraceuticals via TPC, DPPH, and ABTS assays. Three ingredients (bromelain, quercetin, and L. edodes) were chosen for the final supplement formulation because they had better antioxidant profiles. The in vivo phase consisted of a 28-day randomized, double-blinded, placebo-controlled trial with 30 healthy adult female dogs, allocated into treatment and control groups. Dogs were given either the supplement or a placebo along with a standard commercial diet. The gastrointestinal health biomarkers, including calprotectin, cortisol, N-Methylhistamine (NMH), SCFAs, indole/skatole, were evaluated in the fecal samples and underwent 16S rRNA-based microbiome profiling. The microbiome analysis focused on sequencing the 16S rRNA gene to evaluate changes in gut microbiota composition, diversity, and abundance of specific beneficial or pathogenic bacterial taxa following supplementation. Results: Fecal calprotectin, cortisol, NMH, and indole/skatole levels were significantly reduced in the dogs receiving the supplement, indicating reduced intestinal inflammation and stress. Additionally, SCFA levels increased significantly, indicating improved colonic fermentation and metabolic activity. Favorable shifts in gut microbial composition, with an increased abundance of beneficial genera such as Lactobacillus, Bifidobacterium, and Pediococcus, were revealed through microbiome analysis. Importantly, no adverse effects on body weight, condition scores, or overall behavior were observed during the study.

Conclusion: This study’s findings demonstrated that a supplement formulated with quercetin, bromelain, and Lentinula edodes effectively improved gastrointestinal health in dogs by reducing markers of inflammation and stress, while enhancing beneficial microbial activity and SCFA production.  Importantly, the supplement modulated the gut microbiota without disrupting overall diversity, highlighting its potential as a safe and natural dietary strategy for promoting gut health, particularly under kennel conditions. Further research, including male dogs and longer treatment periods, is recommended to broaden the applicability of these results.

Research activities

Study 1:  Initial Investigations and In Vitro Antioxidant Chemical Analyses

1. Objective: To evaluate the antioxidant properties of bromelain, quercetin, and Lentinula edodes (shiitake mushroom).
2. Activities:
   • I conducted extensive literature review to select natural ingredients with potential antioxidant properties.
   • I performed in vitro assays to measure Total Phenolic Content (TPC), DPPH Free Radical Scavenging Activity, and ABTS Radical Cation Decolorization.
   • I formulated a dietary supplement based on the results of the individual and combined antioxidant capacities of the selected ingredients.

Study 2:  In Vivo Trials and Initial Analysis

1. Objective: To assess the safety and efficacy of the formulated dietary supplement on GI health in dogs under breeding conditions.
2. Activities:
   • I conducted a double-blind, placebo-controlled trial involving 30 dogs, divided into treated and control groups.
   • I collected fecal samples at weekly intervals over a 35-day period.
   • I measured fecal markers including calprotectin, histamine, indole/skatole, pH, and Short-Chain Fatty Acids (SCFA).

Study 3:  Microbiota Analysis and Advanced Insights

1. Objective: To investigate the impact of the dietary supplement on the gut microbiome and its broader implications.
2. Activities:
   • I extracted and quantified microbial DNA from fecal samples.
   • I conducted 16S rRNA sequencing and bioinformatics analysis using QIIME 2 and R software.
   • I analyzed microbial community richness and evenness (Alpha diversity) and community composition (Beta diversity).
   • I identified specific taxa significantly affected by the supplement.

Publications