PhD in Veterinary Sciences for Animal Health and Food Safety

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Curriculum vitae

Phd thesis

Title: Cricket meals impact on porcine gut health: pathological and immunological perspectives

Scientific background/state of the art

Gut health plays a pivotal role in regulating nutrient absorption, immune function, and overall productivity. The gastrointestinal (GI) tract functions not only as a site for digestion but also as an immune barrier, interacting dynamically with diet and microbiota to modulate physiological and pathological responses (Pluske et al., 2018; Malematja et al., 2023). In recent years, attention has turned to sustainable, alternative feed ingredients such as insect-derived active compounds. Among these, cricket (Acheta domesticus) meal (CM) has emerged as a promising candidate replacing soyabean meal, due to its high protein content, antimicrobial peptides (e.g.,cecropin, mellitin, attacin), polyunsaturated fatty acids, phenolic compounds and chitin a component with immunomodulatory properties (Belleggia et al., 2023; Boontiam et alet al., 2022; Udomsil et al., 2019). Furthermore, it is authorized as a novel food and feed in the European Union, reflecting its safety and potential for use in human and animal nutrition (IPIFF, 2021). Several studies suggest that insect meals can beneficially modulate gut health (morphology), mucin production, cytokine expression, and microbiota composition, suggesting potential benefits for animal nutrition (Biasato et al., 2023; Schiavone et al., 2017; Van Huis, 2013). However, despite growing interest, detailed investigations focusing on CM’s impact on swine gut health remain scarce. Comprehensive investigations on morphological, biochemical, and immunological aspects of gut health in response to CM intake are necessary to establish its dietary viability for pigs.

Aims

This study aims to investigate the effects of dietary CM supplementation on gut health in pigs, focusing on in-vitro and in-vivo investigations. Particularly the project will evaluate the effects of CM on i) porcine intestinal cell lines (IPEC-J2) (integrity, permeability and potential antioxidant, and antiinflammatory effects due to phenolic, flavonoid and immunomodulatory compounds) and (ii) on pigs fed with a diet containing different percentages of inclusion of CM (systemic and intestinal health). In order to achieve this goal blood profile, histopathology of the main organs, and intestinal evaluations (histomorphometry of the small intestinal wall; mucin, and tight junctions’ expression; immunological responses, and microbiota composition changes) will be done.

Materials and methods

 In-Vitro analyses

Enzymatic Digestion: CM will undergo an enzymatic in-vitro gastrointestinal (GI) digestion to mimic the digestive process that takes place in-vivo, following a protocol adapted for monogastrics (Lo et al., 2022). After digestion digested CM will be centrifuged and supernatant will be stored at -80 °C until cell treatment. The antioxidant activity of digested CM will be assessed using a DPPH radical scavenging assay (Mercatante et al., 2022). Viability of IPEC-J2 exposed to digested CM will be evaluated through MTT assay. Intracellular ROS levels in cells treated with CM will be quantifie using DCFDA assay (Hsu et al., 2025). Immunohistochemistry using commercial antibodies will permit to localize tight junction proteins such as ZO-1 and occludin, and qRT-PCR will be used to measure gene expression of relevant cytokines ((IL-1, IL-6, TNF-α, IL-4 and IL-10) respectively (Xu et al., 2012). To assess epithelial barrier integrity and tight junction functionality, transepithelial electrical resistance (TEER) measurements will be performed on cells cultured on Transwell inserts. Once cells reach confluency and establish monolayers, an electric current will pass through layer and electrical resistance will be measured across the cell layer by voltohmmeter. (Geens & Niewold, 2010; Srinivasan et al., 2015).

 In-Vivo assessments

An in vivo trial was done in autumn 2024 regularly authorized by the Ethical Committee of Torino University (prot. 0454961.25/07/2024). A total of 48 postweaning pigs were divided in 3 groups (4 pigs/pen and 4pens/group): a control group with standard diet and two groups receiving enzymatically processed 5% CM and 5% CM. Animal health and zootechnical parameters were regularly monitored throughout the trial. After 6-week of trial, animals were regularly slaughtered and samples for post-mortem investigations were collected. Briefly, a small sample of, liver and spleen and different intestinal tracts were sampled for histopathological assessment, and a further sample of jejunum was frozen for biomolecular investigations. (Loi et al., 2021). Histomorphometrical measurements of the small intestine will be done. Then, evaluation of mucins production, tight junctions and anti and pro-inflammatory cytokines (IL-1, IL-6 & TNF-α and IL-4 & IL-10 respectively) will be done by histochemistry, immunohistochemistry and qRT-PCR respectively (Kochetkov et al., 2023). Microbiota profiling will be performed through 16S rRNA gene sequencing using Illumina platform from feces samples collected and stored at -80°C (Bolyen et al., 2019).

Data quality and completeness will be analyzed using appropriate statistical methods and compared among groups and with the literature.

Expected results

The in vitro phase is expected to demonstrate how digested CM influence intestinal barrier function, immune response, and oxidative stress in IPEC-J2 cells. Improvements in TEER, reductions in ROS levels, and modulation of cytokine expression are anticipated. Increased/preserved expression of tight junction proteins will indicate enhanced barrier integrity and anti-inflammatory potential. The in vivo trial is expected to improve or not negatively impact animal health, gut morphometry, immune balance, and microbiota composition. These changes have the potential to enhance growth performance, and nutrient absorption.

Bibliography

1. Belleggia, L., Foligni, R., Ferrocino, I., Biolcati, F., Mozzon, M., Aquilanti, L., ... & Harasym, J. (2023). Morphotextural, microbiological, and volatile characterization of flatbread containing cricket (Acheta domesticus) powder and buckwheat (Fagopyrum esculentum) flour. European Food Research and Technology, 249(11), 2777-2795.
2. 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.
3. Bolyen, E., et alet al. (2019). Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology, 37, 852–857. https://doi.org/10.1038/s41587-019-0209-9

4. Boontiam, W., Hong, J., Kitipongpysan, S., & Wattanachai, S. (2022). Full-fat field cricket (Gryllus bimaculatus) as a substitute for fish meal and soybean meal for weaning piglets: Effects on growth performance, intestinal health, and redox status. Journal of Animal Science, 100 (4), skac080.
5. Geens, M. M., & Niewold, T. A. (2010). Optimizing culture conditions of a porcine epithelial cell line for trans-epithelial electrical resistance measurements. Veterinary Research Communications, 34(5), 379–388. https://doi.org/10.1007/s11259-010-9425-4
6. Hsu, M. C., Wang, H. T., & Chen, C. Y. (2025). Carvacrol Protects IPEC-J2 Cells from Oxidative Stress by Suppressing Autophagy. International Journal of Molecular Sciences, 26(8), 3495.
7. International Platform of Insects for Food and Feed (IPIFF). (2021, January 13). FAQs: Insects as novel foods in the European Union. https://ipiff.org/wp-content/uploads/2021/01/FAQs-Insects-as-Novel- Foods-in-the-European-Union-13-01-final.pdf
8. Kochetkov, N., Smorodinskaya, S., Vatlin, A., Nikiforov-Nikishin, D., Nikiforov-Nikishin, A., Danilenko, V., ... & Marsova, M. (2023). Ability of Lactobacillus brevis 47f to alleviate the toxic effects of imidacloprid low concentration on the histological parameters and cytokine profile of zebrafish (Danio rerio). International Journal of Molecular Sciences, 24(15), 12290.
9. Lo, S. H., Chen, C. Y., & Wang, H. T. (2022). Three-step in vitro digestion model for evaluating and predicting fecal odor emission from growing pigs with different dietary protein intakes. Animal bioscience, 35(10), 1592.
10. Loi, F., Pilo, G., Franzoni, G., Re, R., Fusi, F., Bertocchi, L., ... & Nicolussi, P. (2021). Welfare assessment: correspondence analysis of welfare score and hematological and biochemical profilesof dairy cows in Sardinia, Italy. Animals, 11(3), 854.
11. Malematja, E., Manyelo, T. G., Sebola, N. A., & Mabelebele, M. (2023). The role of insects in promoting the health and gut status of poultry. Comparative Clinical Pathology, 32(3), 501-513.
12. Mercatante, D., Ansorena, D., Taticchi, A., Astiasarán, I., Servili, M., & Rodriguez-Estrada, M. T. (2022). Effects of in vitro digestion on the antioxidant activity of three phenolic extracts from olive mill wastewaters. Antioxidants, 12(1), 22.
13. Pluske, J.R., Turpin, D.L., & Kim, J.C. (2018). Gastrointestinal tract (gut) health in the young pig. Animal Nutrition, 4(2), 187–196. https://doi.org/10.1016/j.aninu.2017.12.004
14. Schiavone, A., De Marco, M., Martínez, S., et alet al. (2017). Nutritional value of a partially defatted and a highly defatted black soldier fly larvae meal for broiler chickens: apparent nutrient digestibility, apparent metabolizable energy and apparent ileal amino acid digestibility. Journal of Animal Science and Biotechnology, 8, 51. https://doi.org/10.1186/s40104-017-0181-5
15. Srinivasan, B., et alet al. (2015). TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation, 20(2), 107–126.
16. Udomsil, N., Imsoonthornruksa, S., Gosalawit, C., & Ketudat-Cairns, M. (2019). Nutritional values and functional properties of house cricket (Acheta domesticus) and field cricket (Gryllus bimaculatus). Food Science and Technology Research, 25(4), 597-605.
17. Van Huis, A. (2013). Potential of insects as food and feed in assuring food security. Annual Review of Entomology, 58, 563–583. https://doi.org/10.1146/annurev-ento-120811-153704.
18. Xu, S., Lee, J., & Miyake, M. (2012). Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids. Zygote, 20(2), 147-158.

Research activities

• Animal feeding behaviour evaluation e.g., histopathological, histomorphometrical, gut microbiota, inflammatory responses
• Animal gut health and immunity

• Insect-based diets for animal nutrition

• In vitro digestion and Cell culture models

• Lipidomics

International and National oral Presentation

(presenter *)

Muhammad Adnan Arif*, Talal Hassan, Simona Bavaro, Silvia Mioletti, Muhammad Irfan Malik, Stefano Bagatella, Irene Ferrara, Massimiliano Tursi, Giovanni Perona, Zaira Loiotine, Vladimiro Cardenia, Ilaria Biasato, Ilario Ferrocino, Maria Teresa Capucchio. Exploring the bioactive potential of cricket (Acheta domesticus) meal for gut health. 3rd International – 8th National Veterinary Pharmacology and Toxicology Congress, 11-13th September, 2025 Ankara, Turkey.  

Muhammad Adnan Arif*, Stefano Bagatella, Muhammad Irfan Malik, Talal Hassan, Irene Ferrara, Massimiliano Tursi, Giovanni Perona, Zaira Loiotine, Vladimiro Cardenia, Ilaria Biasato, Ilario Ferrocino, Maria Teresa Capucchio. Fifty shades of crickets: exploring the impact of meals on gut health in monogastric animals.  Game of Research, 4th Edition , University of Turin, Grugliasco, Turin, Italy, 2024, December 12.

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.

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. p396. https://www.espn2025.eu/wp-content/uploads/2025/06/ESPN-2025-Abstractbook-final.pdf

Samanta Mecocci*, Daniele Pietrucci, Stefano Bagatella, Marta Castrica, Gabriele Acuti, Katia Cappelli, Talal Hassan , Muhammad Irfan Malik, Giulio Curone, Muhammad Adnan Arif, Vincenzo Ferrulli, Antonio Boccardo, Silvia Grossi, Carlo Angelo Sgoifo Rossi, Massimo Trabalza-Marinucci, Maria Teresa Capucchio, Claudio Forte, Davide Pravettoni. Gut health effects of hazelnut skin dietary supplementation in Limousine bulls. The 26th Congress of the Association for Science and Animal Production-ASPA, Turin, Italy, 2025, June 17–20.

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 - Turin, Italy, 2024, July 4-5. p34. https://ecsrhm.org/wp-content/uploads/ECSRHM_IZS-Turin-2024-Final-Proceedings.pdf

[Internation Poster Presenttations]

Begum Yurdakok-Dikmen, Muhammad Adnan Arif*, Talal Hassan, Stefano Bagatella, Lucia Simona Bavaro, Emilia Ivanova, Salome Robbert Prieto, Jesus De la Osada Garcia, El Akrem Hayouni,Ayhan Filazi,Maria Teresa Capucchio. Olive Bioactive Compounds in Circular Feed Systems: In Vitro Evaluation within the OLIWA Project. 3rd International & 8th National Veterinary Pharmacology and Toxicology Congress, 11-13th September, 2025 Ankara, Turkey.

Bagatella Stefano*, Muhammad Irfan Malik, Talal Hassan, Muhammad Adnan Arif, Alessandra Sereno, Valentina Bongiorno, Sara Bellezza Oddon, Zaira Loiotine, Ilaria Biasato, Laura Gasco, Capucchio Maria Teresa. Effects of live larvae of Tenebrio molitor as feed ingredients on poultry gut health: histomorphometric and histological investigations-47th European Society of Veterinary Pathology congress-ESVP- Turin, Italy, 2025, August 27-30.

Publications