Assessing the health of farmed rainbow trout (Oncorhynchus mykiss): use of an open-source software for intestine histology

Open-source software are providing opportunities and advantages to operators in several fields as fish welfare. This study is part of a 90-days feeding trial where three diets were tested in rainbow trout (Oncorhynchus mykiss). By substituting guar meal protein concentrate (MYCOPRIME®) for 0% (CD), 5% (D5), and 15% (D15) of traditional protein sources, the diets were formulated to have a protein level of 43% and a lipid content of 25.3%. The proximal intestines of 15 rainbow trout per diet were sampled and Haematoxylin and Eosin staining was applied to histological sections, performing a blind, semi-quantitative assessment. Every section was thoroughly examined at low (10×) and medium (20×) magnification, considering as pathological traits: inflammation (eosinophilic granular cells, goblet cell hyperplasia, and infiltration of macrophages); intraepithelial lymphocytes; steatosis in enterocytes. A score from 0 to 3, corresponding to the severity degree of the microscopical tissue structure modification, has been allocated to all the parameters evaluated. Additionally, the histological sections were stained using Alcian Blue kit to study the goblet cells. For each sample, four random microscopical fields were chosen and 20x magnification images were taken. Consequently, the opensource program ImageJ (Fiji) was used for a quantitative assessment of the goblet cells. The algorithm was then trained to recognize Alcian blue-stained vacuoles, omitting smaller areas than 20 pixels2, since these areas would have been too small to be a true vacuole (Figure 1). For the statistical analysis, GraphPad Prism 9 was used and Kruskal–Wallis and a Dunn's multiple comparisons tests performed (p-value<0.05 significant). The CD group had the highest histological score, while the D15 group had the lowest (p<0.001). Goblet cell hyperplasia showed significant differences between the CD group and the D15 group and even the goblet cell count difference resulted to be significant (p<0.001). According to a recent study, the proximal intestine of healthy rainbow trout has much larger vacuoles and significantly more goblet cells [1], suggesting a potential function in gut health. The smaller number of goblet cells in the D15 group may be associated to the animals' poorer zootechnical performances when no other pathological alterations are present. The quantitative histological assessment by means of the software permitted a more comparable and clear differentiation of the effects of the diets. In conclusion, guar meal protein concentrate was shown to be suitable for use in rainbow trout feed formulation during the growing period, although the effect on the intestinal mucosa should be further developed.