THE ASSESSMENT OF FEED EFFECTS ON LIVER AND INTESTINE OF REARED FLATFISH: WHAT IS THE BEST STANDARD ANATOMOPATHOLOGIC PROTOCOL?

AIM OF THE STUDY : This presentation highlights the importance of using standardized methods by the fish pathologists to assess the effects of feed on reared flatfish. The liver and intestine are the most important organs for the digestion and absorption of nutrients from feed, and monitoring their histological structure is of fundamental importance in assessing the effects of nutrient mixtures.1 A critical analysis of the histological methods on feed effects w ill be presented, aiming to suggest a standardized protocol that should be adopted, on the basis of the advantages and disadvantages that have been encountered in the course of our recent research trials. MATERIAL AND METHODS : Six feeding trials have been carried out on juvenile common sole (Solea solea), Senegalese sole (Solea senegalensis) and turbot (Psetta maxima) (fed diets w ith increasing dietary protein levels;2,3 increasing dietary plant protein in replacement of fishmeal;4 increasing lipid levels5 and increasing mussel meal6, respectively). Routine histology (haematoxylin-eosin stain on formalin-fixed and paraffin-embedded sections) w as carried out to check degenerative and/or inflammatory changes. In the liver, as w ell as the graduation of the lipid content, further histochemical techniques have been applied in order to characterize the type of intracellular accumulation (OilRedO, Toluidine blue, PAS) and transmission electron microscopy (TEM), w hile in intestine in situ techniques to assess the cellular turnover and trophism of epithelial mucosa (anti-PCNA immunohistochemistry and TUNEL method for apoptosis) evaluated by image analysis and TEM w ere employed. RESULTS : The routine histological evaluation is helpful only to demonstrate degenerative-inflammatory changes and its usefulness increases w hen combined w ith a grading system for the lipidic content of the liver that revealed the most frequent degeneration assessed. To confirm the lipidic content, Toluidine Blue on semi-thin sections revealed better than OilRedO w hich run on frozen sections that require a more difficult management of the samples. IHC on intestinal sections and the evaluation of cellular kinetics and mucosal turnover are necessary tools to assess the influence of a diet on mucosal trophism. For both tissues TEM is the elective method to further characterize the type of intracellular accumulation and the early signs of cell damage. CONCLUSIONS : On the basis of our experience it is necessary to adopt a standardized approach to evaluate the effects of experimental diets on liver and intestine; in particular, investigations in liver require Toluidine Blue on semithin sections and TEM, w hile for intestine IHC together w ith digital image analysis and morphometry are suggested in addition to routine histology. BIBLIOGRAPHY : 1Raskovic et al, 2011; 2Gatta et al., 2011; 3Bonaldo et al., 2011; 4Mandrioli et al., 2012; 5Bonvini et al., 2014 (Proceedings of ISFNF); 6Bonaldo et al., 2014 (Proceedings of ISFNF).