Francisco Javier Moyano

Soybean and lupin seed meals as protein sources in diets for gilthead seabream (Sparus aurata) : nutritional and histological implications

The use of vegetable protein sources in diets for freshwater fish has been studied in more detail than for marine fish species. Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different vegetable protein sources, soybean and lupin seed meals. Mean feed intake and growth were not significantly influenced by type or level of plant protein in the diet. Feed utilization indexes such as feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the type of plant protein in the diet, although a general reduction of these values was observed with increased inclusion of soybean meal. Histological studies showed an increased deposition of lipid and decreased glycogen deposits in the liver with increased levels of dietary soybean meal. Protein digestibility coefficients for lupin seed meal diets were similar to the control and 10% higher than those for the soybean meal diets. A significant reduction in trypsin activity was observed in fish fed the lupin seed meal diets, and for soybean meal diets when the substitution level reached 30%. Diets including plant protein showed a higher peak of ammonia excretion rate, which appeared 2 h later than that of the fish meal diet. Highest values of dissolved ammonia were registered in fish fed a soybean meal-based diet. These results suggest that properly treated lupin meals could be an important alternative dietary protein source for gilthead seabream.

Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)

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.

Corn gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): Nutritional and histological implications

Abstract Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different protein sources, corn gluten (CGM) and meat and bone meals (MBM) in diets for juveniles gilthead seabream. Growth, feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the source of dietary protein, although higher values were observed with diets containing increasing levels of meat and bone meal. No differences were found in fish proximate composition at the end of the experiment. Histological studies of liver tissue showed no liver alteration in fish fed diets including increasing levels of corn gluten meal. On the contrary, an increased deposition of lipids, nuclei polarization and isolated necrotic focus were found in hepatocites of fish fed diets exceeding 20% of meat and bone protein. Apparent protein digestibility (ADC) in diets containing CGM showed similar results to those obtained with the control diet. However, a significant reduction in digestibility was observed when MBM was used as partial substitute of fish meal protein. A negative correlation between dietary ash content and protein digestibility was observed. Higher amounts of nitrogen were excreted as levels of CGM and MBM increased in the diets, being significantly higher in the case of 40% substitution either with CGM or MBM protein.

Digestive enzyme activity during larval development of the Senegal sole (Solea senegalensis)

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.

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.

Characterization of α-amylase activity in five species of Mediterranean sparid fishes (Sparidae, Teleostei)

Abstract 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.

Increase of the dietary n - 3/n - 6 fatty acid ratio and addition of phosphorus improves liver histological alterations induced by feeding diets containing soybean meal to gilthead seabream, Sparus aurata

Abstract In a previous study, several histological alterations were found in the liver of gilthead seabream fed with a diet containing 30% soybean meal (SBM). In the current study, SBM containing diets were supplemented with either potassium phosphate, zinc sulfate or phytase ( Aspergillus ficuum ), or increasing the dietary n −3/ n −6 fatty acids ratio to meet that of a fish meal based diet. Diet composition did not affect fish growth, feed efficiency (FE) or protein productive value (PER). Phosphorous supplementation significantly reduced hepatosomatic indexes (HSI), although it did not alter liver lipid content. Both phosphorous supplementation and correcting the dietary n −3/ n −6 fatty acid ratio significantly altered the lipid and protein content in fish muscle. Only these two treatments and principally the corrected dietary n −3/ n −6 fatty acid ratio, improved the liver histological alterations observed in fish fed with SBM based diets.

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

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.

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

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.

Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

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.