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Trabajo presentado en Aquaculture Europe 19 (Our future, growing from water), celebrado en Berlín del 7 al 10 de octubre de 2019., The optimization of feeding management is essential to improve the key performance indicators such as growth rates, feed efficienc , mortality and quality traits of the Mediterranean aquaculture. Optimal feeding frequencies under farming condition may contribute to maximise feed utilization reducing wastes and production costs. Several effects of the feeding frequency on performances have been observed in different fish species mainly depending on the fish size and on the interactions between feeding frequencies and type of administered diets (Yúfera et al., 2014; Enes et al., 2015; Guo et al., 2015; Zeytin et al., 2016). This study was undertaken in order to assess the effects of different feeding frequencies on growth, feed digestibility, somatometric indexes, fish plasma biochemistry, and digestive condition of gilthead sea bream fed different fishmeal and fish oil dietary levels with the final aim to provide guidelines designed for the daily management of feeding practices under farming condition., This research was supported by ERC (European Research Council) in MedAID project (Mediterranean Aquaculture Integrated Development), Call H2020-SFS-2016-2017 (Sustainable Food Security – Resilient and resource-efficient value chains), Grant Agreement n. 727315.

Trabajo presentado en Aquaculture Europe 19 (Our future, growing from water), celebrado en Berlín del 7 al 10 de octubre de 2019., The optimization of feeding strategy in relation to the environmental condition needs further investigation in order to maximise performance, fish health and fish quality of Mediterranean farmed species. Environmental temperature during seasonal changes may affects fish metabolism, digestive enzymes activity and gut bacterial community which may exert an effect on performance and tissue composition (Couto et al., 2012; Guerreiro et al., 2016). Data relating interaction between different dietary protein and energy ratio and water temperature changes on digestion condition, gut bacterial community and fat deposition of gilthead seabream fed current aquafeed formulations are scarce. For this reason, the purpose of this study was to evaluate the effects of two dietary protein and dietary energy ratio (DP/DE) during temperature changes on growth, feed efficienc , fat deposition, plasma biochemistry, digestive enzyme activity and gut bacterial community of gilthead sea bream (Sparus aurata)., This research was supported by ERC (European Research Council) in MedAID project (Mediterranean Aquaculture Integrated Development), Call H2020-SFS-2016-2017 (Sustainable Food Security – Resilient and resource-efficient value chains), Grant Agreement n. 727315.

Feed ingredients are known to possess different buffer capacities which might influence pH ranges along the digestive tract. Fishmeal (FM) have one of the highest buffer capacities among raw materials and its decreasing content in aquafeed formulations might affect the ideal gastrointestinal environment for digestive enzyme action. In addition data on gastric and intestinal pH in carnivorous marine fish species under different feeding regimes are scarce. A study was undertaken, therefore, to assess growth response, gastric and intestinal pH of European sea bass (Dicentrarchus labrax L.) fed diets with low fishmeal levels added with or without buffering agents in comparison to a standard fishmeal diet. Three isonitrogenous (45% protein) and isolipidic (16% lipid) extruded diets containing 10% FM with or without buffering agent (FM10b, FM10, respectively) and one control diet containing 20% FM (FM20) were fed to triplicate fish groups of 80 individuals (initial weight: 23 g) to overfeeding over 64 days. At the end of the trial there were no significant differences between diets for final body weight, feed intake and feed conversion rate even if specific growth rate was slightly higher in FM10 compared to FM10b. Hepatosomatic index was higher in FM20 compared to FM10 while no significant differences were observed in the viscerosomatic index. pH was measured in the stomach and along the intestinal tracts (anterior, middle, posterior) of three fish per tank at 0, 4, 8 and 12 hours post prandial and correlated to the different diets. At the same time enzymatic activity of digestive enzymes and gut microbiota by next generation sequencing were also analysed. In conclusion, low FM diet (FM10) led to equal growth and feed utilization in comparison to standard FM level (FM20) and the inclusion of buffering feed agent did not seem to promote the overall growth performance., Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF. 40 years of research in fish nutrition), celebrado en Las Palmas de Gran Canaria del 3 al 7 de junio de 2018.

The in vitro effect of a Yucca schidigera extract (YSE) and tannins from chestnut wood on composition and metabolic activity of canine and feline faecal microbiota was evaluated. Four treatments were carried out: control diet, chestnut tannins (CT), YSE and CT + YSE. The YSE was added to canine and feline faecal cultures at 0.1 g/l, while CT were added at 0.3 g/l for a 24-h incubation. A total of 130 volatile compounds were detected by means of headspace-solid phase microextraction gas-chromatography/mass spectrometry analyses. Several changes in the metabolite profiles of fermentation fluids were found, including a decrease of alcohols (−19%) and esters (−42%) in feline and canine inoculum, respectively, which was due to the antibacterial properties of tannins. In canine inoculum, after 6 h, YSE + CT caused lower cadaverine concentrations (−37%), while ammonia (−4%) and quinolone (−27%) were reduced by addition of CT. After 24 h, the presence of CT resulted in a decrease of sulphur compounds, such as dimethyl sulphide (−69%) and dimethyl disulphide (−20%). In feline faecal cultures, after 6 h, CT lowered the amount of indole (−48%), whereas YSE tended to decrease trimethylamine levels (−16%). Both in canine and feline inoculum, addition of CT and, to a minor extent, YSE affected volatile fatty acids patterns. In canine faecal cultures, CT exerted a marginal inhibitory effect on Escherichia coli population (−0.45 log 10 numbers of DNA copies/ml), while enterococci were increased (+2.06 log 10 numbers of DNA copies/ml) by YSE. The results from the present study show that YSE and tannins from chestnut wood exert different effects on the composition and metabolism of canine and feline faecal microbiota. In particular, the supplementation of YSE and tannins to diets for dogs and cats may be beneficial due to the reduction of the presence of some potentially toxic volatile metabolites in the animals’ intestine.

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).

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Salmonids, gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) play a lead role in the European fish production; however, other farmed fish species are required to differentiate and widen the market supply. Flatfish have long been of interest for aquaculture in Europe. In particular, turbot (Psetta maxima) is the most important cultured flatfish species in Europe, widely reared also in other countries such as East Asia, whereas the sole species common sole (Solea solea) and Segenegal sole (Solea senegalensis) represent an interesting alternative for the diversification of the European and Italian aquaculture, due to the high price and high market demand. To date, the production of these species in Europe and Italy are still minimal compared to other species and further improvement are needed to achieve a full sustainability of the production cycle. Above all, pursuing the ideal feed formulation and an adequate nutrient utilization are of particular relevance for the success of the aquaculture production of any given species. In this context, a critical review of our studies aimed at optimizing the diets for these flatfish species and conducted during the last ten years will be presented. Results indicated that soles species require innovative ingredients and have specific nutritional needs to improve feed intake and growth. In particular different dietary protein levels demonstrated a considerable influence on growth, feed utilization and nitrogen excretion with the highest growth and feed utilization achieved with a diet containing 57 % of crude protein (1). A feeding trial with different dietary energy levels evidenced that increasing dietary lipid level lead to a substantial decline in performance and affect gut health. Dietary lipid levels higher than 12 % depress growth and lipid utilization. High lipid diets lead to moderate to severe intestinal steatosis and ultrastructural evaluations display cellular suffering due to a lipid overload. Soybean meal (SBM) seems to be a good protein source in diets for Egyptian sole. A level of 30 % of SBM can be added in the diet without any reduction in growth rate and any effect on gut histology. Soy products may be promising protein sources for inclusion in sole diets (2). The substitution of fish meal by mussels meal improved performance and feed utilization. The mussels meal could mimic the characteristics of the sole’s natural prey, in terms of attractiveness and nutrient utilization. It is advisable to consider mussel meal, rather than fish meal as a reference diet to determine the growth potential of common sole. Turbot showed low tolerance to high plant protein inclusion, since vegetable ingredients negatively affected feed intake, growth and fish welfare. A substitution of a mixture of plant protein for up to 52 % of fish meal protein did not reduce feed intake, and at 39 % substitution, turbot maintained optimal growth rate and nutrient utilization. Worsened feed utilization in diets containing higher plant protein levels was not associated with a reduced digestibility of ingredients or alterations of gut histology (3). The administration of diet containing 35 % of fish meal ensured growth performance close to that containing 50 %, without significant effects on health and welfare of turbot juveniles. Diet containing 20 % of fish meal produced sub-optimal growth performance, metabolic stress and an immune response with consequences on health and welfare status. A 5 % fish meal diet caused a worsening of growth performance and fish welfare, probably due to an insufficient feeding and nutrients intake (4). The results of the different experiments will be provided and discussed in order to identify new perspectives for optimizing practical diets for turbot and sole in aquaculture.