## 4 Discussion
### Liver Histology  
In the present study, the inclusion of poultry by-product meal or hydrolyzed feather meal caused no to severe alterations in the hepatic tissue of _S. aurata_ and these alterations were dependent on the level of fishmeal protein replacement. Neither PBM nor HFM altered the liver histomorphology of seabream when these animal proteins replaced fishmeal at 25%. However, at higher replacement levels more lipid droplets and increased hepatic vacuolization were observed, and these changes were more pronounced in fish fed HFM diets. In general, high inclusion levels of PBM caused mild to moderate hepatic alterations compared to the high inclusion levels of HFM that caused severe alterations, particularly in the case of total fishmeal replacement. Although there were no signs of steatosis, which may be caused by the increased lipid vacuolization, the total FM replacement by HFM, contrary to PBM, led to haemorrhage, pancreatic islet necrosis and cirrhosis in a substantial number of fish that were examined. The dietary supplementation of PBM and HFM with essential amino acids, such as lysine and methionine, seemed to improve the digestive physiology. Fish fed these diets showed fewer hepatic alterations and abnormalities compared to fish fed diets of a similar replacement level but without amino acid supplementation.  
The present findings contradict with those reported by [PERSON] et al. [42] in which the 100% replacement of FM by PBM did not cause any liver alteration in _S. aurata_. Although in both studies the total FM replacement by PBM did not lead to any clear signs of steatosis, the mild increase in lipid vacuolization with the increase of PBM dietary inclusion observed  
Figure 8: Significantly different (\(p<0.05\)) predicted functional pathways of the bacterial relative abundances between fish fed the FM, PBM50 and HFM50 diets.  
in the current study may indicate a lower lipid digestibility of the PBM fat. This indication is supported by the fact that fish fed diets with high inclusion levels of PBM had increased fat in their livers (Table 1), although this cannot be clearly said for the HFM fed fish. It has been suggested that that a hepatic lipid accumulation may occur because the excessive dietary intake of lipids that surpasses the physiological capability of the liver to \(\beta\)-oxidize them, thus leading to larger lipid droplets and subsequent steatosis [43]. However, in our study the dietary lipid intake of PBM and HFM fed fish was lower than that of FM fed fish see [7,8], which indicates that dietary lipids, specifically of PBM and HFM lipids, inducehepatic lipid accumulation in _S. aurata_.  
Increased lipid droplets and hepatic vacuolization with signs of inflammation have also been reported in _Lates calcarifer_[22,44] and in _Tinca tinca_[45] when the FM in the fish fed diets were completely replaced with PBM. Moreover, [PERSON] et al. [21] reported an induced steatosis in the hepatocytes of hybrid grouper even when fish were fed diets with 50-70% FM replacement levels by PBM. On the other hand, the total replacement of FM by PBM did not cause any increased vacuolization or alterations in the hepatocytes of _Salmo salar_[14] and of _Oreochromis niloticus_[46]. The effects of dietary HFM as a sole FM replacement on fish liver histomorphology are not well studied. [PERSON] et al. [14] stated that a diet with total FM replacement by HFM did not reveal signs of steatosis in _Salmo salar_ with hepatocytes having even a lower fat accumulation than to those fed on FM. Studies using HFM in a blend with other animal proteins, including PBM, for FM replacement have reported that high substitution levels induce hepatic lipidosis and steatosis in _Lateolabrax japonicus_[19] and in hybrid grouper [20].