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Numerous studies evaluating the effects of the incorporation of microalgae in feeds have reported favourable impacts on different physiological aspects of aquacultured fish. Although productivity is the major goal in terms of profitability in fish farming, however, qualitative aspects are gaining the attention of producers, given the relevance of quality attributes related to organoleptic parameters, proximal composition, and shelf life on the commercial value of fish. Indeed, microalgae are acknowledged for their richness in substances with potential positive effects on all those quality attributes. In this context, this study assesses the effects of finishing diets enriched with the microalga Nannochloropsis gaditana, either crude or enzymatically hydrolysed, on several quality parameters of gilthead seabream (Sparus aurata) fillets. Two inclusion levels (2.5 and 5%) of raw and enzymatically hydrolysed microalgal biomass were incorporated into diets, plus a microalgae-free control diet, and a 42-d feeding trial was carried out on fish of commercial size (approx. 500 g body weight). The influence of the experimental diets on fish biometry, fillet quality parameters, and shelf life was evaluated. The results indicate, overall, that microalgae-enriched diets yielded favourable, dose-dependent effects on several objective quality parameters of fillets, namely, improved fatty acid profile, reduced microbial counts, enhanced lipid oxidative status, and improved textural and skin colour attributes. Although the enzymatic pre-treatment of the microalgal biomass was expected to impact positively its functional effects on all quality parameters, however, no general trend was observed.

The aim of this work was to evaluate the effects of the dietary inclusion of Arthrospira sp. enzyme hydrolysate on gilthead seabream (Sparus aurata) juveniles in a 128-day feeding trial. Algal hydrolysate was tested at low inclusion level, namely, 2 and 4%, against a control diet without Arthrospira sp. supplementation. At the end of the feeding trial, fish body weight was recorded for growth evaluation. No significant differences were found among the experimental groups regarding growth performance or nutrient utilization, despite the fact that those animals fed with diets enriched with Arthrospira displayed higher trypsin, chymotrypsin, and leucine aminopeptidase enzyme activities, compared to fish fed with control diet. The ultrastructural study of the intestinal mucosa also revealed increased microvilli length and absorptive capacity in fish fed with Arthrospira sp. diets, especially at 4% inclusion level. Arthrospira supplementation was also responsible for lower lipid oxidation in muscle tissue, as well as for remarkable colour differences in skin, compared to control animals. These results suggest, overall, that low dietary supplementation with this microalgal hydrolysate might improve not only the intestinal ultrastructure and functionality but also muscle pigmentation and antioxidant capacity of juvenile gilthead seabream.

This study evaluates the potential of different algae extracts (Crassiphycus corneus, Cc, Ulva ohnoi, Uo, Arthrospira platensis, Ap, Haematococcus pluvialis, Hp) as additives for the preservation of rainbow trout fillets. The extracts were prepared with different water to ethanol ratios from the four algae species. The highest ferric reducing antioxidant power (FRAP) was observed in Uo extracted in 80% ethanol. Ap aqueous extract also had considerable FRAP activity, in agreement with a high total phenolic content. Radical scavenging activity (DPPH) was higher in Cc 80% ethanol extract, in agreement with a high total carotenoid content. In fact, when the algae aqueous extracts were assayed on the fish fillets, their antioxidant activity exceeded that of ascorbic acid (ASC). All algae extracts delayed microbial growth and lipid oxidation processes in trout fillets throughout the cold storage period compared to controls, and also improved textural parameters, these effects being more evident for Ap and Hp. With respect to the color parameters, the Hp extract prevented the a* values (redness) from decreasing throughout cold storage, a key point when it comes to colored species, not least salmonids. On the other hand, the Ap extract was not as effective as the rest of treatments in avoiding a* and b* decrease throughout the storage period, and thereby the color parameters were impaired. The results obtained, together with the natural origin and the viability for large-scale cultivation, make algae extracts interesting fish preservative agents for the food industry.

This work evaluates the effects of the dietary inclusion of crude or hydrolysed Arthrospira platensis (Cyanobac- teria) biomass on growth, muscle composition, digestive functionality and immune activities in gilthead seab- ream (Sparus aurata) fry (20.32 mg mean body weight). A 40-day feeding trial was conducted, aimed at assessing four experimental diets that included 5 or 10% (w/w) A. platensis, either crude or hydrolysed, plus a microalgae- free diet as control batch. Overall, none of the dietary treatments caused negative impacts on fish growth, body composition, muscle fatty acid profile, or innate immune response. Thus, the dietary inclusion of both crude and hydrolysed A. platensis reduced significantly the oxidation of muscle lipids, especially when using hydrolysed biomass, regardless of the dietary inclusion level. In relation to digestive enzymes, significantly higher levels of trypsin, chymotrypsin and leucine aminopeptidase activities were measured in fish fed on A. platensis-supple- mented diets compared to control fish. In addition, within each inclusion level (5 or 10% w/w), those animals fed with diets that included the hydrolysed biomass yielded consistently higher digestive enzyme activities than those receiving the crude biomass. Microalgae dietary inclusion also induced favourable changes in fish gut morphology, according to the increase in microvilli length and diameter observed. This fact might well have contributed to reinforce the role of the intestinal mucosa as a protective barrier against microorganisms, as well as to enhance the absorptive capacity of the intestinal mucosa. Finally, 10% inclusion of microalgae hydrolysate enhanced lysozyme activity in liver, this fact suggesting improved protection against infectious diseases. In conclusion, the positive effects observed in fish fed with the diets including A. platensis up to 10% (not least the hydrolysed biomass) with regard to the different parameters assessed (digestive enzyme activities, intestinal epithelium ultrastructure, muscle lipid oxidation, and lysozyme activity) suggest the benefits of including this product in starter feeds for gilthead seabream fry., Evaluación de los efectos de las microalgas, incluidas en dietas de dorada, sobre el crecimiento, supervivencia y resistencia a infecciones., ALGAFISH, SI

A 90-d feeding trial was conducted in which five groups of gilthead seabream (11.96 g initial body weight) were fed with a microalgae-free diet (control group, C) or four diets containing the microalgae Nannochloropsis gaditana at two inclusion levels (2.5% or 5%), either raw (R2.5 and R5 batches) or cellulose-hydrolyzed (H2.5 and H5 batches), to study their effect on the body and muscle growth. At 40 days, the highest values of body length and weight were reached in R5 group, but at 64 and 90 days, these were reached in R2.5. However, feed conversion rate, specific growth, daily intake, and survival (100%) were similar in all the groups. The acquisition of a discoid body shape was accelerated depending on the inclusion level of N. gaditana in the diets. Moreover, H5 diet affected the fish geometric morphology compared to R5 diet. The white muscle transverse area was similar in all groups at 40 days, with the exception of H2.5 group, which showed the lowest area. At day 90, C and R2.5 displayed the highest muscle growth, attributable to increased hyperplasia in C, and higher hypertrophy in R2.5. However, the highest proportion of small and medium fibers was observed in R5 and H5, ALGAFISH, Evaluación de los efectos de las microalgas, incluidas en dietas de dorada, sobre el crecimiento, supervivencia y resistencia a infecciones., SI

Macroalgae are a promising source of a vast variety of bioactive compounds with potential interest in aquaculture. In addition to their sustainable and renewable nature, numerous evidences have been reported pointing to positive effects on fish growth, nutrient utilization, and disease resistance. Nevertheless, the effects on fish quality objective parameters have been scarcely explored. In this study we assessed the influence of Ulva ohnoi included at low level (5%) in a diet for Senegalese sole (Solea senegalensis) on several quality attributes of fish (proximate analysis, texture profile analysis, lipid oxidation, fatty acid profile, pH, water holding capacity, and colour parameters). A 270-d feeding trial was carried out, in which juvenile animals were fed with an Ulva-enriched diet only for 90 d out of the total 270-d study. This enabled to evaluate whether the possible effects on fish were transitory or persistent at the end of the assay. Besides modest effects on fish growth at early stages, the differences attributable to Ulva supplementation were mostly focused on the lipid fraction, texture, and colour parameters. Thus, Ulva diet reduced muscle total lipid content and favoured muscle selective retention of n-3 polyunsaturated fatty acids (PUFA), not least eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) acid, and also diminished lipid oxidation, especially after a period of frozen storage. In terms of consumers' acceptability, seaweed supplementation impacted favourably on fillet texture and colour attributes, namely by increasing lightness (L*) and yellowness (b*), whilst reducing redness (a*). These results are particularly remarkable taking into account that low inclusion level was considered (5%), and above all, that Ulva supplementation was interrupted after 90 d, but the effects mentioned persisted at least up to 270 d. These deferred long-term effects might well be of practical interest when it comes to modifying quality attributes of edible fish.

The combined effect of farm management practices, transport time, and ageing time on the electrophoretic changes of sarcoplasmic (SPP) and myofibrillar (MFP) protein fractions of goat kids was studied. A total of 64 suckling goat kids were withdrawn from two farms with &ldquo, high&rdquo, (GW) and &ldquo, low&rdquo, (DW) welfare-friendly management practices, and they were transported for 2 or 6 h immediately before slaughtering. Longissimus lumborum samples were obtained at 3, 8, and 21 days post-mortem, and muscle proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE. Both protein extracts displayed significant changes attributable to meat maturation. Managing conditions of kids in DW farms increased the post-mortem susceptibility of muscle proteins. Some MFP of Longissimus lumborum muscle, such as troponin T, as well as 26&ndash, 30 and 35&ndash, 37 kDa fractions were influenced significantly by deficient on-farm management, and therefore, these protein fragments might be considered as indicators of low-welfare on-farm management in goat kids.

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver&ndash, Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

The aim of this work was to evaluate two functional feeds for the gilthead seabream, Sparus aurata, containing low inclusion of two microalgae-based products (LB-GREENboost, LBGb; and LB-GUThealth, LBGh). Fish (12–13 g) were fed for 13 weeks a control diet or one of the four diets supplemented with both products at 0.5% or 1%. LBGb and LBGh did not affect specific growth rate or survival, but increased feed efficiency by decreasing feed intake and enlarging the intestines. LBGb increased hepatosomatic index and reduced cortisol levels in plasma, while both products lowered plasma lactate. Extensive metabolite and metabolic enzyme profiling revealed that microalgae supplementations, especially 1% LBGh: (i) decrease plasma lactate and increase hepatic glycogen, (ii) reduce hepatic gluconeogenesis, (iii) enhance hepatic lipogenic activity and lipid secretion, (iv) led fish to double triglyceride content in muscle and to stimulate its lipid oxidative capacity, and (v) increase the content of monounsaturated fatty acids and the omega-3 alpha-linolenic acid in muscle. This study demonstrates that both microalgae-based products are suited to improve feed efficiency and orchestrate significant changes in the intermediary metabolism in gilthead seabream juveniles.

Microalgae are a source of diverse high-value compounds, such as carotenoids and fatty acids, which have a potential application in aquafeeds. Some microalgae species present complex cell wall structures, which make them poorly digestible, thus limiting their use as a feed ingredient. Consequently, applying extracted compounds to aquafeeds instead of using the whole algal biomass is advantageous as this is expected to increase the bioavailability of these nutrients. The aim of this study was to evaluate the effect of the dietary inclusion (2%) of two extracts obtained from the microalga Nannochloropsis gaditana (one composed of saponifiable lipids and the other of non-saponifiable lipids), alone or in combination, on growth, muscle composition, skin color and lipid oxidation in juvenile gilthead seabream, Sparus aurata, following a 39-day trial. Overall, the inclusion of 2% of the saponifiable lipid extract did not affect the growth performance but fish muscle presented a lower percentage of docosahexaenoic acid (DHA) and a higher eicosapentaenoic acid (EPA) than that of the fish fed the diets lacking this lipidic fraction. Despite no effect being observed in fish growth performance, the inclusion of 2% of the non-saponifiable lipid extract enhanced the carotenoid content of the fish muscle, which prevented lipid oxidation, and modulated the skin pigmentation towards a yellow-greenish color. The present study confirms the success of applying both high-value microalgae lipidic extracts, alone or in combination, as feed additives for practical diets in juvenile gilthead seabream.

The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVβ) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme β-galactosidase (β-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme β-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against β-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (β-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.

The development of non-parenteral delivery systems allowing efficient intestinal absorption in fish is a current research topic drawing much interest. Different aspects would benefit from such oral delivery, especially those related to immunization, owing to the convenience of this route in terms of time saving, safety, and lack of stressful effects among others. In the last years, DNA vaccines are evidencing their suitability for fish immunization, although commercial preparations currently available require parenteral administration. In this regard, this work was aimed at assessing a chitosan-based oral delivery system for plasmid DNA (pDNA) in fish. Plasmid pCMVβ was encapsulated in chitosan nanoparticles with the aim of evaluating the ability of inert capsules to retain and protect pDNA from in vitro conditions mimicking the fish gut environment. Furthermore, the feasibility of in vivo oral gene delivery was also assessed in fish by administrating pDNA nanoparticles comparing oral and intramuscular routes. The results indicated that lacZ gene expression (β-galactosidase activity) could be detected in fish tissues following both oral and i.m. administration of pDNA-chitosan nanoparticles up to 60 days. However, organ distribution of the reporter gene expression was more evident after oral (β-gal activity measured in gut, liver and muscle) than after parenteral administration (mostly restricted to adjacent tissues). The results suggest that chitosan nanoparticles enabled efficient oral delivery of pDNA, followed by organ migration and further sustained expression of the reporter gene encoding for β-galactosidase. The intensity of such expression was similar to that obtained by intramuscular injection, a fact that might suppose valuable practical applications in terms of on-farm administration of DNA-based vaccines to fish.

Studies aimed to assess up to what extent farming and transport previous to slaughtering might affect physiology and meat quality in young goat kids are needed, with the ultimate purpose of promoting practices that minimize stress in these animals. In this regard the effects of on-farm management and transport duration on some physiological responses and meat quality parameters in goat kids were assessed. Two farms representing 'high' and 'low' welfare-friendly management practices were selected. In total, 32 suckling kids were withdrawn from each farm, transported by road for 2 or 6 h, and then slaughtered. Blood samples were collected both on-farm and in the slaughterhouse, and biochemistry, cell counts and haematocrit were determined. After slaughtering, carcass quality parameters were measured. Longissimus dorsi muscle was dissected and pH, colour parameters, water holding capacity and shear force were measured throughout 8-day ageing period. Results indicate that, regardless its duration, transport caused significant effects on some blood parameters suggesting stress in live animals, like glucose, cortisol or creatine kinase. Despite the marked stress status in animals, this condition was not decisively reflected on L. dorsi quality parameters, but some effects were observed regarding fat cover in carcasses and colour parameters. The results suggest that postmortem changes throughout ageing were more decisive in terms of meat quality than stressful management either on-farm or during transport.

This study assesses the potential of two tannins, tannic acid (TAN) and quebracho tannin (QUE), to preserve objective quality parameters of rainbow trout fillets throughout a 15-d cold storage period, applied either alone or combined with alginate coating. Untreated fillets (C), and a set of fillets treated with ascorbic acid (ASC) were also included. All the additives reduced microbial counts and lipid oxidation of fillets, being the effects of TAN and QUE at least comparable to those of ASC, the most widely used antioxidant for fresh fish. The results also indicate that alginate by itself reduced lipid oxidation at late stages of the storage period, and increased fillet lightness (L*) as well, but caused scarce effects on other parameters involved in fillet deterioration. However, alginate and additives applied together enhanced the effects of the additives alone, especially regarding microbial and lipid oxidative parameters, this enabling prolonged shelf life of fillets. These valuable properties of commercial tannins, together with their natural origin and large-scale availability, might well enable further applicability of these substances to fish fillets preservation, not least with regard to coloured species, such as rainbow trout.

Encapsulation efficiency of protein in alginate or a combination of alginate and chitosan hydrogels was studied with the aim of ascertaining the adequacy of these carriers to deliver exogenous protein to fish orally. Bovine serum albumin (BSA) was chosen as model protein, and sea bream (Sparus aurata L.) juveniles as in vivo fish model. A three step approach was followed, according to the next scheme: (i) evaluation of different encapsulation protocols in terms of their efficiency to retain BSA; (ii) assessment of the retaining capacity of capsules under in vitro systems, including those that simulate gastric and intestinal conditions, and (iii) in vivo assay, in which the fate of encapsulated BSA during its transit throughout the fish gut was assessed. The results indicate that a combination of alginate and chitosan polymers (30 g L−1 and 1 g L−1, respectively) yielded the best balance among encapsulation efficiency, BSA protection against gastrointestinal proteolytic enzymes, and further sustained release of protein in the gut lumen of gilthead sea bream.

Summary The aim of this work was to determine the seasonality of nutrient profiles of dusky grouper (Epinephelus marginatus Lowe, 1834) cultured in a cage to improve knowledge on its nutritional composition. Fish (n = 735) were reared in a circular polyethylene floating cage for a 19-month experimental period and provided a commercial pelleted feed once a day with a total daily ration of 1% wet body weight. Fish were sampled every 3–4 months, and biometric parameters as well as muscle composition were determined. The edible proportion of fish increased slightly with culture time. The flesh showed low lipid content, similar to those reported for wild grouper. Biometric indices, muscle proximate composition and fatty acid profiles were significantly affected by seasonality, that is lipids and monounsaturated fatty acids increased during summer, in parallel with the decline of polyunsaturated fatty acids.

Ultraviolet (UV) light (λ = 250 nm) was assessed to increase shelf life of sea bass ( Dicentrarchus labrax ). Fillets were exposed to different doses: i) 7.9 kJ/m 2 , designed as UV 8 , ii) 15.8 kJ/m 2 , UV 16 and iii) control, untreated (C). After the treatments, the fillets were stored in a cold-room (4 ± 1 °C) up to 11 days and sampled at 0, 2, 4, 7, 9 and 11 days. The UVL treatments significantly decreased microbial counts during the first 4 days post-mortem (dpm), independently of the doses, compared to C fillets. A delay in rigor process in both UV 8 and UV 16 fillets was observed, as well as consistent improvement of textural parameters (hardness, gumminess and chewiness) at 11 dpm. These findings correlate with the lower proteolysis in myofibrillar protein fraction observed in UV 16 fillets, confirmed by electrophoretic separation. On the other hand, UV treatments decreased collagen content and increased lipid oxidation in fillets. Industrial relevance Continuous ultraviolet light is an interesting technology for food preservation due to its ability on the destruction of microorganisms mainly used in fluid and vegetal sanitation but with a proven effect in animal origin food. The advantages of treatment with continuous ultraviolet light for the food industry could be the absence of negative effect on food quality observed with other preservation treatments. The results of the present study showed that UV treatment causes a clear inhibition of microbial growth up to 4-day cold storage, independently of the doses. A consistently higher hardness throughout cold storage and a protective action on myofibrillar protein degradation were also evidenced. Future studies must be performed to minimize the negative effect observed in the present study.

The effects of freezing and freezing-thawing cycles during cold storage have been studied in meagre (Argyrosomus regius) fillets. Fillets were subjected to three conservation protocols: fresh, freezing at –20°C, and repeated freezing-thawing cycles. Fresh fillets were stored (4°C, 15 days), and the same protocol was followed for freezing and freezing-thawing after the freezing period. Freezing and freezing-thawing fillets were softer and presented lower water holding capacity than fresh, mostly attributable to collagen solubilization, and partial myofibrillar protein degradation. Cold storage (4°C) during 15 days caused softening in fresh and frozen fillets attributable to myofibrillar protein hydrolysis. Freezing-thawing cycles increased proteolysis, this leading to unacceptable softening even from early stages of further cold storage, and this was also revealed by SDS-PAGE.

The rising costs of fishmeal worldwide promote great research effort aimed to find alternative and renewable ingredients for aquafeeds. In the last years considerable attention has been paid to the use of algae as a potential ingredient for fish feeds. The objective of this work was to evaluate the effects of the microalgae Scenedesmus almeriensis incorporated to diets intended to feed sea bream (Sparus aurata) juveniles in a 45-day trial. Growth, body composition, and intestinal functionality of fish were studied. Microalgae meal was tested in triplicate at different inclusion levels (0%, 12%, 20%, 25% and 39%). At the end of the trial, individual body weight was recorded for evaluation of growth and nutrient utilization, and biological samples were obtained for proximate analysis, determination of digestive enzyme activities, intestinal histology, and microvilli morphological evaluation. Results indicated that S. almeriensis did not cause negative effects on fish growth or nutrient utilization efficiency. Growth rate and protein utilization in fish fed on a diet with 20% Scenedesmus meal tended to be higher, although not significantly different from those fed on control diet. None of the treatments caused adverse effects on body composition. Fish fed 12% S. almeriensis showed higher trypsin than animals fed on control diet. Leucine-aminopeptidase and alkaline phosphatase activities showed a quadratic response with increasing levels of Scenedesmus biomass. Ultrastructural study revealed that the dietary inclusion of algal biomass, especially at 20% level, significantly increased the absorptive capacity of intestinal mucosa in both the anterior and posterior intestinal regions. This study confirmed the usefulness of S. almeriensis to partially replace fishmeal in practical diets for gilthead sea bream juveniles.

The influence of two preservation strategies (vacuum [V] and modified atmosphere [MA] ) on microbiological counts, lipid oxidation and color changes during cold storage of meagre (Argyrosomus regius) fillets was studied. Meagre fillets were stored in a cold room under aerobic (control, C), V and MA package systems. Samples were withdrawn at six sampling points throughout 15-day storage, and postmortem changes were assessed. Bacterial counts and lipid oxidation increased consistently with storage time, also changes in color parameters of fillet skin were observed, L* decreased, and a* and b* increased. V-stored fillets showed significantly lower mesophilic (P

The Senegalese sole, Solea senegalensis, is a flatfish with increasing interest in the European aquaculture. However, one of the main limitations found in this species is related to the scarce attraction to commercial aquafeeds, thus determining low feed intake in fish farms. The aim of this study was to evaluate four ingredients of marine origin (fish meal hydrolysate [FMH], polychaete meal [POM], mussel meal [MUM], and squid meal [SQM]) as potential attractants for juvenile Senegalese sole. Four experimental feeds containing 10% of the potential attractants, plus a control diet without these ingredients, were evaluated. A total of 150 fish were distributed into ten 40-L tanks (duplicate tanks per feed). A Latin square design was carried out, and hence all the experimental diets were offered to each tank in successive periods of 7d. Fish were fed twice a day (morning and afternoon), and the feeding behavior of the animals was assessed by direct observations. Behavioral pattern assessed was classified as distant orientation, near orientation, or continuation of intake. The acceptability index for each behavioral phase and experimental feeds used was established for each observation. No behavioral differences could be noticed between the morning and the afternoon feedings. FMH showed the greatest attractant potential than among the ingredients assessed, and consequently this ingredient yielded the higher feed intake rates. On the other hand, POM was not the best attractant for juvenile Senegalese sole. Marine aquaculture in southern European countries is mainly focused on few fish species, such as sea bream, Sparus aurata, and sea bass, Dicentrarchus labrax. Because of the high volume of production of these species, markets have begun to show saturation. The investigation of new species for aquaculture is a necessary strategy for the future with the aim of increasing farm profitability and expanding market opportunities (Dinis et al. 1999).

BACKGROUND This paper deals with the consequences of dietary restriction or complete starvation before slaughtering on the biochemical and textural characteristics of sea bass muscle. RESULTS Results showed that only severe feed restriction influenced negatively total body and individual organ weights, and these animals showed lower condition factor as well. Neither moderate feed restriction (up to 50% of the standard ration) kept for 30 days nor total starvation up to 12 days caused significant effects on fish weight and fillet yield. Muscle lipid content was lower in feed-restricted fish, although this parameter was not altered by starvation time. Differences between the two feeding strategies studied were observed in muscle textural and biochemical parameters, and the results point to an influence of the nutritional status on the post-mortem evolution of collagen and myofibrillar proteins, although firmness was not modified. CONCLUSIONS Moderate feed restriction prior to slaughtering could be advisable in sea bass culture, given that no detrimental effects on fish quality or fish performance were noticed, whereas substantial amounts of feed can be saved. © 2013 Society of Chemical Industry

Senegalese sole is one of the most promising fish species cultivated in the Southern European countries. This study was aimed at assessing the effects of microalgae biomass added to diets for Senegalese sole juveniles on fish growing and condition status. Three isoproteic (52%) and isolipidic (10%) were formulated containing 15% Tisochrysis lutea (TISO), Nannochloropsis gaditana (NAN), or Scenedesmus almeriensis (SCE) biomass, respectively. An experimental microalgae-free diet (CT) and a commercial diet (COM) were used as controls. Fish were fed at 3% of their body weight for 85 days. Final body weight of fish fed microalgae-supplemented diets did not differ from group fed CT diet. Fish-fed CT, TISO, NAN, and SCE showed higher growth performance and nutrient utilization figures than specimen-fed COM diet. The highest carcass lipid content was found in COM group (141 g kg−1), and no differences were observed in body protein content. Ash was significantly higher in TISO, NAN, and SCE groups compared to fish-fed CT. Muscle EPA and DHA contents were not modified owing to the different dietary treatments. The n3/n6 and EPA/DHA ratios in muscle were similar in all the experimental groups. The quantification of digestive proteolytic activities did not differ among experimental groups, although differences in the protease pattern in digestive extracts by zymography were revealed in those fish fed on COM diet. Both α-amylase activity in the intestinal lumen and leucine aminopeptidase in the intestinal tissue were significantly lower in COM fish. Specimens fed on SCE diet showed a higher leucine aminopeptidase activity associated to the intestinal tissue compared to NAN-fed fish (0.40 and 0.25 U g tissue−1, respectively). The ultrastructural study revealed that the dietary inclusion of algal biomass, especially T. lutea and N. gaditana, had a positive impact on the absorptive capacity of the intestinal mucosa. The highest values for the parameters microvilli length and microvilli absorption surface were observed in fish fed on NAN diet (1.99 μm and 45.93 μm2, respectively). Even though further studies aimed at optimizing commercial formulas for Senegalese sole are required prior to any large-scale practical utilization, the results obtained clearly suggest the potential of microalgae as dietary ingredients for this fish species.

In the present study, the nutritional value of Tetraselmis suecica and Tisochrysis lutea (previously known as Isochrysis aff galbana T-ISO strain) freeze-dried biomass for feeding Sparus aurata fry was evaluated. A total of 25,500 fry (3.7 mg body weight) were fed for 55 days on diets containing 5 and 10%(w/w) Tetraselmis or Tisochrysis, as well as on a microalgae-free diet. Fish fed 5 % Tetraselmis showed higher growth performance, nutrient utilization, and survival values than fish fed Tisochrysis. The use of microalgae significantly decreased the body lipid content in fry fed the highest microalgae level. Fry fed Tisochrysis-supplemented diets increased the DHA content in muscle, and consequently the EPA/DHA ratio decreased significantly, whatever dietary level considered. In general, digestive protease activities were not adversely affected by dietary microalgae inclusion, although slight variations were observed during fish development. Microalgae utilization causes a positive effect on intestinal mucosa ultrastructure owing to an increase of total enterocyte absorption surface that was observed in fish fed microalgae-supplemented diets. Cluster analysis of data separated clearly fish fed Tisochrysis-supplemented diets from the rest of experimental groups. This study confirms that Tetraselmis freeze-dried biomass can be used as dietary ingredient in started feeds for S. aurata fry, although an inclusion level of 5 % is recommended., SI

Four lots of sea bream were fed with different ration levels (R100, R75, R50, and R25, corresponding to 100, 75, 50 and 25%, respectively, of the standard ration fed in the fish farm), in order to assess the effects of a 30-day pre-slaughtering period of feed restriction on the textural characteristics of the edible muscle of farmed sea bream (Sparus aurata). Results showed that food restriction over 50% influenced negatively total body and individual organ weights. Fish in a good nutritional condition presented lower muscle pH and poorer muscle characteristics, showing soft texture and decreased water holding capacity (WHC). When the intake was moderately restricted (R75), a positive influence on pH and WHC of muscle was found. A 50% reduction of the ration size also delayed muscle softening during cold storage; whereas, a more severe restriction (R25) did not improve this parameter. Electrophoretic studies showed minor changes in the relative optical density of sarcoplasmic and myofibrillar protein fractions attributable to the different ration sizes assayed. These results suggest that the main contributing factor to the observed enhancement in muscle texture due to feed restriction is likely the decrease of lipid deposits in the structural components of the muscle.

This study assesses the effects of storage temperature on postmortem changes in textural parameters (firmness, water holding capacity), and their relationship with biochemical and electrophoretic determinations (muscle collagen content and solubility, amount and integrity of sarcoplasmic and myofibrillar proteins) in sea bream muscle. Storage at 1C resulted in a prolongation of the firmness compared with 4C, with no noticeable changes in water holding capacity. This delay in muscle softening at 1C could be partially explained by a limited proteolysis of some myofibrillar proteins, although inconsistent tendency was observed for other fractions. In contrast, the rate of muscle collagen degradation was higher at 4C compared with 1C, this becoming the main contributing factor to firmness losses. The great variety of protein fractions separated electrophoretically, and the relatively minor and contradictory changes observed suggest the unfeasibility of electrophoresis as a routine procedure for studying freshness in stored sea bream. PRACTICAL APPLICATIONS According to the results obtained, it is strongly recommended that the storage of sea bream (Sparus aurata) throughout the commercial chain is carried out at 1C, instead of the usual temperature of 4C. This modification clearly delays the process of muscle softening, thus improving objective quality in this species. The electrophoretic separation of muscle proteins has been previously proposed as a suitable procedure for assessing the freshness of fish during cold storage. However, the results obtained here indicate that the attempts aimed to the development of an electrophoretic procedure for detecting modifications in muscle proteins that could be correlated with flesh textural properties were unsuccessful. This is especially true if short-term deterioration is assessed.

Our objective was to determine if condensed tannin extract from quebracho trees (Schinopsis quebracho-colorado; red quebracho) could be used to reduce enteric methane emissions from cattle. The experiment was designed as a repeated 3 x 3 Latin square (4 squares) with 3 treatments (0, 1, and 2% of dietary DM as quebracho tannin extract) and 3 28-d periods. Six spayed Angus heifers (238 +/- 13.3 kg of initial BW) and 6 Angus steers (207 +/- 8.2 kg of initial BW) were each assigned to 2 squares. The measured condensed tannin content of the extract was 91%, and the basal diet contained 70% forage (DM basis). Feeding quebracho tannin extract had no effect on BW, ADG, or nutrient intakes. Furthermore, it had no effect on DM, energy, or fiber (ADF and NDF) digestibility, but apparent digestibility of CP decreased linearly (P < 0.001) by 5 and 15% with 1 and 2% quebracho tannin extract, respectively. There were no effects of quebracho tannin extract on methane emissions (g/d, g/kg of DM, % of GE intake, or % of DE intake). Feeding up to 2% of the dietary DM as quebracho tannin extract failed to reduce enteric methane emissions from growing cattle, although the protein-binding effect of the quebracho tannin extract was evident.

BACKGROUND: The impact of glyphosate on ruminal fermentation, selective pressure on ruminal bacteria and horizontal transfer of the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (epsps) to ruminal bacteria was studied using batch culture with glyphosate-tolerant (Roundup Ready®) canola meal as substrate. RESULTS: A glyphosate concentration × time interaction (P < 0.05) occurred when glyphosate (0–100 mmol L−1) was included in in vitro ruminal incubations with a diet containing 150 g kg−1 Roundup Ready® canola meal (Experiment 1). Glyphosate at 50 and 100 mmol L−1 inhibited fermentation. In Experiment 2, epsps fragments were detected in plant debris for up to 16 h of incubation using primer sets that amplified three fragments (62, 108 and 300 bp) of DNA spanning the transgenic construct. Persistence was affected by fragment size but not by glyphosate concentration (0, 10 or 60 mmol L−1). Extensive polymerase chain reaction assays provided no evidence of acquisition of epsps by feed- or fluid-associated bacteria during fermentation. A glyphosate concentration × time interaction (P < 0.05) was observed for all fermentation parameters measured, and glyphosate caused a general inhibition of fermentation. CONCLUSION: The presence of glyphosate did not increase selective pressure for gene transfer of DNA encoding glyphosate resistance from Roundup Ready® canola meal to ruminal bacteria. Copyright © 2007 Society of Chemical Industry

Current understanding of the effect of moderate amounts of tannins on the utilization of protein contained in forages indicates beneficial effects when fed to ruminants. Nevertheless, there is little knowledge about the effects of tannins on the ruminal hydrolysis of starch-rich concentrates. Modulation of the hydrolysis of starch may reduce the occurrence of metabolic disorders, such as acidosis, in ruminants consuming high-grain diets. The effects of commercial tannic acid (hydrolysable tannins) and quebracho tannins (condensed tannins) (50 g kg−1 DM) on the in vitro fermentation of ground wheat and corn grains by mixed ruminal bacteria was examined. The architecture and chemical composition of wheat and corn endosperms might explain differences in fermentation rates, as well as in response to similar concentrations of tannins. Regardless of the source of tannin, microbial fermentation was inhibited in both grains, as demonstrated by a decline in gas production, DM disappearance, volatile fatty acids and ammonia production. However, these effects were more pronounced for wheat than corn grain, mostly during the initial stages of the incubation. Scanning electron microscopy revealed that both sources of tannins inhibited the microbial hydrolysis of the endosperm protein matrix. Tannins did not prevent bacterial attachment to starch granules, but starch hydrolysis was slowed indirectly as a result of a tannin-mediated reduction in the degradation of the surrounding protein matrix. Tannins are likely to be more effective at modulating the rate of starch digestion in grains that possess a readily degradable protein matrix. Copyright © 2006 Society of Chemical Industry

To identify simple screening tools for selecting condensed tannin (CT)-containing forages as candidate sources for further study, CT were isolated from nine legumes, and their molecular weights (MW), chromophore production, capacity to precipitate bovine serum albumin (BSA) and Fraction 1 protein (Rubisco) isolated from alfalfa, and inhibition of filter paper digestion were compared. Sources were as follows: leaves of sericea lespedeza (Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), and sainfoin (Onobrychis viciifolia Scop.); stems of hedysarum (Hedysarum alpinum L.); seeds of alfalfa (Medicago sativa L.); and whole plants of birdsfoot trefoil (Lotus corniculatus var. corniculatus L.) and three varieties of big trefoil (Lotus pedunculatus Cav.), viz., Lotus uliginosus Schkuhr, L. uliginosus var. glabriusculus, and L. uliginosus var. villosus. Molecular weights and sizes (degrees of polymerization) of the CT varied considerably within and among plant species. Average MW ranged from 3036 Da (crown vetch) to 7143 Da (lespedeza). All CT exhibited greater capacity (w/w basis) to bind alfalfa Rubisco than BSA. Relative astringencies (microg CT required to precipitate 1 mg protein) against BSA ranged from 262.5 for CT from lespedeza to 435.5 for CT from L. corniculatus, and against Rubisco, from 49.6 (sainfoin) to 108.2 (alfalfa seed). Including CT at 300 microg/ml in cultures of Fibrobacter succinogenes reduced digestion of cellulose filter paper by 19.8% (sainfoin) to 92.4% (crown vetch) and increased the specific activity of cell-associated endoglucanase. There were no correlations between inhibitory effects of CT on filter paper digestion and (1) chromophore formation during CT assay by butanol-HCl, vanillin-HCl, or H2SO4; (2) precipitation of BSA or alfalfa Rubisco; and (3) MW of CT. The most inhibitory CT for cellulose digestion included those with broad and with narrow MW distributions. Sainfoin was the most desirable source of CT, as it had the highest capacity to bind alfalfa protein and was least inhibitory to cellulose digestion by F. succinogenes. This study suggests that these properties are not easily defined via chemical means, and that biological assays using rumen bacteria may help identify those CT with properties of nutritional interest.

The inefficiency of starch utilisation by ruminants fed readily fermentable cereal grains, such as barley, decreases the metabolic potential of such feeds and can cause serious dysfunctions related to acidosis or pre-acidosis status in animals with higher energy requirements. The rate and extent of ruminal disappearance of grain dry matter (DM) are determined largely by the morphological features of the seed endosperm, but the presence of polyphenols may also constitute a limiting factor in ruminal hydrolysis. To assess the impact of tannins on the rate and extent of ruminal fermentation of barley grain, commercial grade tannic acid (TA) was included at 0, 1.0, 2.5 or 5.0% (wt/wt, DM basis) with ground barley grain in incubationin situ, and disappearance of DM and crude protein were monitored over 48h. A dose-dependent effect of TA on in situ degradation of barley meal was observed; significance was attained (p < 0.05) at the 5% treatment level. Scanning electron microscopy revealed limited microbial hydrolysis of endosperm cell walls in TA-treated samples, although TA did not prevent microbial attachment to or hydrolysis of starch granules. Tannins may be effective for slowing ruminal disappearance of barley to improve starch utilisation by ruminants.  2005 Society of Chemical Industry

The inefficiency of protein utilisation by ruminants fed protein concentrates (based on legume meals) causes serious economic loss and environmental damage owing to their rapid hydrolysis and deamination in the rumen. Thus efforts aimed at slowing the ruminal fermentation of such feeds are needed, and recent studies have observed potentially positive effects of tannins on ruminant nutrition under certain circumstances. Tannins are a complex group of naturally occurring plant polyphenols characterised by their ability to bind with proteins. This property of tannins is considered responsible for the decreased ruminal digestibility of forages both in vivo and in vitro. Under that perspective, commercial tannic acid was added at three proportions (10, 25 and 50gkg −1 on a dry matter basis) to four different legume meals (horse bean, kidney bean, soybean and pea), and the effect on in situdry matter and crude protein ruminal disappearance was assessed. The results confirmed the dose-dependent (although not persistent after 48h) slowing of in situ digestibility, this effect being significant at the highest tannin treatment when compared with untreated samples. Scanning electron microscopy revealed that soybean seed endosperm cell walls were protected from digestion by the ruminal microbiota, while the digestion of starch granules was relatively unaffected by tannic acid. Electrophoresis of the protein fractions confirmed the lower digestibility of tannin-treated seeds as well as the relative lack of alteration of the electrophoretic profile of individual proteins. Implications for the digestion of concentrates in ruminants are discussed.  2004 Society of Chemical Industry

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

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 influence of two preservation strategies (vacuum package and modified atmosphere package) on the post-mortem changes of textural parameters, pH, water holding capacity, sarcoplasmic and myofibrillar proteins, and collagen content of meagre ( Argyrosomus regius) fillets was studied. Fillets were stored in a cold room in aerobic (control, C), vacuum (V) and modified atmosphere (MA) package. Samples were withdrawn at six sampling points throughout 15-day storage, and post-mortem changes were assessed. The textural parameters were significantly enhanced in V and MA compared to C. Both V and MA treatments reduced the intensity of a group of myofibrillar protein fractions (140–195 kDa) and increased insoluble collagen compared to C. Consequently, the post-mortem flesh softening in C was attributed to increased proteolysis in both intracellular and extracellular structural proteins. The preservation of the textural and biochemical characteristics of meagre fillets subjected to V and MA treatments makes these two treatments highly recommendable for the commercialization of meagre fillets.

Studies of tannin concentrations in forage plant species in arid south-eastern Spain were conducted to assess the relationship between tannins and use of plants by domestic livestock. Tannin concentrations were measured in the most important forage plants (28 shrub species and 12 herbaceous species) in each of the four seasons of the year. Twenty-five percent of the shrubs were rich in tannins. The shrubs, Helianthemum almeriensis and Dorycnium pentapyllum , had the highest concentrations of tannins (4·5% and 4·0% in dry matter, respectively). With the exception of Sedum sediforme , herbaceous species had no tannins.

In the present study, amylase activity existing in the gut of different species of sparid fish was measured and characterized. The study was conducted on five species of Mediterranean sparids, (F. Sparidae), some of which coexist in the same coastal waters; Pagrus pagrus, Pagellus erytrhinus, P. bogaraveo, Boops boops and Diplodus annularis. Main differences were found in the optimal pH for amylase activity as well as in sensitivity to temperature, with resistance to heating very low in B. boops and very high in D. annularis. PAGE zymograms revealed the presence of two isoforms in all the species, while the analysis of extracts using IEF showed a range from one to three active bands. It is suggested that differences in amylase activity are related to variations in feeding habits of the studied fish, and could also represent an ecological advantage for those species showing a more diversified amylase equipment.

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.

The presence of digestive carbohydrases was determined in seabream intestines, and the main activity identified was amylase. Optimum activity for this enzyme was found at pH 8.0 at 40 °C. Amylase activity was highly sensitive to extreme pH, and temperatures exceeding 50 °C. The use of SDS–PAGE zymograms allowed identification of amylase in the form of a high molecular mass fraction exceeding 100 kDa. Results confirm the existence of a well developed amylase equipment in this species which supports the possibility of increasing the amount of carbohydrates in the formulation of its commercial feeds.

An improved method, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

This paper deals with the consequences of dietary restriction or complete starvation before slaughtering on the biochemical and textural characteristics of sea bass muscle.Results showed that only severe feed restriction influenced negatively total body and individual organ weights, and these animals showed lower condition factor as well. Neither moderate feed restriction (up to 50% of the standard ration) kept for 30 days nor total starvation up to 12 days caused significant effects on fish weight and fillet yield. Muscle lipid content was lower in feed-restricted fish, although this parameter was not altered by starvation time. Differences between the two feeding strategies studied were observed in muscle textural and biochemical parameters, and the results point to an influence of the nutritional status on the post-mortem evolution of collagen and myofibrillar proteins, although firmness was not modified.Moderate feed restriction prior to slaughtering could be advisable in sea bass culture, given that no detrimental effects on fish quality or fish performance were noticed, whereas substantial amounts of feed can be saved.

The aim of this study was to assess the potential effects of replacing part of the dietary protein by lipid or carbohydrate on the characteristics of dentex muscle. Dentex were hand-feed four isoenergetic diets (22.6 MJ kg − 1 gross energy) with protein:lipid:carbohydrate ratios 43/16/28 (D1); 43/24/4 (D2); 38/19/28 (D3), and 38/24/13 (D4). At the end of the feeding period, biometric parameters, textural properties of muscle, as well as protein, lipid and collagen contents, and electrophoretic protein profiles of this tissue were studied. The decrease in dietary protein content improved textural parameters (increased firmness and water holding capacity). Within each dietary protein level, diets with high carbohydrate and low lipid contents resulted in lower values of muscle firmness than diets with low carbohydrate and high lipid contents. This increase in firmness matched with a lower muscle lipid and higher muscle collagen contents. SDS-PAGE separation of muscle extracts showed a higher relative contribution of myosin to total optical density in muscle from those animals fed lower dietary protein, although no significant difference in fish texture could be attributable to this finding. Results indicate a favourable effect of replacing protein by alternative energy sources (lipids or carbohydrates) on the rheological properties of muscle in this species, this improvement was more noticeable when protein was replaced by lipid than by carbohydrate.

A novel end-point fluorimetric procedure based on the use of rhodamine-110-labeled specific substrate was developed to determine trypsin activities in biological samples. We evaluated the ability of trichloroacetic acid and acetic acid to stop the enzymatic reaction without hindering the detection of the fluorescence of rhodamine-110 released into the reaction mixture from the specific substrate (CBZ-L-alanyl-L-arginine)(2)-rhodamine-110. Trichloroacetic acid decreased markedly the fluorescence of rhodamine-110, even at low concentrations. On the other hand, the addition of 50 mmol/l acetic acid inactivated efficiently trypsin activity, causing minor effects on rhodamine-110 fluorescence. The proposed procedure was more sensitive than the spectrophotometric end-point method using N-alpha-benzoyl-DL-arginine-p-nitroanilide as substrate. The possibility of carrying out end-point fluorimetric assays improves the performance of monocell fluorimeters by setting specific conditions optimal for each enzyme activity independently of the fluorimeter. This method also allows replicate assays to be conducted simultaneously, resulting in considerable time saving and in increased performance of low-cost equipment.

Periploca laevigata is an abundant and palatable tannin-containing shrub present in the south-east coastal area of Spain. Sheep and goats usually consume this species, which represents 7·2% of total dry matter (DM) intake per year. Periploca laevigata is an important grazing species in the dry season, despite its high tannin content. The objective of the present work was to examine the tannin level of P. laevigata in relation to the extent of utilization by small ruminants, as well as in response to simulated browsing. Periploca sprouts were collected in three areas from plants exhibiting either medium (0·51 animals/HA), low (0·34 animals/HA) or no (0 animals/HA) browsing by small ruminants. These sprouts were also collected in plants with simulated herbivory twice (in two successive months), once, and with no simulated herbivory. A greater concentration of tannins (p

This study evaluates the in vitro protein hydrolysis of four microalgae (Tisochrysis lutea, Nannochloropsis gaditana, Tetraselmis suecica and Scenedesmus almeriensis) by intestinal proteases of gilthead sea bream (Sparus aurata) and Senegalese sole (Solea senegalensis). The hydrolysis of protein was monitored at different sampling times by electrophoretic techniques, and the quantification of the free amino acids released by proteases. Overall, S. aurata or S. senegalensis proteases hydrolysed microalgae protein in a similar way. The highest hydrolysis values (coefficient of protein degradation, CPD > 70%) were obtained for Tisochrysis and Nannochloropsis biomasses, which showed a progressive and almost complete proteolysis at the end of the in vitro assay. Tetraselmis and Scenedesmus protein was also hydrolysed, but SDS-PAGE revealed that two protein fractions remained virtually intact at the end of the in vitro assay. The final amount of free amino acids released in vitro by the fish digestive enzymes ranged from 9 to 25 g 100 g protein−1. A linear relationship between CPD and the amount of free amino acids released was found, a fact that suggests that microalgae protein is hydrolysed efficiently by the digestive proteases of both fish species. The present study provides information about the protein availability from selected microalgae, which will aid in the initial evaluation of the microalgae as potential protein sources in feeds of two important farmed fish species.