- Scientific context / state of the art
Oxidative stress has been implicated in the pathogenesis of many Gastrointestinal (GI) disorders and inflammatory conditions in dogs such as dysbiosis, intestinal bowel diseases, acute diarrhea, etc. It occurs when redox homeostasis within the cell is altered due to either an overproduction of reactive oxygen species (ROS) or a deficiency of counteracting antioxidant system (Semba and Tang, 1999; Panda et al., 2009; Bhattacharyya et al., 2014; Candellone et al., 2022). ROS are highly reactive molecules that are generated from oxygen metabolism as toxic by-products. ROS are mostly produced by the gastrointestinal (GI) tract (Bhattacharyya et al., 2014). ROS can damage cells through the oxidation of some important molecules such as proteins, lipids, or DNA (Romao, 2015). The GI tract is vulnerable to ROS because it is exposed to the external environment, which includes immune cells that are already present, intestinal microbiota, and dietary nutrients. Treatment of GI disorders such as dysbiosis involves the use of anti-inflammatory drugs or enzyme-inhibiting drugs and antibiotics (Rosenfeld, 2017). However, antibiotics can induce antibiotic resistance. Studies in human and animal models have hypothesized the use of antioxidants as a possible alternative and/or support to antimicrobial drugs, considering the global need to reduce antibiotic use (Ferri et al., 2017). Furthermore, antioxidants help prevent oxidative damage in dogs which, in turn, improves their immunity in the gut and immune system (Jewell et al., 2000). In turn, the use of fecal parameters has been already proved to be relevant in evaluating the effects of antioxidants added to the diet on the intestinal health and the influence of the gut/brain axis (Jewell et al., 2000). The aim of this study is to evaluate the antioxidant power of natural substances tested individually and in combination, for their use in the management and improvement of gastrointestinal health in dogs. The dogs included in the study will be healthy, however, being kept in breeding conditions. Some studies report that dogs in breeding conditions are more susceptible to dysbiosis and intestinal problems than dogs living with their owner. The breeding condition, therefore, leads breeders to antibacterial prophylactic treatments on their dogs. (Beerda et al., 2000, Gazzano et al 2008; Broom and Kirkden, 2004). In fact, hierarchical relationships between subjects, repeated pregnancy conditions and the consequent change in the animal behaviour may induce stress that affect the composition of the intestinal microbiota and increase the vulnerability to inflammatory stimuli of the GI tract (Collins and Bercik, 2009). Furthermore, the condition of dysbiosis often causes diarrhea (Ziese and Suchodolski,2021).
This Ph.D will be structured in two consecutive phases one in vitro and one in vivo in order to evaluate the efficacy of a newly formulated dietary supplement with antioxidant proprieties in healthy dogs ( under breeding conditions) and its impact on their wellbeing.
Phase 1-in vitro
The first phase of the project consists in the selection of five natural ingredients with antioxidant function, according to the literature. The selected ingredients will be tested in the laboratories of the Department of Drug Science and Technology, Unito. They will be tested individually and then in combination (mix), to evaluate their individual and synergistic antioxidant capacity, total phenolic content and the proximate chemical analysis. Then, a food supplement will be formulated on the basis of the results obtained from this in vitro study.
Phase 2-in vivo
The second phase consists in a double blinded case-control study on healthy dogs in breeding condition. The study will be performed in a breeding centre (Mein Staffi, Cumiana (TO), Italy) following the approval of the ethical commission, to test the newly formulated food supplement. Key faecal parameters will be used to estimate the efficacy of the newly formulated supplement rich in antioxidants, to highlight the efficacy in preventing/treating dysbiosis and intestinal inflammation, and to improve the modulation of the immunological intestinal barrier. The effects of the antioxidants in vivo will be evaluated in an indirect way using fecal parameters of intestinal inflammation and immunity. We expect that the increasing intake of antioxidants would reduce the inflammatory and immunological factors in treated compared to untreated dogs.
Materials & methods
- Phase 1
1.1 Laboratory analysis on the five natural ingredients individually tested
The five natural ingredients used in this project were selected and then provided by the Candioli Pharma srl (Beinasco, TO, Italy) and they are present in the document: COMMISSION REGULATION (EU) 2022/1104 of 1 July 2022 amending Regulation (EU) No 68/2013 on the Catalogue of feed materials.
1) Lactoferrin, an iron-binding protein, primarily found in mammalian milk and has anti-inflammatory and immunomodulation properties in dogs (Pope et al., 2006; Hellweg et. al., 2008; Handl et al., 2009);
2) Superoxide Dismutase, is an enzyme with antioxidant properties derived from melons, capable of effectively removing superoxide anions from inflamed colon tissues (Romao, 2015);
3) Quercetin, a polyphenolic antioxidant substance found in many plants and foods, such as grapes, onions, and green tea, able to stimulating immune response (Reinboth et al., 2010);
4) Lentinula edodes, a mushroom that improve the intestinal barrier function with high antioxidant properties (Sande et al., 2019; Costa et al., 2012; Finimundy et al., 2013);
5) Bromelain, derived from pineapple and contains proteolytic enzymes with anti-inflammatory, antimicrobial, and immunomodulatory effects (Chakraborty et al., 2021).
The following laboratory analyses will be carried out on each of the five nutraceuticals to see their individual characteristics at the Department of Drug Science and Technology, Unito. In the literature, it is possible to find similar available analysis but we think it is necessary to perform them in this project as the raw materials used can vary from previously tested ones and can affect the final formulation of the feed supplement.
Each of the analyses listed below will be repeated three times for the five natural ingredients tested individually:
- Proximate chemical analysis such as: moisture, crude protein, ether extract, crude fiber, and ash (AOAC official methods, 2019).
- Total Phenolic Content (TPC) determined using the method described by several authors (Lopez-Mejra et al. 2014; Karamac et al., 2019).
- Scavenging Activity (Both DPPH and ABTS methods) determined using the method described by Brand-Williams and colleagues, 1995.
1.2 Laboratory analysis of the mix of the five natural ingredients
During this step, the five natural ingredients are combined together and tested with the same analyzes described in 1.1.
1.3 Evaluation of the antagonist or synergic effect of the five natural ingredients
The combination of all the five natural ingredients together will be evaluated and, in case no synergistic effect will be found, a new round of the same analysis will be performed to see which combination of ingredients would give the best result in terms of antioxidant capacity.
1.4 Formulation of the complementary feed
A complementary feed will be formulated with the supervision of an expert veterinary nutritionist (Prof. Meineri) taking into account the previously obtained results. Furthermore, the dosage of new complementary feed will respect the nutritional needs of dogs according to Nutritional Guidelines (FEDIAF, 2021).
1.5 Proximate chemical analysis of the basic commercial diet
In order to take into account the composition of the commercial complete feed that will be used (as a basis) in Phase 2, for both control (CRT) and treated (TRT) groups of dogs, a proximate chemical analysis will be performed on this complete feed (as previously described 1.1). (AOAC official methods, 2019)
2) Phase 2
2.1. Case-control study on breeding dogs to evaluate the effect of the complementary feed
The case-control will be conducted on healthy adult American Staffordshire Terrier dogs in a kennel located in Turin province (Mein Staffi, Cumiana, Italy). Subjects under current or recent antibiotic therapy or with evident systemic diseases will be excluded from recruitment.
A total of 24 adult dogs will be randomly assigned to two groups: control (CTR, n = 12) and treated (TRT, n = 12) groups. Both groups will be fed a basic commercial diet for at least 7 days before the beginning of the study. The amount of daily food will be calculated based on the equation:
ME (kcal / day) = 110 × kg BW 0.75 (NRC, 2006).
(ME= metabolic energy; BW = body weight)
A placebo (composed of maltodextrin and appetite stimulants) or the newly formulated complementary feed will be added to the food of dogs belonging to the CTR or TRT group, respectively. All subjects will be provided with water ab libitum.
The duration of the trial will be 35 days divided into weekly testing points: T0 (day 0 –recruitment), T1 (day 7), T2 (day 14), T3 (day 21), T4 (day 28) and T5 (day 35).
2.2 Laboratory Analysis
A set of parameters will be recorded at each time point (from T0 to T5) to estimate the effect of the administration of the newly formulated supplement in comparison to the placebo:
- Health and nutritional status (BW -body weight, BCS -body condition score, MCS -muscle condition score) (WSAVA, 2013a).
- Fecal analysis:
Fecal samples will be stored in special sterile containers and immediately refrigerated (-20 ° C) and used for the following analyses:
- Microbiota (microbial composition of the gastrointestinal tract and the relationship between the bacterial species) will be characterized by using DNA Extraction and Amplicon Target Sequencing (Klindworth et al., 2013).
- Calprotectin concentration (a well-known intestinal inflammatory parameter) will be determined by immunosorbent assay (ELISA) developed and analytically validated at the Gastrointestinal Laboratory at Texas A&M University.
- IgA concentration (a parameter related to the immune status) will be determined by immunosorbent assay (ELISA).
- Indole and Skatole (Putrefactive Fecal Compounds) will be determined by gas chromatography (GC) according to Flickinger et al.,2003.
- Short Chain Fatty Acids (SCFA) (bacterial metabolites) will be measured using a gas chromatography mass spectrometry (GC-MS) assay.
- Cortisol concentration (a stress marker) will be determined by Enzyme Immune Assay (EIA).
The statistical analysis that will be adopted will be a one-way analysis of variance, with the molecule variable (quercetin, bromelain, mushroom, mix ...) and the value of the five molecules will be compared for each single analysis (DPPH, ABST, TPC). The differences between the values will be considered significant with a p-Values <0.05. The reported values will represent not an arithmetic mean, but the least squares mean (LSM). The standard error of the mean (SEM) will also be calculated.
Sequencing data on the microbiota analysis will be evaluated by the Quantitative Insights into Microbial Ecology (QIIME) 2 (Kuczynski et al., 2011).
The comparison of data from the CTR and TRT group and related to fecal markers and nutritional parameters will be evaluated by analysis of variance (ANOVA) using the SPSS statistical software (Version 11.5.1 for Windows, SPSS Inc., USA).
The project is expected to evaluate a new supplement based on the combination of natural ingredients with the highest antioxidant capacity previously tested in the laboratory. This product will be tested to see improvement in the general health of dogs under breeding conditions. The effects of the antioxidants will be evaluated indirectly by testing the intestinal health. The oxidative stress can cause inflammation/dysbiosis. Fecal parameters of inflammation and immunity can be used to see benefits of the antioxidant activity of the ingredients on the oxidative stress. In particular, the results of this study will be useful to promote the use of a safe and well tolerated diet supplement, to contain the phenomena of dysbiosis associated with diarrhea that often occurs in dogs subjected to the stress induced by the breeding condition.
The experimental protocol was approved by the Ethical Committee of the Department of Veterinary Sciences of the University of Turin (Italy) (Protocol number: 2741).
- Beerda, B.; Schilder, M.B.; Van Hooff, J.A.; De Vries, H.W.; Mol, J.A. Behavioural and hormonal indicators of enduring environmental stress in dogs. Anim. Welf.-Potters Bar 2000, 9, 49–62
- Bhattacharyya, A., Chattopadhyay, R., Mitra, S., & Crowe, S. E. (2014). Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiological reviews, 94(2), 329-354. https://doi.org/10.1152/physrev.00040.2012
- Broom, D.M.; Kirkton, R.D. Welfare, stress, behaviour and pathophysiology. Vet. Pathophysiol. 2004, 337–369.
- Candellone, A., Girolami, F., Badino, P., Jarriyawattanachaikul, W., & Odore, R. (2022). Changes in the Oxidative Stress Status of Dogs Affected by Acute Enteropathies. Veterinary Sciences, 9(6), 276. https://doi.org/10.3390/vetsci9060276
- Chakraborty, A. J., Mitra, S., Tallei, T. E., Tareq, A. M., Nainu, F., Cicia, D., ... & Capasso, R. (2021). Bromelain a potential bioactive compound: a comprehensive overview from a pharmacological perspective. Life, 11(4), 317. https://doi.org/10.3390/life11040317
- Costa Orsine, J. V., Vinhal da Costa, R., & Garbi Novaes, M. R. (2012). Mushrooms of the genus Agaricus as functional foods. Nutr. hosp, 1017-1024. https://doi.org/ 10.3305/nh.2012.27.4.5841
- Ferri, M., Ranucci, E., Romagnoli, P., & Giaccone, V. (2017). Antimicrobial resistance: A global emerging threat to public health systems. Critical reviews in food science and nutrition, 57(13), 2857-2876. https://doi.org/10.1080/10408398.2015.1077192
- Finimundy, T. C., Gambato, G., Fontana, R., Camassola, M., Salvador, M., Moura, S., Hess, J., Henriques, J. A. P., Dillon, A. J. P., & Roesch-Ely, M. (2013). Aqueous extracts of Lentinula Edodes and pleurotus sajor-caju exhibit high antioxidant capability and promising in vitro antitumor activity. Nutrition Research, 33(1), 76–84.
- Gazzano, A.; Mariti, C.; Alvares, S.; Cozzi, A.; Tognetti, R.; Sighieri, C. The prevention of undesirable behaviors in dogs: Effectiveness of veterinary behaviorists’ advice given to puppy owners. J. Vet. Behav. 2008, 3, 125–133. [CrossRef
- Grellet, A., Chastant-Maillard, S., Robin, C., Feugier, A., Boogaerts, C., Boucraut-Baralon, C., ... & Polack, B. (2014). Risk factors of weaning diarrhea in puppies housed in breeding kennels. Preventive Veterinary Medicine, 117(1), 260-265. https://doi.org/10.1016/j.prevetmed.2014.07.016
- Handl, S., Wehr, U., Zentek, J., & Krammer‐Lukas, S. (2009). Histological and immunohistochemical evaluation of duodenal and colonic biopsies after oral bovine lactoferrin supplementation in beagle puppies. Journal of animal physiology and animal nutrition, 93(1), 76-82. https://doi.org/10.1111/j.1439-0396.2007.00781.x
- Hellweg, P., Krammer-Lukas, S., Strasser, A., & Zentek, J. (2008). Effects of bovine lactoferrin on the immune system and the intestinal microflora of adult dogs. Archives of Animal Nutrition, 62(2), 152-161. https://doi.org/10.1080/17450390801892575
- Jewell, D. E., Toll, P. W., Wedekind, K. J., & Zicker, S. C. (2000). Effect of increasing dietary antioxidants on concentrations of vitamin E and total alkenals in serum of dogs and cats. Vet Ther, 1(4), 264-272.
- Karamać, M., Gai, F., Longato, E., Meineri, G., Janiak, M. A., Amarowicz, R., & Peiretti, P. G. (2019). Antioxidant activity and phenolic composition of amaranth (Amaranthus caudatus) during plant growth. Antioxidants, 8(6), 173. https://doi.org/10.3390/antiox8060173
- Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., & Glöckner, F. O. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic acids research, 41(1), e1-e1. https://doi.org/10.1093/nar/gks808
- Muegge, B. D., Kuczynski, J., Knights, D., Clemente, J. C., González, A., Fontana, L., ... & Gordon, J. I. (2011). Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science, 332(6032), 970-974. 1126/science.1198719
- Nutritional guidelines. FEDIAF (email@example.com)
- Official methods of ANALYSISTM, 21st edition (2019). AOAC INTERNATIONAL. (2022, May 5).
- Panda, D., Patra, R. C., Nandi, S., & Swarup, D. (2009). Oxidative stress indices in gastroenteritis in dogs with canine parvoviral infection. Research in veterinary science, 86(1), 36-42. https://doi.org/10.1016/j.rvsc.2008.05.008
- Pope, L. L., Flickinger, E. A., Karr-Lilienthal, L. K., Spears, J. K., Krammer, S., & Fahey Jr, G. C. (2006). Effects of lactoferrin supplementation on ileal and total tract nutrient digestibility, gastrointestinal microbial populations, and immune characteristics of ileal cannulated, healthy, adult dogs. Archives of Animal Nutrition, 60(1), 10-22. https://doi.org/10.1080/17450390500353390
- Reinboth, M., Wolffram, S., Abraham, G., Ungemach, F. R., & Cermak, R. (2010). Oral bioavailability of quercetin from different quercetin glycosides in dogs. British Journal of Nutrition, 104(2), 198-203. https://doi.org/10.1017/S000711451000053X
- Romao, S. (2015). Therapeutic value of oral supplementation with melon superoxide dismutase and wheat gliadin combination. Nutrition, 31(3), 430-436. https://doi.org/10.1016/j.nut.2014.10.006
- Rosenfeld, C. S. (2017). Gut dysbiosis in animals due to environmental chemical exposures. Frontiers in Cellular and Infection Microbiology, 7. https://doi.org/10.3389/fcimb.2017.00396
- Semba, R. D., & Tang, A. M. (1999). Micronutrients and the pathogenesis of human immunodeficiency virus infection. British Journal of Nutrition, 81(3), 181-189. https://doi.org/10.1017/S0007114599000379
- Sande, D., de Oliveira, G. P., e Moura, M. A. F., de Almeida Martins, B., Lima, M. T. N. S., & Takahashi, J. A. (2019). Edible mushrooms as a ubiquitous source of essential fatty acids. Food Research International, 125, 108524. https://doi.org/10.1016/j.foodres.2019.108524
Half first Year achievements: During this first half year of my Ph.D, I focused my studies on the first part of Phase 1: the selection of the ingredients suitable to be tested for their antioxidant capacity through an extensive literature review and the experience in the Candioli Pharma srl. I learned new laboratory techniques at the Department of Drug Science and Technology, Unito (i.e. Organic extraction techniques, method development, antioxidants in food and plant extract analysis, etc.).
- Full first Year achievements:
Laboratory analysis on the selected substances and formulation;
The following laboratory analyses were carried out on each of the five nutraceuticals to see their individual characteristics and then on the mix of the three that showed good antioxidant activity. These analysis were carried out personally, under the guidance of Dr. Annalisa Costale in the Laboratories of the Department of Pharmaceutical Sciences, UNITO.
- a) The chemical composition (protein, fats, ash, fiber) of individual ingredients and the mixture. AOAC methods.
b)The total phenolic content (TPC) analysis of individual ingredients and the mixture was determined spectrophotometrically after a reaction with Folin–Ciocalteu reagent (FCR), following the Karamać et al. (2019) method with slight modifications. The results were expressed as mg of gallic acid equivalents (GAE, mg/g) (Karamać et al., 2019). Each determination was performed in triplicate.
- c) The DPPH scavenging activity was evaluated according to the procedure described by Brand-Williams et al. (1995) with slight modifications.
- d) The ABTS radical cation activity was evaluated following the Karamać et al. (2019) with few modifications (Karamać et al., 2019).
Each determination was performed in triplicate.
National Stays (For internships & training)
- 7 months stay at a research and development industry called Candioli Pharma srl, Italy (09/11/21 – 31/05/22).
- 4 months stay at the research laboratories of the Department of Pharmaceutical Technology, UNITO, Italy (04/06/22 – 31/09/22).
- Full articles in ISI journals (as co-author):
- Meineri, G.; Martello, E.; Radice, E.; Bruni, N.; Saettone, V.; Atuahene, D.; Armandi, A.; Testa, G.; Ribaldone, D.G. Chronic Intestinal Disorders in Humans and Pets: Current Management and the Potential of Nutraceutical Antioxidants as Alternatives. Animals 2022, 12, 812. https://doi.org/10.3390/ani12070812
- Meineri, G.; Martello, E.; Atuahene, D.; Miretti, S.; Stefanon, B.; Sandri, M.; Biasato, I.; Corvaglia, M.R.; Ferrocino, I.; Cocolin, L.S. Effects of Saccharomyces boulardii Supplementation on Nutritional Status, Fecal Parameters, Microbiota, and Mycobiota in Breeding Adult Dogs. Vet. Sci. 2022, 9, 389. https://doi.org/10.3390/vetsci9080389
- Congress attendance (with active participation)
- Terra Madre Congress in Pollenzo and Turin, 23rd – 25th September 2022 (With active participation as a presenter)
Atuahene, D.; Costale, A.; Martello, E.; Ribaldone, D.; Stefanon, B.; Sandri, M.; Meineri, G. New Nutraceuticals derived from natural sources for improving gastrointestinal health in Animal Nutrition. Terra Madre Congress 2022.