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The objective of the present study was to assess if trypsin, a key enzyme involved in protein digestion, presents some kind of functional adaptations to seasonal changes in water temperature in freshwater fish. In order to test this hypothesis, individuals of two fish species Carassius gibelio (agastric) and Perca fluviatilis (gastric) were sampled in the basin of Chany Lake (Siberia, Russia) at two different seasons (spring and summer). Apparent kinetic parameters (Km and Vmax) were determined for both species and seasons at the actual pH values in fish guts, and at actual temperatures. Results showed a significant effect of both the species and sampling season on the apparent kinetic parameters of trypsin. In the case of Prussian carp, Km and Vmax were lower for each assayed temperature (for 5 and 15 °C the differences were significant) for fish sampled in summer when compared to those sampled in spring. In contrast, values of Km in perch tended to be lower in spring at 5 and 25 °C but these differences were not significant, while Vmax showed a significant decrease in summer samples. This suggests a sort of functional adaptation of the same trypsin enzymes to seasonal changes, oriented to maximize protein digestion under variable conditions.

The aim of this research was to evaluate the possible positive effects of feeding Nile tilapia (Oreochromis niloticus) with a diet containing a commercial probiotic AQUA-PHOTO® [Lactobacillus plantarum (1.51 × 106 CFU g−1) and Bacillus subtilis (1.34 × 107 CFU g−1)] on digestive enzymes’ activity and gut microbiome profile. The experimental design was completely randomized, with two treatments [control and probiotic diets (0.08 g.100 g−1 of feed)] and six replicate tanks per group (n = 8 animals per experimental unit, mean weight 8.18 g ± 1.85 g). The 96 fish were distributed into 12 aquaria and fed with the probiotic and control diets for 7 days. After this period, intestine samples (anterior, middle, and posterior portions) were collected from two individuals per tank. The samples were analyzed for assessment of DGGE profile and digestive enzymes’ activity (amylase, alkaline protease, and alkaline phosphatase). The results were subjected to analysis of variance (ANOVA) and the means compared by t test (P

Global warming and changes in nutrient supply can alter the functioning of headwater stream ecosystems, where leaf-shredding invertebrates are pivotal for the transfer of nutrients through ...

Brewers spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds. © 2021, The Author(s)., Te authors thank UNICER, a sof-drink company (Porto, Portugal), for providing the brewer’s spent grain used in this study. Tis study was supported by the project “SPO3-Development of innovative sustainable protein and omega-3 rich feedstufs for aquafeeds, from local agro-industrial by-products”, reference POCI-01- 0145-FEDER-030377, funded by European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT) and by strategic funding of UIDB/04423/2020 by UIDB/04469/2020 unit, the BioTecNorte operation (NORTE-01-0145-FEDER-000004) through national funds provided by FCT, and under the scope of the strategic funding of UIDB/04469/2020 unit. Te H.F. and C.C were supported by grants SFRH/BD/131219/2017 and SFRH/BPD/114942/2016, respectively, from FCT, MCTES, FSE, and UE under the North Portugal Regional Operational Programme (NORTE2020). José Manuel Salgado was supported by Beatriz Galindo contract of Ministry of Education (Spain)., info:eu-repo/semantics/publishedVersion

Understanding the daily digestive function could lead to more efficient feeding practices in the farmed fish species. In this work, the effect of several daily feeding protocols was assessed on the daily expression of the genes related to the circadian system (arntl1, clock, per1, and cry1), appetite (cck), nutrients hydrolysis (try1, ctrb1, pga, atp4a, cel, and amy2a), peptides transport (slc15a1), pH, and activity of key digestive (trypsin, chymotrypsin, amylase, 4C- and 7C-like lipases) and absorptive (alkaline phosphatase) enzymes in the gastrointestinal tract (GIT) of Senegalese sole (Solea senegalensis) juveniles. Some of the tested feeding protocols demonstrated a significant influence on the entrainment of the peripheral oscillator. The synchrony of slc15a1 and some digestive proenzymes with per1 in some of the experimental treatments could underline the role of clock genes in the regulation of these biological functions. Unlike gene expression of the digestive proenzymes wherein feeding protocol occasionally affected the temporal organization, the influence of this factor was determinant on the daily average production and/or circadian pattern of their activities. Daily pH was maintained at approximately neutral levels in all the GIT segments, and no pepsin activity was detected. Fish fed a single diurnal meal showed the lowest activity for all the studied enzymes, except for amylase, suggesting an insufficient utilization of the ingested nutrients. Higher amylase activity in this group, on the other hand, implies the possible activation of alternative compensatory metabolic pathways. Overall, our results pointed to the suitability of higher feeding frequencies, especially during dark hours, for the aquaculture production of this species., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to MY and WISEFEED funded by the European Union's H2020 Program (Marie Skłodowska-Curie Grant No. 691150). NG was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

Fishmeal (FM) possesses one of the highest buffering capacities in comparison to most alternative vegetable aquafeed ingredients and its decreasing content in current formulations might affect the ideal gastrointestinal environment for digestive enzyme action and gut bacterial community of carnivorous fish species. A study was undertaken, therefore, to assess growth response, gastrointestinal pH, digestive enzyme activity and gut bacterial community of European sea bass (Dicentrarchus labrax L.) juveniles fed low FM diets (10% FM) with or without a feed buffering additive, calcium carbonate (FM10 + B and FM10, respectively) in comparison to a standard FM diet (20% FM, FM20). Three isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups of 80 individuals (initial weight: 23 g) to overfeeding over 64 days. No significant differences due to low FM dietary levels were observed in final body weight, specific growth rate, feed intake, feed and protein efficiency. Low FM diet did not affect gastrointestinal pH in the stomach, anterior intestine, mid-intestine and hindgut at 0, 4, 8 and 12 hours post meal (hpm) while the inclusion of calcium carbonate in low FM diet seems to slightly increase the pH in the hindgut at 12 hpm. The absence of significant differences of pepsin, chymotrypsin, amylase and lipase activity suggests a comparable digestive efficiency among treatments although trypsin activity was slightly reduced in low FM diets after 4 hpm. Decreasing FM content seems to exert an effect on the overall gut bacterial community analysed by next-generation sequencing even if no significant effects on specific bacterial component were detected. The gut bacterial community in all the treatments was particularly rich in lactic acid bacteria such as Lactobacillus spp. which may provide important beneficial functions for the host and be associated with a healthy intestinal epithelium. According to the results, increasing the feed buffering capacity does not seem to improve digestive conditions while it is feasible to include 10% FM dietary level in practical formulation for European sea bass juveniles without negatively affecting growth, feed efficiency and digestive luminal conditions. info:eu-repo/semantics/publishedVersion

Rendered terrestrial animal by-product meals (ABPM) are commonly used in aquafeeds for fish and shrimp. However, the nutrient composition and digestibility of these products is often variable due to differing processing conditions. As such, the aquafeed industry could benefit from technologies that rapidly assess the nutritional quality of ABPM prior to purchase and their incorporation into aquafeeds. To be useful, new technologies would need to accurately reflect the results of traditional assays such as in-vivo apparent digestibility. This study presents a comparison of three methods for assessing the protein digestibility of poultry by-product meals (PBMs) that were subjected to different cooking temperatures (between 110 °C to 145 °C) and pre-rendering storage times (between 0 and 8 h). The first method was a traditional in-vivo assessment of digestibility based on indirect techniques and the collection of faecal material from Barramundi (Lates calcarifer). The second was the in-vitro pepsin nitrogen digestibility assay applied at pepsin concentrations of 0.2% and 0.0002%, respectively. The third method employed a two-step, membrane bioreactor (also known as a gastro intestinal model or GIM), based on digestive enzymes collected from the gut of Barramundi. In-vivo protein digestibility of PBMs ranged from 57% to 83%, being lowest in PBM that had been purposely overheated (145 °C) and highest in PBM subjected to the lowest cooking temperature (110 °C). In-vivo amino acid digestibility followed similar trends. Estimates of protein digestibility and protein hydrolysis based on the pepsin assays and the GIM were highly correlated with in-vivo protein ADCs. Estimated protein digestibility based on the pepsin assays was similar in magnitude to those determined in-vivo. Estimates of protein digestibility based on the GIM (protein hydrolysis) were highly correlated with the other methods, but the values estimated using GIM were much lower. Despite this discrepancy the GIM data could be modelled with a high degree of certainty using quadratic functions, and these functions could be used to reliably estimate digestibility of protein from PBMs, at least for the data generated in this study. All methods were sensitive enough to distinguish between PBMs that had been carefully treated prior to rendering or PBM that had been purposely overheated. The use of GIM technology requires further development, however it offers a great deal of promise and the opportunity to rapidly assess the protein quality of many different raw materials.

Simple Summary The use of seaweeds in aquafeeds is receiving increasing attention due to their potential nutritional and functional benefits. However, several green seaweeds such as Ulva presents nutritional limitations because of the undigestible polysaccharides, although these may exert a positive effect on the immunological status of the fish. The present study developed three different experiments aimed to re-evaluate the presence of protease inhibitors described for Ulva ohnoi, to assess its nutritional value as an ingredient and also to evaluate its potential protective effect on the oxidative metabolism of fish, being experiments developed in two different fish species (European sea bass Dicentrarchus labrax and gilthead sea bream, Sparus aurata). Results indicate the absence of negative effects of U. ohnoi on protein digestion of sea bream but a limited value as a feed ingredient. In contrast, its contents in bioactives seem to be correlated to the observed positive effects on the immune status and oxidative metabolism when fish are challenged by the consumption of highly oxidized dietary oil. This study evaluated the use of Ulva ohnoi as an ingredient in feeds for aquaculture in three different experiments. Experiment 1 was oriented to confirm the negative effect of U. ohnoi on fish digestion. Experiment 2 assessed the effect on growth, feed efficiency, and immune status of juvenile sea bass (Dicentrarchus labrax) fed on diets including U. ohnoi, previously treated or not with carbohydrases used to partially hydrolyze indigestible polysaccharides. Experiment 3 was aimed to evaluate the potential protective effect of U. ohnoi on the oxidative status of sea bream (Sparus aurata) challenged by the consumption of a feed formulated with the oil fraction completely oxidized. Results show a negligible effect of U. ohnoi meal on protein digestion when included in feeds at levels of 10% or less. Moreover, results of growth and feed use evidenced the possibility of using up to 5% inclusion of algal meal in feeds without adverse effects on the zootechnical parameters, while the enzyme pretreatment was ineffective to improve its nutritional use. Finally, the inclusion of U. onhoi in feeds determined both an immunostimulatory effect, evidenced by an increase in skin mucus lysozyme in the two mentioned fish species, and a positive influence on the oxidative metabolism of seabream when fed on a diet including rancid oil., This research was funded by the Excellence Campus of Marine Science (CEIMAR) within the "III Call for Innovative Projects in the field of Blue Economy".

A 121-day feeding trial was undertaken to test the effects of two dietary lipid levels (16 and 21% L16, L21) in triplicated gilthead sea bream groups (initial weight: 67.5 g) reared at two different water temperatures (high, H 23°C and low, L 17°C) in the same recirculation system but exposed to a switch in temperature after 58 days. Fish kept at H were transferred to L (HL transition, autumn shift), and the fish kept at L were exposed to H (LH transition, summer shift), while continuing to receive the same diet to apparent satiation in each group. At the end of the trial, no significant diet effect on specific growth rate (SGR), feed intake (FI), and feed conversion rate (FCR) were detected in fish exposed to HL transition compared with those exposed to LH transition, while gross lipid efficiency (GLE) and lipid efficiency ratio (LER) were higher in L16. After temperature changes, L16 displayed higher SGR, FI, GLE, and LER, while mesenteric fat index was reduced. After temperature changes, the combined effects of low lipid diet and low temperature conditions resulted in higher pepsin activity, while trypsin, chymotrypsin, and lipase activities were generally higher at high lipid content. The combined effect of diet and temperature did not alter the metabolic plasma profile, except for the observed final higher aspartate aminotransferase (AST) and alkaline phosphatase (ALP) values when combining high dietary lipid (L21) and temperature changes. Different diets showed a significantly different gut microbiome layout, only at high temperature with L16 diet resulting in a higher load of Lactobacillus. On the contrary, no dietary impact on ecosystem diversity was observed, independently from the temperature. In addition, L16 diet in the HL transition favored an increase in Weissella and Bradyrhizobium genera in the gut microbiome, while in the final condition of LH transition, L21 diet favored a significant increase in Streptococcus and Bacillus. According to the results, the utilization of 16% dietary lipid levels in gilthead sea bream should be preferred during seasonal temperature changes in order to optimize feed utilization and gut health., This research was undertaken under the MedAID (Mediterranean Aquaculture Integrated Development) project, which has received funding from the European Union’s Horizon 2020 Research and Innovation Programme, Call H2020-SFS-23-2016, Grant agreement no. 727315 (http://www.medaid-h2020.eu/). MY and NG received support from the Spanish Ministry of Science, Innovation and Universities (MCIU), the State Research Agency (AEI), the European Regional Development Fund (FEDER/ERDF), and project Thermodigest (RTI2018-096134-B-I00) granted to MY.

A better knowledge of daily digestive function may help to improve the feeding efficiency in farmed fish species. In this study, daily expression profiles of key genes related to the circadian system (arntl1, clock, per3 and cry1), nutrients hydrolysis (pga, atp4a, try, ctrb, cel1b, pla2, and amy2a), appetite control and food intake (ghrl and cck), and nutrient transport (slc15a1) were assessed in the gastrointestinal tract (GIT) of gilthead seabream (Sparus aurata) juveniles, under four different daily feeding frequencies (one, three, five diurnal meals and continuous diurnal feeding). Additionally, the activity of two key proteolytic enzymes (pepsin and trypsin) and variations in luminal pH within the different GIT segments were determined. Clock genes showed significant circadian patterns, being highly synchronized to the light-dark cycle and first daily meal in all the groups. In contrast, most of the genes codifying digestive enzyme precursors did not exhibit relevant changes in expression along the daily cycle. A correlation was detected between the expression of slc15a1 and per3 and, to a lesser extent between arntl1/clock and ghrl, supporting the role of clock genes in daily organization of some digestive functions. Feeding frequency had a poor influence on the expression of all the analysed genes. In contrast to the results obtained for the expression of genes encoding digestive enzymes, the proteolytic activities exhibited circadian rhythms, and their temporal distribution were significantly affected by some of the experimental feeding protocols, being mostly synchronized with the presence of food in the corresponding GIT segment. Fish fed three times a day did not exhibit a significant daily rhythm for trypsin activity, but the daily average of activated trypsin was similar in all feeding frequencies. Gastric pH was maintained neutral, and acidification occurred only after food ingestion although showing different daily pattern for each feeding protocol. Overall, these results indicate that in gilthead seabream the majority of modulations for the hourly profile of digestive functionality occur post-transcriptionally to adapt to different feeding frequencies., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to M.Y. and WISEFEED funded by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). The authors would like to thank Dr. Tomé Silva for his valuable help in revision of the results and statistical advices. Neda Gilannejad was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

Melanoidins are complex molecules usually found within dietary matrixes suffering heating steps during the production process. These compounds are known to exert a number of negative effects on the digestibility of nutrients and digestive enzymes in mammals, but also determines an increase in the antioxidant capacity of the diet with potential effects for animal health. In the present work, digestive and nutritional consequences of the ingestion of glucose-glycine melanoidins were tested in juvenile rainbow trout (Oncorhynchus mykiss), which was fed two non-heated, pelletized diets: a control diet without melanoidins and an alternative diet containing 1.2% of glucose-glycine melanoidins. The results pointed to a small effect on gut pH in interaction with digestion time, and a lack of effect on gastric evacuation and digestive proteases. In addition, melanoidins were associated with a small increase in the digestibility of the diet dry matter, leaving unchanged nitrogen and phosphorus digestibility. On the other hand, melanoidins determined a complex increase in the postprandial antioxidant capacity of blood plasma, particularly apparent in the antioxidant components above 3 kDa. Fil: Serrano, Xavier. Universidad Católica de Temuco; Chile Fil: Hernández, Adrián J.. Universidad Católica de Temuco; Chile Fil: Morales, Gabriel Alejandro. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larson, Majorie. Universidad Católica de Temuco; Chile Fil: Ruiz, Joceline. Universidad Católica de Temuco; Chile Fil: Orellana, Paola. Universidad Católica de Temuco; Chile Fil: Díaz, Manuel. Universidad de Almería; España Fil: Moyano, Francisco J.. Universidad de Almería; España Fil: Márquez, Lorenzo. Universidad Católica de Temuco; Chile

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture, This work was funded by project AGL2010-14876 from MICINN to JMM.

The present work uses a novel approach to develop a mathematical model explaining the effect of three main factors - temperature, total reaction time and enzyme:substrate ratio - in the hydrolysis of protein by the intestinal enzymes of the Senegalese sole. The methodology combines a factorial design based in the Response Surface Methodology and in vitro digestibility assays adapted to the physiology and culture conditions of the species. The model revealed that, within the physiological ranges considered in the study, the efficiency of protein hydrolysis by the intestinal enzymes of the sole was linearly correlated to two of the selected factors; directly to incubation time and inversely to the amount of substrate. The effect of temperature was not linear but quadratic, hence showing a maximum at 23 °C. The information obtained can be used as a tool to maximize the biological response under variable conditions and may orientate some on-farm feeding practices., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to M.Y. and WISEFEED funded by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). Neda Gilannejad was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

Fil: Márquez, Lorenzo. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Hernández, Adrián J. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Carvalho, Edison S. M. Instituto Gulbenkian de Ciência, Telomeres and Genome Stability Group. Oeiras, Portugal. Fil: Morales, Gabriel A. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Buenos Aires, Argentina. Fil: Dantagnan, Patricio. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Díaz, Manuel. Universidad de Almería. Departamento de Biología y Geología. Grupo de Nutrición y Alimentación Animal. Almería, España. Fil: Moyano, Francisco J. Universidad de Almería. Departamento de Biología y Geología. Grupo de Nutrición y Alimentación Animal. Almería, España. The activity of fish acid proteases is strongly dependent on the pH, and many basic and applied works had focused on researching the pH-profile of digestive proteases using different buffered media. Nevertheless, enzymatic activity is also known to be affected by the ionic strength (IS) of the medium. The present work is aimed at researching the combined effects of pH (2.5, 3.0, 3.5, 3.75 and 4.0) and IS (50, 100 and 200 mM) on the protease activity of gastric extracts of juvenile Coho salmon Oncorhynchus kisutch. It is concluded that the activity of Coho salmon gastric proteases is maximal at pH 2.5-3.0, drops towards pH 4.0 irrespective of ionic strength, and they showed a lower activity at IS 50 mM in comparison to IS 100 or 200 mM. The present results point out to the convenience of considering the effect of ionic strength when measuring the activity of fish gastric proteases. tbls.

The present study is focused to elucidate the main characteristics of the digestive function of this carnivorous fast-growing fish living at high temperatures. With this aim, we have examined the effects of an increased temperature from 30 to 34 °C on the daily pattern of gastrointestinal pH, enzymatic proteolytic digestive activity and the feed transit time in early juveniles of cobia (Rachycentron canadum), a species living in tropical and subtropical waters with an increasing aquaculture production. Fish were fed two meals a day. Gastric luminal pH was permanently acidic (mean pH values: 2.76–4.74) while the intestinal pH increased from neutral/slightly acidic to slightly alkaline when the digesta was present, with an increasing alkalinity from proximal to distal intestine (mean pH values: 6.05 to 7.69). The temperature did not affect the gastric pH but a slightly higher acidity was induced in the intestine at 34 °C. Pepsin activity showed a daily rhythm at 30 °C with maximum in the middle of the light period, while at 34 °C some hourly changes coinciding with feed adding without a clear daily trend during the 24-h period were observed. The trypsin activity exhibited a daily rhythm at both temperatures with an increase after morning feeding to reach a maximum several hours later. Average pepsin activity during the daily cycle was slightly higher at 34 °C (6.1 and 7.3 U mg−1 BW at 30 and 34 °C respectively), but values were significantly different only at 8 and 24 h after the morning meal. Similarly, the trypsin activity was significantly affected by the temperature only at 8 and 16 h after the morning meal, but daily activity averages were similar (1.20 and 1.29 U g−1 BW at 30 and 34 °C respectively). The partial transit rates of the first meal in the stomach for each period inter-samplings were higher during the first 4-h period and decreased progressively along the rest of the 24-h cycle at both temperatures, but no significant differences were detected at 30 °C. In addition, the transit was notably faster at 34 °C particularly during the first 8 h after feeding, with rates between 100 and 65% of total volume displaced (intake or released) during each 4-h period. In the intestine the transit rate was relatively constant and similar at both temperatures during 12 h after feeding. Then the rates remained very low during the following 12 h. Residence time of the first meal was longer at 30 than at 34 °C, particularly in the stomach (12 h:02 min vs 4 h:54 min respectively). In the intestine the difference was not so large (8 h:18 min vs 6 h:24 min respectively). In a parallel study under same conditions, cobia reared at 30 °C grew faster and showed a more favorable feed conversion ratio than those at elevated temperature (34 °C). The present results indicate that at 34 °C, a subtle increase of proteolytic activity cannot compensate for the faster gut transit rate. Therefore, 30 °C is more appropriate temperature for the early on-growing of cobia because at higher temperatures the digestion efficiency decrease being one of the causes for a lower growth., The project WISEFEED received funding by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). Additional funding from project EFISHDIGESTAGL2014-52888 (MINECO, Spain + FEDER/ERDF contribution). S.E. acknowledges a Foundation for Science and Technology of Portugal (FCT) investigator grant IF/00482/2014/CP1217/CT0005 funded by the European Social Fund, the Operational Programme Human Potential and FCT. This work also received national funds through FCT through project UDI/Multi/04326/2013 and Norwegian Agency for Development Cooperation NORHED, No. QZA-0485 SRV-13/0010.

Larvae and early juveniles of fast-growing fish species show tremendous growth potential, leading to higher requirements for protein, high-unsaturated fatty acids, and other nutrients. Several commercial weaning diets with relatively high success have been developed for low/moderate-growing species; however, additional challenges are outlined to meet growth potential and energy requirements of such fast-growing species. The objective of the present study was to evaluate two commercial microdiets for marine fish, one having simultaneously a very high protein and high lipid contents (HIGH), and another (MOD) with a high protein and moderate lipid content, in a growth performance trial with greater amberjack post-larvae from 33 to 78 days after hatching. Moreover, histological preparations of liver, anterior and posterior intestine were assessed for hepatic and intestinal lipid inclusions quantification and gut epithelial brush height measurement. Activities of the digestive enzymes: pepsin, trypsin, chymotrypsin, lipase and amylase were also analyzed. Post-larvae fed HIGH microdiet exhibited higher final weight and lower feed conversion ratio that those fed on MOD microdiet. Liver displayed a higher level of lipid inclusions for the MOD diet than for HIGH diet. Moreover, enterocytes of posterior intestine presented a much higher level of supranuclear vacuoles for the HIGH diet compared to MOD diet. The lower Trypsin/Chymotrypsin ratio observed at the end of the experiment in larvae fed on MOD diet may indicate a deficiency in protein of this diet. Together, these results support that larvae of greater amberjack, and likely other fast-growing marine fish species, require high protein–high lipid microdiets. The use of microdiets developed targeting slower growing marine species may lead to sub-optimal performances in fast-growing larvae., This study was undertaken by Project WEANTUNA supported by Eurostars (E! 7994) and co-financed by FEDER through PO Algarve 21 in the framework of QREN 2007-2013 (30200).

The activities of some of the main digestive enzymes of long snouted seahorse (Hippocampus reidi; Ginsburg, 1933) measured during the initial 20 days after birth were used to assess the nutritional response to different enrichment of the Artemia salina used as live food. Some significant differences in the activities of trypsin total protease and esterases were observed among the experimental treatments, changes in the activities of acid and alkaline phosphatases, as well as in the trypsin/estaerase ratio, were linked to the different dietary treatments. This provided information both on some characteristics of the digestion process in newborns and the influence of the diet on the maturation of the intestinal epithelium during their early development. The use of a multivariate analysis combining the information of all the enzyme activities highlighted differences between treatments. Results obtained confirm the interest of using digestive enzymes as valuable indicators of nutritional condition in larvae of this species and may help to optimize feeding protocols required for mass-scale production of seahorses in captivity. Statement of relevance Overexploitation, habitat loss, and incidental bycatch during commercial fishing operations, make seahorses susceptible to decline. The high global demand of seahorses exceeds supply from specimens breeding in captivity, and in this unsustainable situation, the long snouted seahorse, H. reidi, is one of the most frequently commercialized species. Larval rearing remains the bottleneck for successful cultivation of seahorses. In this sense, the design of a suitable feeding regime based on food adapted to the nutritional needs of the species is highly desirable. Due to their key role in nutrient digestion and assimilation, changes in the production patterns of the main digestive enzymes can be used as indicators of the nutritional status helping to assess the adaptation to foods with different content in macronutrients and therefore contributing to optimize feeding strategies for this species. The biochemical parameters proposed as indicators of nutritional status might be good analytic tools, not only to establish the time of change of live prey during the weaning but also to formulate the most digestible biochemical composition of live prey. Phosphatases activities, trypsin/esterase ratio and the clusters of enzyme activities, were consistent in the results and might be considered as good indicators of nutritional status in this species.

The morphological and functional development of the digestive system in meagre ( Argyrosomus regius, Asso, 1801) was described by means of histological and enzymatic approaches in order to provide insight into the digestive physiology of the species at early life stages of development and evaluate whether the acquisition of digestive capacities matched the morphological development of digestive organs. For this purpose, A. regius were reared from hatching to the juvenile stage at 18 °C using a standard feeding sequence of enriched rotifers (2–15 days posthatching, dph), Artemia metanauplii (14–51 dph) and microdiet (35–51 dph). Histological and enzymatic analyses indicated that A. regius larvae had at the onset of exogenous feeding (2 dph, 3.2 ± 0.1 mm in standard length, SL) a well differentiated exocrine pancreas, and bile salt-activated lipase and alkaline proteases being the main pancreatic enzymes involved in food digestion. Ontogenic changes in pancreatic enzymes occurred between 3.2 and 3.9 mm in SL, coinciding with the resorption of the oil globule and the complete transition to exogenous feeding, and between 4.2 and 5.8 mm in SL (20–25 dph). Regarding acid digestion, pepsin was not detected in A. regius until 6.0 and 6.8 mm in SL (31 dph), coinciding with notochord flexion and the progressive decrease in the activity of alkaline protease and leucine-alanine peptidase (intestinal cytosolic enzyme) activities, indicating a shift in the mode of digestion. The detection of pepsin activity in A. regius occurred after the appearance of the first gastric glands (4.6 and 5.8 mm SL). Thus, authors need to be cautious when extracting conclusions about the beginning of acid digestion and the onset of weaning of larvae just using histological data, since it has been shown that morphology does not match functionality with regards to the stomach. The comparison of present results with other studies conducted in this species using different rearing conditions ( e.g. mesocosm technique, larval density, water temperature, feeding sequence) indicated that the functional development of the digestive system in A. regius assessed by the activity of alkaline and acid proteases is a well-conserved process that generally occurs within a range of body size regardless of larval age and rearing conditions. Thus, authors recommend using morphometric and/or morphological variables ( e.g. length, developmental stage) together with the age of larvae expressed in days after hatching for comparative purposes among different studies. Statement of relevance The morphological and functional development of the digestive system in meagre ( Argyrosomus regius ) was described by means of histological and enzymatic approaches in order to provide insight into the digestive physiology of the species at early life stages of development. Combining histological and enzymatic analytical procedures we evidenced that stomach's functionality did not match its morphological organization, since pepsin activity was not detected until some days after the appearance of the first gastric glands. This indicates that authors need to be cautious when extracting conclusions about the beginning of acid digestion and the onset of weaning of larvae just using histological data.

The products resulting from the non-enzymatic browning in processed diets are known to exert negative effects on the digestive enzymes of vertebrates. In addition, browning heavy products (BHP's) are known to co-precipitate with proteins depending on the pH and ionic strength of the medium and on the isoelectric point of the protein. As the manufacture of aquafeeds as well as aquafeed ingredients frequently implies heating processes, the effects of BHP's from a well-known model mixture (glucose + glycine) on the gastric proteases of the rainbow trout Oncorhynchus mykiss were investigated in the present work. The results support that BHP's interact with gastric proteases most probably to form a co-precipitate, thus potentially reducing the protease activity in the fish stomach. The maximal loss of activity found in the experiments was above 20%. The interaction is pH and ionic strength-dependent and relatively stable against mechanical perturbation. In the range of BHP concentration 500-1000 μg/mL, the interaction was dose-dependent at pH 3.5 with a maximum above 20%, and apparently independent of the dose at pH 4.0. Above an ionic strength of 100 mM due to monovalent ions (NaCl), the intensity of the interaction is reduced. On the other hand, divalent cations such as Ca2+ and Mg2+ in the range 30-80 mM disturb the interaction between BHP's and gastric proteases even at ionic strengths below 100 mM, thus alleviating the inactivating effect of BHP's. Fil: Serrano, Xavier. Universidad Católica de Temuco; Chile Fil: Hernández, Adrián J.. Universidad Católica de Temuco; Chile Fil: Larson, Majorie. Universidad Católica de Temuco; Chile Fil: Morales, Gabriel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal; Argentina Fil: Díaz, Manuel. Universidad de Almería; España Fil: Moyano, Francisco J.. Universidad de Almería; España Fil: Márquez, Lorenzo. Universidad Católica de Temuco; Chile

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24 h cycle in larvae reared under a 12 h light/12 h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60 days post-hatch. In each sampling day, larvae were sampled every 3 h during a complete 24 h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution. J.A. Mata-Sotres was supported by a doctoral grant (ID 215473) from the Mexican National Council for Science and Technology (CONACYT).

Activities of pepsin, trypsin, chymotrypsin, alpha-amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick-lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS-PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha-amylase and no lipase activity recorded in pre-adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.

Melanoidins, a product of advanced browning reactions due to the thermal treatment of the diet, has been previously demonstrated to inhibit the activity of trypsins from mammalian origin, but no data exists in relation to fish trypsins. The present work is devoted to the investigation of the effects of glucose-glycine melanoidins on the trypsin-like activity contained in intestinal crude extracts (ECIs) prepared from juvenile individuals of the rainbow trout Oncorhynchus mykiss. A turbidity assay was conducted to test for the existence of precipitation or aggregation phenomena in mixtures of ECIs and melanoidins, but no consequences were observed. Three experiments were conducted to test for the effects on the trypsin-like activity on the hydrolysis of the substrate BAEE (Nα-Benzoyl- L -arginine ethyl ester). Glucose-glycine melanoidins can inhibit the intestinal trypsin-like activity of the rainbow trout, but the effect is modulated by the pH and ionic strength of the reaction medium. The inhibition dropped when the pH was changed from 7.5 to 8.5, also when the ionic strength of the buffer was raised from 40 to 140 mM, while it was undetected at 180 mM. It is concluded that the effect of this type of melanoidins on the trypsin-like activity from the rainbow trout is probably low in the ionic conditions prevailing in the intestine of this species.

The impact of deoxynivalenol (DON) on rainbow trout, Oncorhynchus mykiss, is mainly characterised by impaired growth performance and reduced feed intake, usually with the total absence of any visible clinical signs. Despite the high concentrations of DON in the present study (up to 11,412 ± 1141 μg kg−1), no clinical signs (except anorexia at the higher DON dosage) were observed, which confirms the difficulties of diagnosing DON ingestion. Compared to the control group, the proteolytic enzyme activities (pepsin, trypsin and chymotrypsin) in trout were altered by DON ingestion. However, it was not clear if the observed impact on digestive enzymes was due to the direct action of DON, or a consequence of the lower feed intake determined for DON-treated animals. The impact of DON on the abundance of specific measured mRNA transcripts was unexpected with higher expression levels for insulin-like growth factors, igf1 and igf2, which are directly related to elevated insulin levels in plasma. This can also in part be influenced by the trypsin activity and by npy, given its higher mRNA expression levels. The apparent digestibility of dry matter, protein and energy was not affected by dietary levels of DON, however, nutrient retention, protein, fat and energy retention were significantly affected in animals fed DON. Adenylate cyclase-activating polypeptide (PACAP) expression seems to play an important role in controlling feed intake in DON fed trout. In the present study, we have shown for the first time that DON is metabolized to DON-3-sulfate in trout. DON-3-sulfate is much less toxic than DON, which helps to explain the lack of clinical signs in fish fed DON. Being a novel metabolite identified in trout makes it a potential biomarker of DON exposure. Suppression of appetite due to DON contamination in feeds might be a defense mechanism in order to decrease the exposure of the animal to DON, therefore reducing the potential negative impacts of DON., The present work was performed under the WiseFeed project (European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 691150) being Biomin Holding GmbH, a partner of the project, represented by Rui A. Gonçalves in his secondment to Instituto de Ciencias Marinas de Andalucía, Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), Puerto Real, Cádiz, Spain.

There is a large amount of scientific literature on digestive enzymes activity during fish larval development. Nevertheless, most of this information refers to descriptive aspects of the ontogeny of hydrolyzing capacities, and only few studies looked into functional aspects and its relation with feeds and feeding activity. A comparison of published data normalized by days-degrees reveals at least three groups of species (precocious, medium and late) in relation to the time required for reaching maximum activity of digestive enzymes capacities, irrespective of the taxonomic group. Recent progress has allowed enlarging our knowledge on fish larvae digestive function. Such progress is partially based on new methodologies, but also on extended experimental designs and sampling protocols. On one side, the molecular basis of the onset of the digestive functionality has been examined in several species, cloning the mRNAs codifying the precursors of the main digestive enzymes and determining the gene expression profiles. On the other side, the daily rhythms coupling feeding and digestive enzyme production activities started to be investigated, giving a new dimension to our understanding of the digestive capacity during these early stages. Comparison between molecular expression and actual activity of the different enzymes is providing new insights on the regulation mechanisms. The connection between feeding behavior and digestive response both at genomic and biochemical levels is a required step towards the rationalization of feeding protocols with both live and inert diets, as a way to increase the nutrients utilization and the developmental performance at these early stages.

Models simulating the in vitro digestive hydrolysis of nutrients by different animal species are frequently used to obtain a better understanding of factors affecting this process. Optimization algorithm of a model may be used to prospect the more favourable combination of selected factors resulting in the higher performance. This study was conducted to determine the combination of factors (pH, enzyme:substrate ratio, and reaction time) leading to highest bioavailability of proteins and carbohydrates in the gilthead seabream gastrointestinal tract. Besides, a novel multi-objective algorithm, desirability function, was introduced for optimization of the digestive hydrolysis of nutrients within the simulated gut of the species, using models based on the Response Surface Methodology. Design of experiment was defined based on the physiology and culture conditions of the species, and in vitro assays were performed in a two-phase (stomach ad intestine) digestion process, using the species-specific enzyme extract. According to results, intestinal phase of digestion makes the major contribution to the total protein hydrolysis, being the efficiency of the process directly correlated to all the three studied factors. In contrast, the efficiency of carbohydrate hydrolysis was directly correlated to the amount of substrate and inversely to the pH, while reaction time did not exert a significant effect. The physiological range of the factors studied in the assays favoured the hydrolysis of proteins over carbohydrates, a similar scenario to that observed in the live fish. Results from the mathematical models and their simultaneous optimization obtained from this work may have practical applications in design of feeds for this species., This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) with FEDER/ERDF contribution by project EFISHDIGEST (AGL2014-52888-R) to MY. Additional funding from project WISEFEED funded by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). NG is supported by a grant from MINECO (BES-2015-071662).

A basal isonitrogenous and isoenergetic plant-based diet (Control) was supplemented with either 10 g kg−1 sodium diformate (NaDF), 4000 FTU kg−1 phytase (Phy) or a combination of both additives (NaDF + Phy). Three hundred juvenile rainbow trout with an average weight of 120 g were randomly distributed into 12 fibreglass tanks (300 L). After 65 days of trial, fish fed diets containing phytase, NaDF or the combination of both additives showed a higher growth rate (P

Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology., Most of the work outlined on this topic was supported by the International Foundation for Science (No. A/4306-1 and No. A/4306-2) granted to EP, the Agencia Española de Cooperación Internacional/Asociación Universitaria Iberoamericana de Postgrado (AUIP/AECI) from Spain, and the Program ʻDoctorado Iberoamericano en Ciencias’ at the University of Cadiz, Spain.

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects RIDIGEST (AGL2011-23722) and AQUAGENOMICS (Consolider-Ingenio Program CDS2007-0002) with FEDER/ERDF contribution.

In vitro assays simulating the digestion process have been extensively used in the evaluation of the nutritional quality of foods and feeds for humans and terrestrial animals. Such techniques have been comparatively less used in nutrition of aquacultured species, although in recent years the interest in developing alternatives to the conventional in vivo digestibility assays has increased. Using 65 published papers as a reference, this review summarizes the different approaches and configurations used to date in the simulation of the digestion of food by aquatic animals, as well as the main limitations detected in the different methods and the potential advantages derived from their application. Some recommendations are suggested to develop suitable in vitro assays oriented either to the nutritional evaluation of feed ingredients or to get a more detailed knowledge on the interactions among digestive enzymes and feed components.

The Mugilidae are a group of fish with a great interest for aquaculture due to their omnivorous profile, rapid growth, and resistance to environmental variations. The selection of feed ingredients for these species is currently focused on an extensive use of plant by-products, with this being limited by their content in anti-nutritive factors (mainly phytate and non-starch polysaccharides, NSPs). Nevertheless, specific enzymes can be used to counteract some of those negative effects. In the present study, the effect of pretreating two high-plant feeds with a mixture of enzymes (glucanases + phytase) on the digestive use of protein and phosphorus by juvenile mullets (Mugil cephalus) was assessed using both in vitro and in vivo assays. The enzymatic treatment significantly modified the potential bioavailability of some nutrients, such as a reduction of sugars, pentoses, and phytic phosphorus. Also, it increased the digestibility of protein in one of the feeds but reduced that of phosphorus in both of them. The potential usefulness of enzyme treatment and the information provided by the two types of assays are discussed.

To assess to what extent addition of phytase to a plant-based diet results in spatio-temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant-based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant-based diets used in fish aquaculture. Fil: Morales, Gabriel Alejandro. Universidad de Almería; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marquez, L.. Universidad de Almería; España Fil: Saenz de Rodrigañez, M.. Universidad de Almería; España Fil: Bermúdez, L.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Robles, R.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Moyano, F. J.. Universidad de Almería; España

a b s t r a c t An in vitro assay simulating digestion in fish stomach was used to assess the effect of a bacterial phytase on different variables potentially affecting the digestibility of phosphorus and protein in eight plant ingredients: total protein solubility at different pH, differential solubility of protein fractions and potential bioavailability of amino acids after enzyme hydrolysis. Dephosphorylation of native phytate (IP6) significantly (P

In the present study, the protective effect of nucleotides over damages induced by the consumption of a diet containing a high amount of vegetable ingredients (560 g kg(-1)) in the intestinal epithelia of the meagre (Argyrosomus regius) was assessed by assays performed with an Ussing-type chamber. Two experimental feeds were prepared including or not a commercial mixture of nucleotides (1 g kg(-1)). Nucleotides significantly enhanced fish growth during the experiment. On the other hand, differences in the integrity and functionality of intestinal epithelia were evidenced by a change in the polarity of intestinal trans-epithelial potential. Samples of fish fed on the control diet showed a preferentially secretory short-circuit current, while those of fish receiving the nucleotide-supplemented diet showed a significantly lower and preferentially absorptive negative current. It is concluded that alterations of intestinal physiology juvenile meagre resulting from the intake of high amounts of plant ingredients could be minimized by nucleotide supplementation.

The aim of the present study was to determine the optimum dietary levels of soybean lecithin (SBL) for seabream (Sparus aurata) larvae, and its influence on production performance and digestive enzymes activity. Larvae were fed five formulated microdiets with five levels of SBL. Complete replacement of live preys with the experimental microdiets for seabream larvae at 16 dph produced over 55% survival rates, particularly in fish fed with the highest levels of SBL. Moreover, increase in dietary SBL up to 80 g kg−1 significantly improved larval growth, leading to high final total length and body weight. An increase in alkaline phosphatase activity with the elevation up to 80 g kg−1 SBL was also found denoting a better maturation of the digestive system. Besides, there was a stimulatory effect of dietary SBL on PLA2 activity. Finally, increasing dietary SBL lead to better utilization of dietary highly unsaturated fatty acid, as it was reflected in their higher content in both neutral and polar lipid of the larvae. In summary, elevation of dietary SBL up to 80 g kg−1 in microdiets for seabream significantly improved digestive enzymes activities, enterocyte maturation, utilization and deposition of dietary essential fatty acids and larval growth, as a consequence of a better digestion, absorption, transport and deposition of dietary nutrients.

Cholecystokinin (CCK) is an important regulator of pancreatic enzyme secretion in adult mammals and teleosteans. Although some studies have focused on the interaction between CCK and trypsin in marine fish larvae, little is known about the circadian patterns of the regulatory mechanism involving these two digestive components. In this study, we took advantage of the characteristic change from a diurnal to a nocturnal feeding habit that occurs in Senegalese sole (Solea senegalensis) post-larvae, to conduct an experiment where larvae and postlarvae were submitted to three different feeding regimes from mouth opening: continuous feeding, diurnal feeding and nocturnal feeding. The aim was to establish different daily feeding scenarios to uncover the operating mechanisms of CCK and tryptic enzyme activity over the 24-hour cycle to better understand the regulation of digestion in developing fish larvae. Results show a prevalence of simultaneous and opposing trends of CCK level and tryptic activity as a function of the postprandial time. This finding supports the existence of a regulatory loop between these two digestive components in pre- and post-metamorphic Senegal sole larvae. In addition, CCK level was also modulated by the gut content, tending to be lower when the gut is full and higher when is being emptied. Furthermore, larvae were able to synchronize digestive functions to very different feeding regimes, although it seems to be important having a diurnal feeding phase during pre-metamorphic stages for a proper development., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution granted to M. Yúfera. C. Navarro-Guillén was supported by a doctoral fellowship (BES-2012-051956) from MINECO (Spain).

12 páginas, 2 tablas, 3 figuras, Differences in survival and growth rates in seahorse Hippocampus guttulatus juveniles feeding on Artemia sp. or copepods have been related to specific digestive capacities of seahorse newborn, which are capable of actively forage on available prey from the first day of live. Other seahorse species, such as H. abdominalis and H. hippocampus, show high success feeding on Artemia nauplii suggesting species-specific differences in the digestibility of prey among seahorses. In this study, the profiles of digestive enzyme activity during the initial 15 days after release (DAR) were very low for trypsin, chitinase and α-amylase. In contrast, higher activities towards any of the assayed substrates for lipase (butyrate, octanoate and oleate) were evident from 0 DAR onwards. From 15 DAR onwards, the effect of diet composition became evident in juveniles previously fed on a mixed diet (Artemia + copepods), which showed a clear increase in all the assayed enzymes when compared with juveniles fed on Artemia as a sole prey. As a practical applicability of this study, a feeding schedule ensuring an adequate digestibility of the prey is proposed based on ontogenetic enzymatic activities of seahorse juveniles fed on different prey, The study was financed by the Spanish Government (Plan Nacional, Project CGL2009-08386) and the Regional Government of Galicia (Xunta de Galicia, Project 09MDS022402PR). A. Blanco was supported by PhD JAE-Pre grants (Junta para la Ampliación de Estudios Program) from the Spanish National Research Council (CSIC), co-financed by the European Social Fund

[EN] The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible., This work was supported by the Vicerrectorat d'Investigacio, Innovacio i Transferencia - Universitat Politecnica de Valencia, Project Name: Aquaculture feed without fishmeal (SP20120603). URLs of funder:http://www.upv.es/entidades/VIIT/info/indexnormalc.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.