Etiology of soybean-induced enteritis in fish
Promotor: Prof. J.A.J. Verreth; Co-promotors: Dr. Jan H.W.M. Rombout & Dr. Johan W. Schrama (Collaboration project CBI and Aquaculture and Fisheries)
Recent efforts to successfully substitute fishmeal for soybean meal (SBM) as an important source of protein for aquafeeds continue to be beset by problems and constraints. The inclusion of SBM, especially in the diet of salmonids (i.e. Atlantic salmon), induces an inflammatory response of the distal intestinal mucosa, known as enteritis. The general aim of this study was to understand the etiology of enteritis as well as the underlying mechanisms of its development. To do so, numerous factors (both dietary and non-dietary) were evaluated and the possible mechanisms behind the inflammatory process were studied in both Atlantic salmon (Salmo salar L.) and common carp (Cyprinus carpio L).
In Chapter 2, water temperature was addressed as an important husbandry condition which could influence the speed at which the disorder develops. The study demonstrates that enteritis is indeed affected by temperature, which delays the response to SBM inclusion in the diet by slowing down the appearance of the characteristic signs of the inflammation process when the fish are kept at the lower temperature (8 vs. 12 °C). A semi-quantitative scoring system was developed to assess the extent of the morphological changes observed in this study. The use of this method facilitated the further understanding of the etiology of the disorder, when the different dietary and non-dietary factors involved in the inflammatory process were studied.
Chapter 3 described the influence of SBM on the kinetics of the disorder. This was studied at two different inclusion levels over a period of 57 days. At the higher dose (20% SBM), the signs of enteritis seemed to increase steadily whereas at the lower dose (10% SBM) the disorder displayed a tendency to level off, a process which began at two weeks and continued thereafter. It was concluded that the severity of enteritis and its kinetics are dose-dependent, showing no signs of recovery during feeding SBM-containing diets. Electron microscopy studies suggested a blocking of the endocytosis process.
Nonetheless, as this basic mechanism had only been studied and described in salmonids, it was deemed pertinent that a study of SBM-induced enteritis could be made in another fish species that had different feeding habits to the Atlantic salmon. In Chapter 4, SBM-induced enteritis in the omnivorous common carp was studied and compared to the SBM-induced intestinal inflammation in the carnivorous, Atlantic salmon. For the first time ever reported, the results suggested that the symptoms of enteritis also occur in common carp. Contrary to previous observations made in studies with Atlantic salmon, the common carp started to recover from week four onwards, something that was especially noticeable in the refilling of the supranuclear vacuoles (SNV). The morphological changes and the modulation of the inflammatory process were defined and correlated to the up- and down-regulation of several cytokines that were presumed to influence this process. Also in this study, electron microscopy suggested that endocytosis blocking is directly linked to the disappearance of the SNV.
The potential role of endocytosis in the development of the disorder was further investigated in Chapter 5. Ferritin was used to prove the eventual blocking of endocytosis. After one week of SBM-feeding, ferritin uptake had diminished and the SNV had decreased in size. At week three, ferritin uptake could not be detected and the SNV were not present in the enterocytes. This suggests that the endocytosis block is the first step in the disappearance of the SNV. In addition, the results of this study on young Atlantic salmon (30 g) kept in freshwater, facilitated a comparison with older salmon (300g) kept in seawater, regarding the influence of salinity on the severity of enteritis. It was suggested that SBM-induced enteritis was not strongly influenced by either salinity or age. Nevertheless, further studies are needed to support this observation.
In Chapter 6, SBM obtained from different commercial sources and production plants world-wide were compared, as a means of determining the extent to which enteritis in Atlantic salmon depends on the origin/processing of the grains. The most affected parameter was the disappearance of supranuclear vacuoles in enterocytes. In contrast, the increase in goblet cells showed the smallest variation between the different SBM sources. This study showed that different commercial sources of SBM resulted in differences in the severity of SBM-induced enteritis in Atlantic salmon
Chapter 7 deals with a sub-fraction of SB molasses containing soyasaponins, which were thought to be responsible for the induction of enteritis. The morphological changes observed were induced when soyasaponins were fed to Atlantic salmon alone or in combination with other soybean components that follow the same solubility pattern after the fractionation process. It is supposed that changes in membrane permeability are mechanisms of action of these molecules. Consequently, it is assumed that the intestinal microbiota may have an influential role on the severity of enteritis when access is gained through the disrupted intestinal barrier.
The findings of this thesis are discussed in Chapter 8. It is concluded that the etiology and further development of SBM-induced enteritis is related to the dietary factors rather than to the non-dietary factors. SBM inclusion levels and the commercial source used for the diet formulation have a great impact on the severity of the disorder, mainly affecting the endocytosis process. Nonetheless, the actual causative components present in SB are still issues that require further research. This thesis suggests that the decreased nutrient uptake capacity of the enterocytes is directly related to the disappearance of the SNV as seen in Atlantic salmon. This situation was clearly reversed in common carp as this species began a recovery process. However, the reasons as to why the carp recovered are still unknown.