PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Maria Teresa Capucchio

Phd thesis

Title: Use of novel feed ingredients in monogastric animals: validating porcine in-vitro tools for gut health evaluation in dogs.

Scientific background/state of the art

Gut health, a cornerstone of animal welfare and productivity, encapsulates multifaceted elements including digestion efficiency, intestinal barrier integrity, disease resilience, microbiota stability, and immune function (Colombino et al., 2021). Perturbations in this delicate equilibrium can precipitate diminished feed conversion, increased disease susceptibility, and augmented production costs (Kogut et al., 2020; Colombino et al., 2022).
Functional feeds such as natural extracts (i.e., from olive) and insect derived feed constituents (chitin and chitosan), notably sourced from Hermetia illucens (HI) exhibit promising potential in fortifying gut-health by virtue of their antioxidative properties and capacity to foster beneficial microbial proliferation (EspiN et al., 2013; Sarica & Ürkmez, 2016; Biasato et al., 2023; Domínguez-Oliva et al., 2023). Nevertheless, the optimal utilization of these novel ingredients necessitates further elucidation through ongoing research endeavors. A nuanced comprehension of the interplay between gut physiology, microbiota dynamics, and nutritional inputs is imperative in orchestrating the pinnacle of gut-health in monogastric animals.
Current investigations into these ingredients' impact on livestock and pets have utilized various research methods. However, the detailed in-vitro effects of these functional feeding ingredients using porcine models, along with the validity and translatability of such models to other monogastric animals such as dogs, have yet to be thoroughly investigated.

Aims

Overall aim: to comprehensively assess the potential of functional compounds derived from plant extracts (i.e. oleuropein) and HI insect meal to improve gut-health in monogastric animals, while validating non-invasive ex-vivo investigative methods in comparison with traditional in-vivo approaches.
Specific aims:
▪ To identify preferred natural extracts for pet diets by means of longitudinal studies and literature review.
▪ To determine safe inclusion levels of natural extracts, and HI insect meal in monogastric diets through in-vitro testing.
▪ To investigate the effects of selected functional compounds on intestinal cell lines (integrity and permeability, antioxidant, and anti-inflammatory properties).
▪ To develop intestinal organoids (enteroids) to better study immunometabolic interaction and molecular assessment of crosstalk between gut epithelial, immune cells, and dietary substances thanks to their multicellular composition.
▪ To investigate the effects of these functional feed ingredients on canine gut health by means of in-vivo tests comparing the results of the non-invasive ex-vivo investigation methods with traditional in-vivo animal studies for assessing gut health interventions.

Materials and methods

1. Selection of natural ingredient/ extract:
   ▪ Literature review on bio-refinery waste with gut-health effects.
   ▪ Preparation of pet owner/veterinarian surveys to identify preferred natural extracts for pet diets and pathologies.
2. In-vitro testing:
   ▪ Use of in-vitro static digestion method (Info-digest) with digestive adjustments according to monogastric animals permiting a good absorption of HI meal bioactive compounds.
   ▪ Test digested samples in cell line assays (colorectal adenocarcinoma cells- Caco2, Intestinal porcine epithelail cells aka IPEC-J2, and primary porcine cells) to assess the pro/anti-inflammatory and antioxidant effects of natural extracts and/or HI insect meal.
   ▪ Functional feed will undergo assessment using 2,2-diphenyl-1-picrylhydrazyl assay (DPPH) and fluorescence flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT), transepithelial electrical resistance (TEER) measurements, immunohistochemistry, and RT-PCR. These analyses will evaluate antioxidant activity post-digestion and cell treatment, cell viability, barrier function (tight junctions), and anti-inflammatory activities (expression of cytokines IL-1, IL-6, and TNFα) respectively.
3. Immunometabolic interaction and molecular assessment:
   ▪ Development of ex-vivo 3D porcine organoid (enteroids) and testing for nutrient absorption using Liquid chromatography–mass spectrometry (LC–MS) or High-performance liquid chromatography (HPLC) and anti-inflammatory activities (RT-PCR). 
4. In-vivo trials (dogs):
   Enrollment of healthy dogs (control, one selected extract; two percentages of inclusions). Assessment of clinical health (5 pillars of welfare), digestibility (proximate analysis of feed and fecal sample), antioxidant status (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS (+)) decolonization assay), gut-health (calprotectin, IgA, inflammatory cytokines by means of commercial ELISA kits), and fecal microbiota through fecal immunoassays and 16S rRNA sequencing.

Expected results

▪ Survey-based studies (questionnaires) will identify preferred plant extracts to be analysed.
▪ In-vitro assays will assess the chosen extracts and insect meal for their antioxidant and anti-inflammatory properties. They will also determine safe dietary inclusion levels for in vivo trial.
▪ In-vivo assay should reduce faecal markers of intestinal inflammation and antioxidant capacity; 16S rRNA sequencing of fecal microbiota will anticipate a shift in gut microbiota towards beneficial species (increase in the abundance of commensal bacteria and a decrease in potentially pathogenic species).

Overall, the project is expected to strengthen the antioxidant and anti-inflammatory effects of these novel feed ingredients and validates the use of ex-vivo and in-vitro testing as alternatives or complements to in-vivo animal testing, thus advancing ethical and efficient research practices. 

Reference

1. Biasato, I., Gasco, L., Schiavone, A., Capucchio, M. T., & Ferrocino, I. 2023. "Gut microbiota changes in insect-fed monogastric species: state-of-the-art and future perspectives." Animal Frontiers (13) 4: 72–80. doi:https://doi.org/10.1093/af/vfad025.
2. Chisoro, P., Jaja, I. F., & Assan, N. (2023). Incorporation of local novel feed resources in livestock feed for sustainable food security and circular economy in Africa. Frontiers in Sustainability, 4. https://doi.org/10.3389/frsus.2023.1251179.
3. 
   Colombino, E., Biasato, I., Ferrocino, I., Oddon, S. B., Caimi, C., Gariglio, M., Dabbou, S., Caramori, M., Battisti, E., Zanet, S., Ferroglio, E., Cocolin, L. S., Gasco, L., Schiavone, A., & Capucchio, M. T. 2022. "Effect of insect live larvae as environmental enrichment on poultry gut health: gut mucin composition, microbiota and local immune response evaluation." Animals (11) 10: 2819. doi:https://doi.org/10.3390/ani11102819.
4. 
   Colombino, E., Prieto-Botella, D., & Capucchio, M. T. 2021. "Gut health in Veterinary Medicine: A Bibliometric Analysis of the literature." Animals (11) 7: 1997. doi:https://doi.org/10.3390/ani11071997.
5. 
   Domínguez-Oliva, A., Hernández-Ávalos, I., Martínez‐Burnes, J., Olmos-Hernández, A., Verduzco-Mendoza, A., & Mota-Rojas, D. 2023. "The importance of animal models in biomedical Research: Current insights and applications." Animals (13) 7: 1223. doi:https://doi.org/10.3390/ani13071223.
6. 
   Espı́N, J. C., Larrosa, M., Garcı́a-Conesa, M. T., & Tomás‐Barberán, F. A. 2013. "Biological significance of urolithins, the gut microbial Ellagic Acid-Derived Metabolites: the evidence so far." Evidence-based Complementary and Alternative Medicine 1-15. doi:https://doi.org/10.1016/j.psj.2019.12.011.
7. 
   Griesinger, C., Desprez, B., Coecke, S., Casey, W., & Zuang, V. (2016). Validation of alternative in vitro methods to animal testing: concepts, challenges, processes and tools. Advances in Experimental Medicine and Biology, 65–132. https://doi.org/10.1007/978-3-319-33826-2_4.
8. 
   Kogut, M. H., Lee, A., & Santín, E. 2020. "Microbiome and pathogen interaction with the immune system." Poultry Science (99) 4: 1906–1913. doi: https://doi.org/10.1016/j.psj.2019.12.011.
9. 
   Sarica, S., & Ürkmez, D. 2016. "The use of grape seed-, olive leaf- and pomegranate peel-extracts as alternative natural antimicrobial feed additives in broiler diets." European Poultry Science (EPS) 80. doi:https://doi.org/10.1399/eps.2016.121.

 

 

Research activities

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Co supervisor

Dr. Michael H. Kogut (USDA-ARS, Southern Plains Agricultural Research Center, College Station, Texas, USA).

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Gut-Health and Animal Health Team: https://veteren.campusnet.unito.it/do/home.pl/View?doc=/research/research_line/reasearch_topic/gut_health.html

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Publications:

Hassan, T., Merlino, V. M., Badino, P., Odore, R., Shahid, M. Q., Amerio, A., & Renna, M. (2024). Milk consumers in Pakistan and Italy: a comparative study on the effects of geographical affiliation, socio-demographic characteristics and consumption patterns on knowledge, attitude and perception of antimicrobial resistance. BMC Public Health, 24(1). https://doi.org/10.1186/s12889-024-21002-w

Battaglia, L., Dianzani, C., Muntoni, E., Marini, E., Bozza, A., Bordano, V., Ferraris, C., Garelli, S., Valsania, M. C., Terreno, E., Capozza, M., Costanzo, D., Capucchio, M. T., Hassan, T., Pizzimenti, S., Pettineo, E., Di Muro, M., & Scorziello, F. (2024). Ultrasmall solid lipid nanoparticles as a potential innovative delivery system for a drug combination against glioma. Nanomedicine, 1–16. https://doi.org/10.1080/17435889.2024.2434452

Malik, M. I., Bilal, M., Anwar, M. Z., Hassan, T., Rashid, M. A., Tarla, D., Dunshea, F. R., & Cheng, L. (2024). Effects of betaine supplementation on dry matter intake, milk characteristics, plasma non-esterified fatty acids, and beta-hydroxybutyric acid in dairy cattle: a meta-analysis. Journal of Animal Science, 102. https://doi.org/10.1093/jas/skae241

Malik, M. I., Li, J., Capucchio, M. T., Hassan, T., & Sun, X. (2024). Effects of distiller’s dried grains with solubles on enteric methane emissions in dairy and beef cattle: a meta-analysis. Frontiers in Veterinary Science, 11. https://doi.org/10.3389/fvets.2024.1480682

Robles-Jimenez, L., Angeles, S., Ramirez-Perez, A., Fuente, B., Velazquez-Ordoñez, V., Cardoso-Gutierrez, E., Renna, M., Rastello, L., Capucchio, M., Hassan, T., Gasco, L., Pino-Moreno, J., Ghavipanje, N., Dominguez-Vara, I., & Gonzalez-Ronquillo, M. (2025). In vitro and in vivo investigations on the use of yellow mealworm (Tenebrio molitor) as a novel protein feed ingredient for fattening lambs. Animal Feed Science and Technology, 116224. https://doi.org/10.1016/j.anifeedsci.2025.116224

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Professional trainings:

Advanced methodologies for microbial monitoring to improve the safety of foods and processes, Department of Veterinary Science, Grugliasco, 13th May, 2024.

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International oral presentation

(* Presenter)

Talal Hassan, Muhammad Irfan Malik, Muhammad Adnan Arif, Elena Diaz Vicuna, Lara Rastello, Laura Gasco, Claudio Forte, Manuela Renna, Maria Teresa Capucchio*. Gut-health evaluation in small ruminants: a multidisciplinary approach to optimize animal health and performance. European College of Small Ruminants Health and Management-ECSRHM- Torino, Italy, 2024, July 4-5.

Talal Hassan*, Elena Diaz Vicuna, Dorotea Ippolito, Francesca Barroero, Muhammad Irfan Malik, Martino Musati, Antonio Natalello, Claudio Forte, Maria Teresa Capucchio. Gut-health histomorphometry in fattening lambs fed with hazelnut skin and linseed. International Congress on Natural Products Research-ICNPR- Kraków, Poland, 2024, July 13-17.

Talal Hassan*, Muhammad Irfan Malik, Chiara Sferra, Marta Gariglio, Ilario Ferrocino, Ilaria Biasato, Laura Gasco, Maria Teresa Capucchio. Can the mixture of Hermetia illucens and Tenebrio molitor meals influence the gut health of broiler chickens? Symposium on Gut Health in Production of Food Animals, Saint Louis, Missouri, USA, 2024, November 10-13.

Talal Hassan, Stefano Bagatella, Muhammad Irfan Malik, Alessandro Bellato, Dorotea Ippolito, Simona Zoppi, Maria Beatrice Boniotti, Giovanni Loris Alborali, Maria Teresa Capucchio*. Pathological and microbiological investigations of enteric disorders in post-weaning pigs: association between pathogen co-infections and enteritis pattern. Symposium on Gut Health in Production of Food Animals, Saint Louis, Missouri, USA, 2024, November 10-13.

Eleonora Erika Cappone*, Valeria Zambotto, Chiara Bianchi, Maria Teresa Capucchio, Talal Hassan, Muhammad Irfan Malik, Stefano Bagatella, Muhammad Adnan Arif, Valentina Bongiorno, Ilaria Biasato, Edoardo Fiorilla, Marta Gariglio, Achille Schiavone. Digestibility and gut health of three endangered Italian chicken breeds: Bionda Piemontese, Bianca di Saluzzo and Millefiori Piemontese. The 24th European Symposium on Poultry Nutrition, Maastricht, The Netherlands, 2024, June 23-26.

 

National oral presentation

Talal Hassan, Elena Diaz Vicuna, Dorotea Ippolito, Francesca Barroero, Muhammad Irfan Malik, Martino Musati, Antonio Natalello, Claudio Forte, Maria Teresa Capucchio*. Hazelnut skin and linseed as partial replacements for maize in lamb diet: impact on growth, ruminal histomorphometry, and gut-health. Italian Society of Veterinary Sciences-SISVET- Parma, Italy, 2024, June 12-14.

Talal Hassan*, Joana Nery, Silvia Mioletti, Luigi Bertolotti, Zaker Rehan Mohammed, and Maria Teresa Capucchio. Bio-refinery natural by-products in pet food formulations to optimize animal health and gut functions. Game of Research, 3rd Edition, University of Turin, Grugliasco, Turin, Italy, 2023, December 14.

 

International poster presentation

Talal Hassan*, Joana Nery, Mohammad Zaker Rehan, Muhammad Irfan Malik, Silvia Mioletti, Luigi Bertolotti, Alba Maria Ramos, Natascia Bruni, José M. Pinilla-Rosas, Maria Teresa Capucchio. A survey-based study on knowledge, attitude, and perception of pet owners towards the use of plant extracts from bio-refinery in pet food formulations. International Congress on Natural Products Research-ICNPR- Kraków,Poland, 2024, July 13-17.

Nicolò Lonigro, Talal Hassan, Mohammed Zaker Rehan, Mauro Bigliati, Maria Teresa Capucchio*. In-vitro investigation of the gastroprotective effects of a natural extract blend: a promising avenue for canine gastritis prevention. International Congress on Natural Products Research-ICNPR- Kraków, Poland, 2024, July 13-17.

Muhammad Adnan Arif, Talal Hassan, Muhammad Irfan Malik, Stefano Bagatella, Alessandra Sereno, Lorena Sanchez, Sara Bellezza Oddon, Ilaria Biasato, Laura Gasco, Maria Teresa Capucchio*. Effects of Alphitobious diaperinus insect meal on histomorphometry and mucin production in Oncorhynchus mykiss. 1st international Congress on the cultivation and processing of alternative biomass- Cloppenburg, Germany, 2025, May 12-14.

 

National poster presentation

Matteo Sina*, Talal Hassan, Francesca Bonvini, Elena Colombino, Dorotea Ippolito, Barbara Colitti, Federica Raspa, Sara Antoniazzi, Luigi Bertolotti, Manuela Renna, Maria Teresa Capucchio. Comprehensive characterization of rumen microenvironment in pre-weaned calves at 77 days of age. Game of Research, 3rd Edition, University of Turin, Grugliasco, Turin, Italy, 2023, December 14.