Discussion

Comprehensive approaches have been used to address the total or partial FM/FO replacement in a wide range of finfish including rainbow trout⁽ Reference Panserat, Ducasse-Cabanot and Plagnes-Juan ^{21 )} and typically marine fish such as European sea bass⁽ Reference Kaushik, Covès and Dutto ^{22 )} and gilthead sea bream. In particular, for gilthead sea bream, the long-term consequences of feeding low FM/FO feeds on growth performance and endocrine status⁽ Reference Benedito-Palos, Navarro and Sitjà-Bobadilla ^{7 ,} Reference Gómez-Requeni, Mingarro and Calduch-Giner ^{23 ,} Reference Benedito-Palos, Saera-Vila and Calduch-Giner ^{24 )}, health and welfare⁽ Reference Saera-Vila, Calduch-Giner and Prunet ^{17 ,} Reference Estensoro, Benedito-Palos and Palenzuela ^{25 –} Reference Pérez-Sánchez, Estensoro and Redondo ^{30 )}, fish quality⁽ Reference Ballester-Lozano, Benedito-Palos and Navarro ^{13 ,} Reference Ballester-Lozano, Benedito-Palos and Mingarro ^{31 ,} Reference Benedito-Palos, Bermejo-Nogales and Karampatos ^{32 )} and food safety⁽ Reference Nácher-Mestre, Serrano and Benedito-Palos ^{33 ,} Reference Nácher-Mestre, Serrano and Benedito-Palos ^{34 )} have been considered in a highly integrated manner. However, knowledge on the specific effects and consequences of a given nutrient or a group of nutrients is mostly lacking. Thus, this is one of the first studies analysing at the same time the effects of dietary deletion or reduction of six different nutrients recognised as essential to fish^{( 10 )}, which contributes to fill the gaps in the diagnosis of the nutritional fish condition under standardised rearing conditions. Of note, we did not have the same degree of deficiency for the six different nutrients tested. Indeed, EPA and DHA contents were reduced to trace levels in the n-3 LC-PUFA diet with the total replacement of FO by VO. Likewise, the main source of P in the CTRL diet was the added calcium phosphate, but it was more difficult to induce a severe Met deficiency while preserving the supply of other essential amino acids.

Adipose tissue (AT) is now recognised as an important target tissue for the diagnosis and treatment of most lipid metabolic disorders arising from an excessive lipid influx⁽ Reference Unger, Clark and Scherer ^{35 )}. Clinically, lipotoxicity not only appears with fattening, but also with hypoxia, blockage of glucocorticoid-sensitive pathways and the acquisition and maintenance of inflammatory phenotypes⁽ Reference Nawrocki and Scherer ^{36 –} Reference Maury and Brichard ^{38 )}. In humans as well as in other animal models, FO and n-3 LC-PUFA of FO are able to reverse these clinical symptoms, decreasing lipolysis and alleviating the inflammatory condition of AT, which in turn reduces the production of lipolytic cytokines, the release of free FA and thereby the risk of hepatic steatosis⁽ Reference Puglisi, Hasty and Saraswathi ^{39 )}. This liver syndrome is the result of a massive synthesis and/or deposition of TG in the form of lipid vacuoles, and it is commonly observed in many fish species challenged with xenobiotics and unbalanced diets⁽ Reference Wolf and Wolfe ^{40 )}. This metabolic derangement is often accompanied by the displacement of the nucleus of hepatocytes and even pyknosis. Relatively little is known about the ultimate mechanism, although the dietary protein source and protein/energy ratio have a major effect on the regulation of lipid metabolism in European sea bass⁽ Reference Dias, Alvarez and Arzel ^{41 ,} Reference Dias, Alvarez and Diez ^{42 )}. The replacement of FM and FO with plant protein and oil sources also has a number of effects on the regulation of intermediary metabolism in trout⁽ Reference Panserat, Hortopan and Plagnes-Juan ^{43 )} and Atlantic salmon⁽ Reference Leaver, Villeneuve and Obach ^{44 )}. In gilthead sea bream, tissue FA uptake⁽ Reference Saera-Vila, Calduch-Giner and Gómez-Requeni ^{45 ,} Reference Saera-Vila, Calduch-Giner and Navarro ^{46 )} and mitochondrial respiration uncoupling⁽ Reference Bermejo-Nogales, Calduch-Giner and Pérez-Sánchez ^{47 )} are highly affected by FM and FO replacement with plant ingredients. In addition, increases in cell size and lipolytic rates are characteristic features of isolated adipocytes from fish fed either plant proteins or VO⁽ Reference Cruz-García, Sánchez-Gurmaches and Bouraoui ^{48 ,} Reference Albalat, Gómez-Requeni and Rojas ^{49 )}, and lipoid liver degeneration is frequently observed as a metabolic disturbance in gilthead sea bream fed high plant ingredient-based feeds⁽ Reference Benedito-Palos, Navarro and Sitjà-Bobadilla ^{7 ,} Reference Gómez-Requeni, Mingarro and Calduch-Giner ^{23 ,} Reference Caballero, Izquierdo and Kjørsvik ^{50 )}. Accordingly, the present study shows that the total replacement of FO by VO in FM-free diets (n-3 LC-PUFA diet) caused a slight reduction of MSI related to a lipodystrophic phenotype with clinical signs of hypolipidaemia and hepatomegaly (high HSI). The loss of mesenteric AT mass, together with low plasma levels of choline and calcium, was even higher in the fish fed diets with reduced vitamin supply. However, abnormal liver lipid deposition rates were not found by light microscopy in this group of fish. Therefore, the absence or modification of AT mass cannot be used as the only criterion for the risk assessment of liver steatosis.

The main signs of P deficiency in fish nutritional studies are poor bone mineralisation and bad growth performance⁽ Reference Antony Jesu Prabhu, Schrama and Kaushik ^{9 ,} Reference Lall and Lewis-McCrea ^{51 )}, which were correlated in the present study with low plasma phosphate concentrations and high plasma activities of ALP in the absence of apparent skeletal deformities. ALP activity is a well-known bone turnover biomarker⁽ Reference Fjelldal, Lock and Hansen ^{52 )}, but here we also observed an increased plasma ALP activity with the reduced dietary supply of trace minerals (iron, magnesium, zinc, copper and selenium) in Min fish. However, the increase in MSI in combination with hyperlipidaemia allows differentiating the effects of deficiency in P from those due to deficiencies in trace minerals. In this sense, it must be noted than an important role of P in the regulation of lipid metabolism has also been reported in other fish species⁽ Reference Albrektsen, Hope and Aksnes ^{53 ,} Reference Vielma and Lall ^{54 )}.

Hct and Hb values are general indicators of health, and these haematological parameters change in response to nutrient deficiencies, environmental conditions, growth status and anti-nutritional factors⁽ Reference García-Garrido, Muñoz-Chapuli and Deandres ^{55 ,} Reference Lim and Lee ^{56 )}. A high incidence of anaemia has been reported in yellowtail and parrot fish fed FM-free diets⁽ Reference Takagi, Murata and Goto ^{57 ,} Reference Maita, Aoki and Yamagata ^{58 )}. This has been attributed, at least in part, to taurine deficiency, and thus its supplementation seems to be required when these fish are fed low levels of taurine in FM- or plant protein-based diets⁽ Reference Takagi, Murata and Goto ^{59 )}. In our study, all the diets were supplemented with taurine (0·3 %), and signs of anaemia, such as low erythrocytes, Hct and Hb values, were found only in fish fed the n-3 LC-PUFA diet, which also showed an increased erythrocyte osmotic fragility, as reported in rats with deficiencies in LC-PUFA⁽ Reference Ehrström, Harmsringdahl and Alling ^{60 )}. Importantly, this clinical sign was not found in the PL fish group, and therefore contributes to better define the sometimes-overlapping signs of nutrient deficiencies in EFA and PL.

Previous studies in gilthead sea bream have already highlighted increased RB of blood leucocytes in fish fed diets with a high replacement of FO by VO⁽ Reference Saera-Vila, Calduch-Giner and Prunet ^{17 ,} Reference Estensoro, Benedito-Palos and Palenzuela ^{25 )}. This has been confirmed in our study, and importantly RB turned out to be a specific criterion for the diagnosis of n-3 LC-PUFA deficiency, as it was notably increased in this group and not with PL or other nutrient-induced deficiencies. Likewise, a close talk between PL and bone metabolism exists, and the decrease in plasma ALP activity is becoming an easy and highly valuable marker of PL deficiencies in gilthead sea bream. Of note, a high PL supply is required during early life stages in fish to improve survival rates and to decrease the incidence of skeletal deformities⁽ Reference Tocher, Bendiksen and Campbell ^{61 )}, but PL requirements in juvenile and adult fish are still controversial⁽ Reference Niu, Liu and Tian ^{62 ,} Reference Daprà, Geurden and Corraze ^{63 )}.

Plasma transaminases and GLDH are commonly used in clinical chemistry as markers of tissue damage⁽ Reference Ozer, Ratner and Shaw ^{64 )}, but the current results indicate that they are poorly informative of nutritionally mediated metabolic derangements in gilthead sea bream. In contrast, overall plasma total antioxidant activity was increased in parallel to the reduced growth performance, indicative of a reduced aerobic metabolism and, therefore, of a reduced production of reactive oxygen species. Strong support for this comes from inbreeding selection of rat strains, which demonstrates that most stressful and oxidative risk factors correlate with the low expression of genes required for mitochondrial biogenesis and oxidative phosphorylation⁽ Reference Wisløff, Najjar and Ellingsen ^{65 )}. Experimental evidence also indicates that plasma antioxidant capacity is increased by hypoxia exposure, probably due to the concurrent decrease of basal metabolism, mitochondrial respiration uncoupling and oxidative phosphorylation⁽ Reference Bermejo-Nogales, Calduch-Giner and Pérez-Sánchez ^{66 )}. However, the magnitude and even the direction of the change is poorly predictable when comparisons are made between this and previous feeding trials with practical diets containing FM⁽ Reference Saera-Vila, Calduch-Giner and Prunet ^{17 )}.

Most growth regulatory events in fish are mediated at the hormonal level by the GH/IGF axis, keeping pituitary GH secretion and hepatic/extra-hepatic IGF production under control⁽ Reference Company, Astola and Pendón ^{67 –} Reference Reindl and Sheridan ^{69 )}. Therefore, circulating GH and IGF-I are one of the most important endocrine determinants of growth in a vast array of stress and nutritional disorders arising from crowding and handling stress⁽ Reference Saera-Vila, Benedito-Palos and Sitjà-Bobadilla ^{70 )}, changes in ration size⁽ Reference Pérez-Sánchez, Calduch-Giner and Mingarro ^{68 ,} Reference Pérez-Sánchez, Martí-Palanca and Kaushik ^{71 )}, dietary protein/energy ratio⁽ Reference Martí-Palanca, Martínez-Barbera and Pendón ^{72 ,} Reference Company, Calduch-Giner and Kaushik ^{73 )} and dietary protein and lipid sources⁽ Reference Gómez-Requeni, Mingarro and Calduch-Giner ^{23 ,} Reference Benedito-Palos, Saera-Vila and Calduch-Giner ^{24 ,} Reference Gómez-Requeni, Mingarro and Kirchner ^{74 )}. This notion was also found here, and importantly a close positive correlation between growth rates and circulating levels of IGF-I was evidenced, regardless of the nutrient deficiency. As expected, an opposite trend was found for GH and growth rates, which would reflect a lowered negative feedback inhibition of IGFs upon pituitary GH release as a result of a transcriptional defect in the signal transduction of GH receptors. This metabolic feature leads to liver GH resistance and reduced hepatic IGF production, in spite of increased plasma levels of GH, as it has been stated previously in a wide range of fish species, including gilthead sea bream⁽ Reference Pérez-Sánchez, Martí-Palanca and Kaushik ^{71 ,} Reference Beckman, Shimizu and Gadberry ^{75 –} Reference Wilkinson, Porter and Woolcott ^{77 )}.

Histological traits also gave interesting information on the possible pathological outcome of nutrient deficiencies. The highest accumulation of hepatic lipids was found in gilthead sea bream fed the n-3 LC-PUFA-deficient diet, followed by SAA, P and Min. However, lipid accumulation did not reach the highest score of steatosis observed by us with other dietary interventions⁽ Reference Benedito-Palos, Navarro and Sitjà-Bobadilla ^{7 ,} Reference Sitjà-Bobadilla, Peña-Llopis and Gómez-Requeni ^{15 )}, or by other authors using diets with an excess of dietary lipids⁽ Reference Caballero, López-Calero and Socorro ^{78 )}, EFA deficiencies⁽ Reference Montero, Robaina and Socorro ^{79 )} and VO⁽ Reference Caballero, Izquierdo and Kjørsvik ^{50 ,} Reference Wassef, Wahby and Sakr ^{80 ,} Reference Alexis ^{81 )}. Glycogen accumulation was also high in the liver of SAA, P and Vit fish, although the observed glycogen deposition did not reach an extreme condition. Massive accumulation of supranuclear lipid droplets in the intestinal epithelial layer is also considered a sign of inadequate/unbalanced diets due to a reduced metabolisation of absorbed lipids, either because they are not needed or because they are absorbed in a higher amount than needed. This accumulation was not observed in the posterior intestine for any diet, but it was a clear feature in the anterior intestine after feeding n-3 LC-PUFA-, PL- and Vit-deficient diets. This epithelial accumulation stands as an early marker of deregulated lipid metabolism compared with lipid accumulation in the liver, as it was visible in PL and Vit fed fish, in which lipoid liver degeneration was not found.

Another histological feature with clear differences among fish was the number of goblet cells and their staining characteristics. In this sense, it is noteworthy that the n-3 LC-PUFA diet induced a strong reduction in the number of goblet cells, and the remaining goblet cells were not stained either by Giemsa or by PAS, indicating that mucin content was not neutral or acidic. Neutral mucins were also absent in the anterior intestine as a result feeding the different diets, except SAA and Vit, and in the posterior intestine, except PL and Vit. Previous gilthead sea bream studies have indicated that other models of nutritional- and parasite-induced enteritis also invoke modifications in goblet cell type and number⁽ Reference Estensoro, Redondo and Salesa ^{26 ,} Reference Baeza-Ariño, Martínez-Llorens and Nogales-Mérida ^{82 )}. In fact, the 66 % replacement of FO by VO in plant protein-based diets produced a significant decrease in GC with neutral and acidic mucins in the anterior intestine and medium intestine as well as in those with carboxylic mucins and sialic acid in the medium intestine, but no significant changes in the posterior intestine. In European sea bass fed mannan oligosaccharides, the number of goblet cells secreting acidic mucins was increased⁽ Reference Torrecillas, Makol and Caballero ^{83 )}. In yellow perch fed wheat–gluten–protein-based diets, even supplemented with free lysine, the number of goblet cells was also decreased⁽ Reference Ostaszewska, Dabrowski and Kamaszewski ^{84 )}. In contrast, Atlantic salmon⁽ Reference Bakke-McKellep, Press and Baeverfjord ^{85 )}, Atlantic cod⁽ Reference Olsen, Hansen and Rosenlund ^{86 )} and carp⁽ Reference Urán, Goncalves and Taverne-Thiele ^{87 )} fed high plant protein-based diets presented goblet cells hypertrophy and hyperplasia.

Rodlet cells are exclusive of teleost epithelial layers and represent a cell type whose function has not yet definitively been established, although considered to be closely linked to the immune system and osmoregulation⁽ Reference Reite ^{88 )}. Many studies consistently report an association between rodlet cells proliferation/hyperplasia and the presence of a variety of parasites, chemicals and environmental stressors⁽ Reference Manera and Dezfuli ^{89 ,} Reference Shimada Borges, Salimbeni Vivai and Branco ^{90 )} and they even have been proposed as biomarkers⁽ Reference Manera and Dezfuli ^{89 )}. However, there is no previous report on the relationship with the diet. In the present study, the number of rodlet cells was increased only in the posterior intestine, notably in fish fed Min diet. The increased presence of rodlet cells could, therefore, be interpreted either as a sign of inflammation or as a sign of osmoregulatory imbalance and reinforce the idea that cell osmoregulation is dependent on trace minerals rather than P uptake. The higher presence of rodlet cells was coincident with other inflammatory markers (intra-epithelial lymphocytes and submucosal granulocytes) in fish fed Min, PL and Vit diets either in the anterior intestine or in the posterior intestine. In any case, the observed cellular inflammation was mild and by no means comparable with that caused by other dietary interventions in Atlantic salmon⁽ Reference Baeverfjord and Krogdahl ^{91 )}, common carp⁽ Reference Urán, Goncalves and Taverne-Thiele ^{87 )} or gilthead sea bream⁽ Reference Sitjà-Bobadilla, Peña-Llopis and Gómez-Requeni ^{15 )}.

In summary, clinical blood biochemistry and tissue histopathology have been proved highly informative to assess the nutritional condition of farmed gilthead sea bream. The final diagnosis outcome might require confirmation by more specific assays, but the generated information is by itself useful for the overall assessment of fish performance and metabolic condition when the measured parameters are referred to a CTRL group or historical data for a given fish strain, life stage and/or rearing condition. All this information is summarised in online Supplementary Table S1 as a set of clinical signs for a given nutritional deficiency in gilthead sea bream. To establish the normal range of variation of these parameters as a function of season and growth performance, the data from this study were combined with those derived from other past and on-going feeding trials with practical diets. The rationale for this is to cover a wide range of variation for marine and plant ingredients without apparent detrimental effects on fish performance through the production cycle. The reference values for these studied biomarkers are shown in online Supplementary Table S2, and will be periodically updated on the basis of the data produced within the ARRAINA project.