Histological evaluation of the intestine of broiler chickens: comparison of three sampling methods

Marielen de Souza, Claudineia Emidio Cicero, Maísa Fabiana Menck-Costa, Larissa Justino, Juliana Rubira Gerez, Ana Angelita Sampaio Baptista, Ana Paula Frederico Rodrigues Loureiro Bracarense

Abstract


After the growth-promoting antibiotics prohibition, intestinal health became an increasing concern worldwide in poultry farming. The intestinal histological evaluation is an inexpensive technique that brings relevant information, but in poultry, the immediate process of intestinal post-mortem autolysis interferes directly on the samples quality for histological analysis hindering a precise diagnosis. This study aimed to standardize a technique for broilers’ intestines sample collection and fixation for histological analysis. Seven broiler chickens received a standard diet until 23 days of age when they were euthanized. Fragments of duodenum, jejunum, and ileum were collected using three methods: intestine strips, transverse section, and Swiss roll and posteriorly fixed in 10% buffered formalin and bouin solution. Tissue samples were submitted for histological (number of villi and viable villi per field) and morphometrical (villi height, crypt depth and villi:crypt ratio) evaluations and the results analyzed statistically. A significant high number of villi and viable villi per field in all regions was observed in the Swiss roll method. In the duodenum (p= 0.0066) and jejunum (p= 0.0058) an interaction between the Swiss roll method and the fixative buffered formalin was observed in the viable and number of villi per field, respectively. Regarding the morphometrical analysis significant differences were observed, in the jejunum villi height sampling by the methods Swiss roll (1,157.66 ± 148.25 µm, p= 0.0015) that showed the highest mean. Deeper crypt depths were observed in the jejunum (156.59 ± 15.68 µm, p= 0.0002) and ileum (131.13 ± 15.01 µm, p= 0.0006) collect by the Swiss roll method. An interaction between the bouin fixative was also observed in the jejunum (p= 0.0223) for this variable. Duodenum sampling by transversal section (12.68 ± 1.45 µm, p= 0.0076) was the only segment that had a significant difference for villi:crypt ratio, showing the highest mean. It can be concluded that the Swiss roll technique was the best method for morphometrical evaluation of the chickens’ intestines, since the highest counts of villi per field and viable villi per field were obtained, while buffered formalin was considered as the best fixative.

Keywords


Post-mortem autolysis; Histology; Intestinal health; Intestinal morphometry; Poultry.

Full Text:

PDF

References


Aviagen. (2018). Ross manual de manejo de frangos de corte. 2018.

Belote, B. L., Tujimoto-Silva, A., Hümmelgen, P. H., Sanches, A. W. D., Wammes, J. C. S., Hayashi, R. M., & Santin, E. (2018). Histological parameters to evaluate intestinal health on broilers challenged with Eimeria and Clostridium perfringens with or without enramycin as growth promoter. Poultry Science, 97(7), 2287-2294. doi: 10.3382/ps/pey064

Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O., & Yang, V. W. (2016). Improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses. Journal of Visualized Experiments, 13(113), 1-8. doi: 10.3791/54161

Biasato, I., Gasco, L., Marco, M., Renna, M., Rotolo, L., Dabbou, S.,... Schiavone, A. (2018). Yellow mealworm larvae (Tenebrio molitor) inclusion in diets for male broiler chickens: effects on growth performance, gut morphology, and histological findings. Poultry Science, 97(2), 540-548. doi: 10.3382/ ps/pex308

Boroojeni, F. G., Männer, K., Rieger, J., Calvo, E. P., & Zentek, J. (2019). Evaluation of a microbial muramidase supplementation on growth performance, apparent ileal digestibility, and intestinal histology of broiler chickens. Poultry Science, 98(5), 2080-2086. doi: 10.3382/ps/pey556

Buesa, R. J. (2008). Histology without formalin? Annals of Diagnostic Pathology, 12(6), 387-396. doi: 10. 1016/j.anndiagpath.2008.07.004

Fletcher, O. J., & Tahseen, A.-A. (2016). Alimentary system. In T. Abdul-Aziz, O. J. Fletcher, & H. J. Barnes (Eds.), Avian histopathology (4nd ed., pp. 271-354). Jacksonville: American Association of Avian Pathologists.

Gava, M., Moraes, L., Carvalho, D., Chitolina, G., Fallavena, L., Moraes, H.,… Salle, C. (2015). Determining the best sectioning method and intestinal segment for morphometric analysis in broilers. Brazilian Journal of Poultry Science, 17(2), 145-149. doi: 10.1590/1516-635x1702145-150

Immerseel, F. V., Rood, J. I., Moore, R. J., & Titball, R. W. (2009). Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiology, 17(1), 32-36. doi: 10.1016/j.tim. 2008.09.005

Ito, N. M. K., Miyaji, C. I., Myaji, S. O., & Lima, E. A. (2009). Fisiopatologia do sistema digestório e anexos. In A. Berchieri, Jr., E. N. Silva, J. D. Fabio, J. D. Fabio, L. Sesti, & M. A. F. Zuanaze (Eds.), Doenças das aves (2a ed., pp. 215-256). Campinas: Facta.

Jones, M. L. (2007). How formalin affects the outcome of routine and special stains. Biotechnic & Histochemistry, 82(3), 155-159. doi: 10.1080/10520290701567908

Kogut, M. H.; Yin, X.; Yuan, J. & Broom, L. (2017). Gut health in poultry. Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, 12(31), 1-7. doi: 10.1079/PAVSNNR201712031 Recovered from https://www.cabdirect. org/cabdirect/abstract/20173301742

Moolenbeek, C., & Ruitenberg, E. J. (1981). The "Swiss roll": a simple technique for histological studies of the rodent intestine. Laboratoy Animals, 15(1), 57-59. doi: 10.1258/002367781780958577

Morgan, N. K. (2017). Managing gut health without reliance on antimicrobials in poultry. Animal Production Science, 57(11), 2270-2279. doi: 10.1071/AN17288

Oviedo-Rondón, E. O. (2019). Holistic view of intestinal health in poultry. Animal Feed Science and Technology, 250(2019), 1-8. doi: 10.1016/j.anifeedsci.2019.01.009

Patil, S., Premalatha, B. R., Rao, R. S., & Ganavi, B. S. (2013). Revelation in the field of tissue preservation - a preliminary study on natural formalin substitutes. Journal of International Oral Health, 5(1), 31-38.

Qi, D., Shi, W., Black, A. R., Kuss, M. A., Pang, X., He, Y.,… Duan, B. (2020). Repair and regeneration of small intestine: A review of current engineering approaches. Biomaterials, 240(2020), 119832. doi: 10. 1016/j.biomaterials.2020.119832

Rajanikanth, M., Ravi, P. A., Sreenath, G., Sonia, B. J. K., & Shyam, N. D. V. N. (2015). Transit fixatives: an innovative study. Journal of Clinical and Diagnostic Research, 9(3), 1-3. doi: 10.7860/JCDR/2015/ 11083.5615

Santos, M. C. L. G., Saito, C. P. B., & Line, S. R. P. (2008). Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods. Pathology - Research and Practice, 204(9), 633-636. doi: 10.1016/j.prp.2008.04.005

Silva, A. P., Lourenço, A. L., Marmello, B. O., Bitteti, M., & Teixeira, G. A. P. B. (2019). Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss Roll versus Intestine Strips. Experimental and Molecular Pathology, 111(2019), 104302. doi: 10.1016/j.yexmp.2019.104302

Smyth, J. A. (2016). Pathology and diagnosis of necrotic enteritis: is it clear-cut? Avian Pathology, 45(3), 282-287. doi: 10.1080/03079457.2016.1158780

Suresh, G., Santos, D. U., Rouissi, T., Hegde, K., Brar, S. K., Mehdi, Y.,… Ramirez, A. A. (2020). In-field poultry tests to evaluate efficacy of bioformulation consisting of enzymes and yeast biomass. Animal Feed Science and Technology, 262(2020), 114398. doi: 10.1016/j.anifeedsci.2020.114398

Swatson, H. K., Gous, R., Iji, P. A., & Zarrinkalam, R. (2002). Effect of dietary protein level, amino acid balance and feeding level on growth, gastrointestinal tract, and mucosal structure of the small intestine in broiler chickens. Animal Research, 51(6), 501-515. doi: 10.1051/animres:2002038

Williams, J. M., Duckworth, C. A., Vowell, K., Burkitt, M. D., & Pritchard, D. M. (2016). Intestinal preparation techniques for histological analysis in the mouse. Current Protocols in Mouse Biology, 6(2), 148-168. doi: 10.1002/cpmo.2

Yamauchi, K. (2007). Review of a histological intestinal approach to assessing the intestinal function in chickens and pigs. Animal Science Journal, 78(4), 356-370. doi: 10.1111/j.1740-0929.2007.00448.x

Yegani, M., & Korver, D. R. (2008). Factors affecting intestinal health in poultry. Poultry Science, 87(10), 2052-2063. doi: 10.3382/ps.2008-00091




DOI: http://dx.doi.org/10.5433/1679-0359.2021v42n6p3247

Semina: Ciênc. Agrár.
Londrina - PR
E-ISSN 1679-0359
DOI: 10.5433 / 1679-0359
E-mail:  semina.agrarias@uel.br
Este obra está licenciado com uma Licença  Creative Commons Atribuição-NãoComercial 4.0 Internacional