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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.

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).

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

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.

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 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.

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.

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

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).

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg−1 (100 g kg−1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg−1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n-3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg−1 KPL.

The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model simulating conditions existing in the digestive tract of rainbow trout ( Oncorhynchus mykiss ). Two commercial phytases, expressed in P. lycii and E. coli , were evaluated. Fungal phytase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P

The hydrolysis of protein in different animal and plant sources by the intestinal proteases of Senegalese sole (Solea senegalensis) was studied using a combination of an in vitro digestibility assay and the evaluation of the protein fractionation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In vitro hydrolysis was performed for 90 min in a closed reactor maintained at constant pH and temperature. Samples of the reaction mixture at different time intervals were developed in SDS-PAGE gels to evaluate the progressive hydrolysis of the different protein fractions in each protein source. A numerical value [coefficient of protein degradation (CPD)], which integrates the information obtained after image analysis of the gels, is proposed for comparison among proteins, according to the intensity of the hydrolysis produced by sole proteases. Additionally, the total free amino acid released from proteins was measured during the in vitro assay. Casein, squid meal and soybean concentrate (SBC) proteins showed very similar degradation patterns, with a quick and almost complete proteolysis within the first minutes of the enzymatic reaction. Fish and krill meals were hydrolysed more progressively. On the contrary, pea meal (PM) and corn gluten meal (CGM) showed scarce changes in their protein profile after 90 min of reaction. For animal protein sources, the final CPD values ranged from 77.6% to 87.0%, showing not significant differences. By contrast, PM (30.5%) and CGM (32.3%) presented significantly lower CPD values (P

8 páginas, 8 figuras., The development of cost-effective and nutritionally adequate formulated diets is a key step in the sustainable expansion of spiny lobster aquaculture. Despite proteins are the major and most expensive component of diets, few studies are available on protein digestibility in spiny lobsters and such assessments have never been performed by in vitro methods. Two techniques were used for studying in vitro protein digestion of some common aquafeed ingredients by the spiny lobster Panulirus argus: i) the digestion cell in which the digestion products are removed by dialysis, and ii) electrophoresis which allows the visualization of the different protein fractions in the tested meals. Since three main trypsin isozyme patterns or phenotypes have been recently described in P. argus, the potential differences in protein digestion between individuals with different trypsin isozyme patterns were assessed. Results herein presented demonstrate for the first time in a crustacean species that the different trypsin phenotypes differ in protein digestion efficiency. Also, the digestion cell method was applied for the first time to a crustacea, proving to be sensitive to small changes in digestion efficiency. This method could be used in further in vitro studies for examining other aspects of spiny lobsters digestive process., This work was supported by IFS grant No. A/4306-1 and AUIP/AECI. E. Perera is a PhD fellow of AUIP at the University of Cadiz, Spain, within the Program “Doctorado Iberoamericano en Ciencias”, whose support is highly appreciated.

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60 dah. Most digestive enzymes were present from yolk absorption (5-6 dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8 dah up to 20 dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5 kDa) were detected at 8 dah using SDS-PAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf = 0.54) for acid proteases (pepsin-like) from 3 dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13 dah.

Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.

Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.

The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.

The effects of starvation and re-feeding on metabolites and tissue composition, GH/IGF-I axis, and digestive enzyme activities in juvenile thick-lipped grey mullet (Chelon labrosus) were evaluated. Fish were divided into three feeding groups (n = 72, 82.00 ± 4.09 g initial body mass). The control group was fed 1% of their body mass once a day throughout the experiment with commercial pellets. The other two groups were deprived of feed for 21 days (starved), or re-fed for 7 days after 14 days of food deprivation (re-fed). Full-length cDNAs from pituitary GH and hepatic IGF-I were cloned by screening a cDNA library or by PCR techniques. Furthermore, changes in their mRNA expressions were assessed by real time PCR in specimens maintained under the different feeding patterns. Results showed a negative growth in starved and re-feeding groups. Starvation induced a significant increase in plasma triglycerides as well as a decrease in liver glucose and glycogen. Re-feeding increased plasma glucose, lactate and protein, as well as liver glucose and glycogen. In addition, starvation significantly increased pituitary GH expression, while re-feeding down-regulated it. No significant changes were observed in hepatic IGF-I expression in any dietary treatment. Digestive enzyme activities were not significantly affected either by starvation or by re-feeding. The results of the present work suggest that juveniles of the thick-lipped grey mullet may easily adjust their metabolism under situations characterized by a short-term starvation and re-feeding., This work was funded by project AGL2010-14876 from MICINN to JMM.

Changes in digestive pH and protease activity have been determined throughout the transition from larvae to the juvenile stage in Sparus aurata in rearing conditions (from 0.04 to 100 g wet weight). Measurements of pH have been taken in the stomach and different segments along the length of the intestine using a pH microelectrode. In starved fish, the gastric pH ranged between 6.0 and 8.0 approximately, except in juveniles of intermediate size (between 1.0 and 7.0 g wet weight), which exhibited a wider pH range of 2.0−8.0. Fed fish with digestive content showed, in general, lower pH values in the stomach. A progressive decrease was observed from a pH range of 5.5–8.0 in the youngest animals (0.04 g) to a pH range of 2.0−6.2 when juveniles were approaching 1.0 g wet weight. Above this weight, the gastric pH remained constant (between 2.0 and 6.0 approximately). The pH values in the intestine ranged between 6.7 and 8.4. They were similar in the different segments and weight classes examined, and there were no significant differences between fed and starved animals. Specific acid protease activity (units per milligram soluble protein) in fed animals increased from small (0.04–1.0 g) to intermediate juveniles (1.0 and 7.0 g), but then remained similar in larger juveniles. On the contrary, specific alkaline protease activity in fed animals decreased from small to intermediate juveniles, and then remained at a similar level in larger juveniles. The results reflect a progressive transition during several months from alkaline digestion in larvae with undeveloped stomachs to the acid digestion in juveniles with fully developed stomachs. Full gastric capacity is developed in seabream juveniles of 1 g wet weight, which represents approximately 100 days post-hatching in cultured populations. Nevertheless, in the following 2.5 months, during which the intestine reaches the appropriate length, juveniles still show a transitional period in the regulatory mechanism of digestion, probably linked to the adaptation to a different feeding habit.

The pH-stat system has been used to assess the effect of tannic acid (TA) on solubility and in vitro enzyme hydrolysis of different proteins. Added TA (from 10 to 50 g kg−1) decreased the extent of hydrolysis of bovine serum albumin. Enzymic hydrolysis of casein, pea meal, soybean meal, and haemoglobin (HB) was increased, as measured by total amino acids released and by the degree of hydrolysis. SDS-PAGE confirmed the results of the in vitro enzymatic hydrolysis. These findings suggest that, under in vitro conditions, when simulating the gastrointestinal environment of domestic mammals, the negative effects of TA described from in vivo experiments are not necessarily due to reduced hydrolysis of proteins. Copyright © 2003 Society of Chemical Industry

This paper presents the results of some experiments oriented to optimise and standardise the measurement of protein hydrolysis by fish proteases using the pH-stat technique. Various factors affecting the degree of hydrolysis (DH) were considered (autohydrolysis of the protein sources, type of enzymes utilised, substrate/enzyme ratio and effect of inhibitors). The crude extracts obtained from fish digestive tissues showed their suitability for the determination of protein hydrolysis, rendering better results than those obtained using mixtures of commercial enzymes. DH values for a given protein were greatly affected by the substrate/enzyme ratio, since small modifications in protein concentration resulted in significant variations in DH. Protease inhibitors present in various plant protein sources produced a reduction in DH values. Cet article presente les resultats de quelques experimentations menees pour optimiser et normaliser la mesure de l'hydrolyse des proteines par les proteases de poissons en utilisant une technique de pH stat. Differents facteurs affectant la degre d'hydrolyse sont consideres: autohydrolyse des sources de proteines, type d'enzyme utilise, ratio substrat/enzyme et effet des inhibiteurs.

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml 1 ); B, Artemia (2 nauplii ml -1 ); C, mixed diet (2 nauplii ml -1 and 3 mg ml -1 microencapsulated diet); and D, microencapsulated diet (3.7 mg ml -1 ) Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0.376 day -1 ; regime B, G=0.253 day -1 ; regime C, G=0.254 day -1 ; regime D, G=0.162 day -1 ). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5.6-5.9 mm L T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8.6-8.7 mm L T (regimes A, B and C), but only 7.3 mm L T with regime D, 4.4-6.2 days were required to complete eye migration under the regimes A, B and C, and 18.3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.

This study was conducted to evaluate the effect that feeding on differently treated soybean meals may have on digestive protease activity of the Nile tilapia (Oreochromis niloticus). Untreated full fat soybean (USB), heated fullfat soybean (HSB), soaked fullfat soybean (SSB) and commercial defatted soybean meal (DSB) totally replaced fish meal (FM) in test diets (30% crude protein) fed to fish juveniles (mixed sexes) for 30 days. After this period, total activity of digestive proteases was determined in each group and the ability of such proteases to hydrolyse the different protein sources and formulated feeds was tested in vitro. Significant differences were observed between treatments, with lower values obtained for fish fed on USB, suggesting a modification in the composition of secreted proteases. In addition, an inhibitory effect produced by wheat bran included in the test diets on protease activity of fish was also shown. The usefulness of using in vitro assays for a preliminary assessment of the nutritive value of aquafeed ingredients is discussed.

The presence of protease inhibitors in different ingredients used in fish feeds may negatively affect their digestive utilisation. The effect of inhibitors present in several protein sources on the activity of digestive proteases of sea bream was assessed using `in vitro' assays. Inhibition produced by extracts of plant proteins ranged from 25 to 50 % of total activity, whereas that obtained using animal protein sources ranged from 1 to 20 %. The exception was ovoalbumin, which showed the highest measured value (62 %). A plot of the inhibition values obtained by changing the relative concentrations enzyme/inhibitor resulted in different curves for different protein sources. The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms allowed visualisation of the aforementioned differences in inhibition. The importance of measuring protease inhibition in the preliminary evaluation of fish feed ingredients is discussed.

The development of artificial feeds for marine fish larvae is demanding and, taking into account the diAculties and costs associated with the rearing of the larvae of some fish, preliminary evaluation of feed ingredients using in vitro techniques may be an alternative to in vivo assays. Some proteins used in microfeeds for marine fish were tested in vitro for examination of their eAect on the proteases of seabream larvae. Casein and cuttlefish meal did not aAect protease activity of crude larval extracts, whereas ovalbumin 1 produced a 60% inhibition. The use of a pH-stat for the assessment of the degree of hydrolysis (DH) of proteins confirmed the low nutritive value of ovalbumin, since a much lower value was obtained for microcapsules prepared using this protein source when compared with those prepared using casein or cuttlefish meal (3.2 vs. 7.3 and 7.6, respectively). Products resulting from such hydrolysis were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐ PAGE). Image analysis of such gels led to the formulation of an index named the coeAcient of protein degradation (CPD) which expressed the extent to which the main protein fractions were hydrolysed by larval proteases in a given time. A significant correlation (r 2 a 0.98, P < 0.05) was obtained between DH and CPD values measured for either protein sources or microcapsules. The combination of protease inhibition assays with measurements of DH and CPD is proposed as a preliminary evaluation of protein ingredients used in the formulation of artificial feeds for larval fish.

The activities of the main digestive enzymes (proteases, amylase, lipase) as well as those of acid and alkaline phosphatases were assessed during the larval development of the Senegal sole, Solea senegalensis. Important variations in specific activities of all the enzymes were observed during the period of study and were mostly related to the beginning or the end of metamorphosis. Both acid and alkaline protease activities were identified at early stages of development. Acid proteases accounted for only 10% of total protease activity in a 9 days-old larva, but exceeded 75% in a 33 days-old individual. Maximum lipase activity was related to the development of exocrine pancreas (days 6 to 10) and to metamorphosis. It is suggested this can be related to the metabolism of lipid reserves taking place during this morphological change. Activity of alkaline phosphatase decreased from day 5 to day 20 but therafter revealed a sharp increase, possibly linked to the development of enterocytes. The pattern of development of the main digestive enzymes found in S. senegalensis is similar to that described in other flatfish species.

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.

An in vitro model designed to assess protein bioavailability in diets for growing Octopus was developed. The model required a previous assessment of some functional features of protein digestion in this species like the main producing organs, optimum pH for activity and total production per g tissue. The main producing organs identified were the salivary glands and the hepatopancreas (HP), being optimum pH for protease activity quite different in both organs (mostly alkaline in the posterior salivary glands and acid in the HP). In spite of the high-specific protease activity measured in the salivary glands, a major role of the HP in protein hydrolysis is suggested due to the much bigger size of this viscera. All this information was used as a basis to develop an in vitro two-step hydrolysis process, which simulated protein hydrolysis performed by these two organs using the Octopus enzymes. The assay was used to evaluate differences in amino acid bioavailability from several protein sources (casein, gelatin, fish meal, squid meal and krill meal) that could be used as feed ingredients for this species. As significant differences were detected both in total amount and in rate of release of the amino acids from such proteins, the model is proposed as a complementary tool in the selection and nutritional evaluation of protein ingredients to be used in diets for Octopus., This research was supported by grants from the Planes Nacionales de Acuicultura (JACUMAR) and Conselleria d'Agricultura, Pesca, Alimentacio I Aigua from the Generalitat Valenciana.

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.

Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices., Spanish Ministry of Science and Innovation (Projects AGL2007-64450-C02-01/02 and Consolider Ingenio 2010 Program (Project Aquagenomics CSD2007-0002) + European Regional Development Fund supported this work.

After the development of the gastric function in juvenile fish, dietary proteins enter a two-phase digestive process comprising an acidic gastric phase followed by an alkaline intestinal phase. However, the main gastric protease, pepsin, is strictly dependent on the existence of a low-enough environmental pH. In 20-g gilthead sea bream, Sparus aurata, the mean minimal gastric pH is close to 4.5, while the mean pH in the duodenal portion of the intestine was nearly fixed at 6.5. The mean maximal gastric content of HCl was approximately 20 microEq for a low-buffering diet. Gastric proteases were more severely affected than intestinal proteases when assayed at actual sub-optimal pH values, 4.5 and 6.5, respectively. When the gastric proteases of juvenile fish were pre-incubated with a citric acid buffer at pH 6.0, the activity at pH 4.5 was very low, whereas when they were pre-incubated with the same buffer at pH 3.0, the activity at pH 4.5 was significantly increased; this fact suggests a deficient activation of zymogens during the gastric digestion and points to a potential approach to improve protein digestion in juvenile gilthead sea bream.

Common snook (Centropomus undecimalis) is one of the most important marine species under commercial exploitation in the Gulf of Mexico; for this reason, interest in developing its culture is a priority. However, larviculture remains as the main bottleneck for massive production. In this sense, our objective was to determine the changes of digestive enzymes activities using biochemical and electrophoretic techniques during 36 days of Common snook larviculture fed with live preys (microalgae, rotifers, and Artemia). During larviculture, all digestive enzymatic activities were detected with low values since yolk absorption, 2 days after hatching (dah) onwards. However, the maximum values for alkaline protease (6,500 U mg protein(-1)), trypsin (0.053 mU × 10(-3) mg protein(-1)), and Leucine aminopeptidase (1.4 × 10(-3) mU mg protein(-1)) were detected at 12 dah; for chymotrypsin at 25 dah (3.8 × 10(-3) mU mg protein(-1)), for carboxypeptidase A (280 mU mg protein(-1)) and lipase at 36 dah (480 U mg protein(-1)), for α-amylase at 7 dah (1.5 U mg protein(-1)), for acid phosphatases at 34 dah (5.5 U mg protein(-1)), and finally for alkaline phosphatase at 25 dah (70 U mg protein(-1)). The alkaline protease zymogram showed two active bands, the first (26.3 kDa) at 25 dah onwards, and the second (51.6 kDa) at 36 dah. The acid protease zymogram showed two bands (RF = 0.32 and 0.51, respectively) at 34 dah. The digestive enzymatic ontogeny of C. undecimalis is very similar to other strictly marine carnivorous fish, and we suggest that weaning process should be started at 34 dah.

Several samples of P. splendida larvae were obtained from eggs until day 60 after hatching (dah) to determine acid and alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase, lipase, and acid and alkaline phosphatase activities using biochemical techniques. Additionally, SDS–PAGE alkaline protease zymogram and PAGE acid protease zymogram were carried out to identify active isoforms during larviculture. Alkaline protease and chymotrypsin were present at the moment of hatching, increased gradually reaching the maximum values at 35 dah. Trypsin and leucine aminopeptidase activities were low from hatching, increasing gradually as larvae grew. Alkaline protease zymogram showed four zymogens, which appears at different days, remaining present until the end of the larviculture (95.2 kDa at 11 dah, 26.4 kDa at 9 dah, 21.4 kDa at 3 dah, and 23.3 kDa at hatching). Pepsin activity was present at day 7 after hatching and increased progressively until the end of the larviculture. Acid protease zymogram only showed one zymogen (0.65 rf), which appear at 6 dah. Lipase was high at the time of hatching and increased until 15 dah, after which decreased gradually. Amylase was high from the beginning and until 15 dah and then decreased rapidly to almost nothing onward. Alkaline and acid phosphatases presented a high activity at the egg stage, fell slightly during the first feeding and increased again from 20 to 30 dah. Results obtained in this study show that larvae can be fed artificial diets starting on day 10 after hatching.

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17–21 kDa) and isochymotrypsin-like enzymes (23–38 kDa) were identified. Amylases (38–47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus . Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

International audience; The aim of the present work was to evaluate to what extent variation in the activity of either trypsin or chymotrypsin could be a sensitive and reliable indicator of nutritional deficiencies during post-weaning stages in a marine fish. Sea bass Dicentrarchus labrax larvae obtained from two different spawns classified as high quality (HQ) or low quality (LQ) on the basis of their hatching variables, were used to evaluate if enzyme production could be conditioned by factors other than those related to the feeding regime. In addition, larvae in each group were either fed normally or received a ration reduced by 60%. Trypsin and chymotrypsin activities were separately measured in larvae at days 3 and 15 after weaning. Changes in mass and final survival were also recorded. Food limitation resulted in a lower final mass only in the HQ larvae. Survival was significantly higher in HQ larvae, but also in larvae suffering food restriction, irrespective of the quality of the spawn. In both groups of larvae, lower food availability was correlated to increased values of trypsin, and particularly, of chymotrypsin. Variations were high enough to support the usefulness of such enzymes as indicators of a modified nutritional status. It is suggested that the noticeable increase in the activity of both enzymes observed in the restrictedly fed larvae could compensate initial deficiencies and explain the good results in growth and survival obtained in LQ larvae.

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high α-amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.