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Abstract

Pseudoplatystoma punctifer posee un gran potencial para la acuicultura, pero la alta tasa de canibalismo existente durante su etapa temprana de vida dificulta por ahora su cultivo a gran escala. Una nutrición adecuada reduce significativamente la incidencia de canibalismo y el conocimiento de la ontogenia funcional del sistema digestivo es esencial para adaptar el protocolo de alimentación a las capacidades digestivas de esta especie y así aumentar la producción de semilla. Por tanto, el objetivo del presente estudio fue analizar la expresión de los principales genes implicados en la digestión (amilasa, tripsina, quimotripsina, pepsinógeno, fosfolipasa, lipoproteína lipasa y neuropéptido Y) durante el desarrollo larvario y juvenil temprano de P. punctifer. Los resultados mostraron que esta especie cuenta con la maquinaria enzimática necesaria para digerir el alimento exógeno antes de la primera alimentación (4 dpf). La expresión de los genes siguió el perfil común de una especie carnívora con un aumento significativo en la expresión de pepsinógeno a partir de los 10 dpf para pasar de una digestión proteica básica en el intestino (tripsina, quimotripsina) a una digestión proteica ácida más eficiente en el estómago (pepsina). Por tanto, P. punctifer está preparada para ser destetada con dietas complejas a partir de 10 dpf. Los resultados también mostraron que el cambio de dieta durante la etapa juvenil moduló la actividad enzimática a nivel transcripcional, por lo que el análisis de la expresión de los precursores enzimáticos digestivos será de utilidad en estudios nutricionales dirigidos a establecer la composición nutricional óptima para esta especie.

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A lipase was purified from the extract of the delipidated powder of red sea bream hepatopancreas to nar homogeneity by fractional precipitation with ammonium sulfate and sequential chromatography on first anion-exchange-, hydrophobic- and second anion-exchange columns followed by gel filtration and anion-exchange HPLC. The final enzyme preparation showed a single band with an apparent molecular mass of approx. 64 kDa by sodium dodecyl sulfate-polyacrylamid e gel electrophoresis. The purified enzyme had a pH optimum in the range of pH 7.0–9.0. Using -nitrophenyl myristate or triolein as a substrate, the enzyme required the presence of sodium taurocholate or sodium cholate for its activity. No activity was observed in the presence of sodium deoxycholate. The enzyme preferentially hydrolyzed ethyl esters of polyunsaturated fatty acid, such as arachidonic acid and eicosapentaenoic acid which were resistant to porcine pancreatic lipase. These results strongly suggest that the enzyme purified from the hepatopancreas of red sea bream is homologous to mammalian bile salt-activated lipase.
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The fish larval stage is a critical step since only those specimens that survive will reach the adult stage in future. Knowledge related to fish larval nutritional requirements and digestive enzymes capacity is still scarce, although necessary to obtain satisfactory survival and growth rates. Trypsinogen is the precursor of trypsin, the main proteolytic enzyme acting during the early larval stage. Bile salt-activated lipase (BAL) is a multi-substrate digestive enzyme that hydrolyzes carboxyl ester bonds of acylglycerols, cholesterol esters and fat-soluble vitamin esters. The goal of this study was to determine the pattern of trypsinogen and BAL expression during larval development in red porgy (Pagrus pagrus, Pisces, Sparidae), reared under standard conditions to provide the basis for future experiments testing the possible transcriptional regulation for this enzyme under different nutritional conditions. Thus, partial cDNAs for trypsinogen and BAL from red porgy were isolated. The putative aminoacid sequences obtained for both precursors showed around 80% identity to other fish sequences from GenBank database. Trypsinogen and BAL were expressed from hatching and specifically located in the exocrine pancreas, revealed by in situ hybridization. The present study shows that this species is being prepared for protein and lipid digestion before exogenous feeding starts, exhibiting an ontogenetically programmed pattern for trypsinogen and BAL expression during the yolk-sac stage.
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Specific activities and mRNA levels of trypsin and amylase were studied in sea bass larvae. From day 20 to day 30, Dicentrarchus labrax were fed two rations of one day old Artemia: satiation (LP) and one-eighth of the satiation ration (LP/8) or two isoenergetic compound diets that varied in protein (30 and 60%) and carbohydrate (37 and 7%) content (FP30 and FP60 respectively). Trypsin mRNA levels and specific activities were mainly influenced by the nature of dietary protein and the Artemia ration. By using fish meal as protein source, dietary protein concentration did not affect either mRNA level nor specific activity of trypsin. These results suggested that the trypsin synthesis was not affected at a transcriptional level by the protein ration, i.e., Artemia ration. Decrease in amylase mRNA observed from day 29 in the four dietary groups suggested that this decrease in amylase expression is genetically programmed during sea bass larvae development. Nevertheless, the composition and the quantity of the diet influenced the amylase specific activities revealing primarily translational regulation of amylase. This study shows for the first time that the molecular mechanisms which control the dietary adaptation of trypsin and amylase an independently regulated, age-dependent and influenced by the composition and the quantity of the diet.
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Assessing the activity of digestive enzymes is a common procedure in many biological, physiological and nutritional studies. After reviewing the available literature on fish digestive tract maturation and enzymatic activity (pancreatic and intestinal enzymes) published between 1994 and 2017, authors detected some possible methodological and/or interpretative inconsistencies in this kind of studies, and concluded that special attention should be paid on: i) the time of conservation of frozen samples prior their analysis, ii) the proper purification of the brush border of enterocytes by a double centrifugation step (Crane et al., 1979) when authors want to evaluate the activity of intestinal brush border enzymes in order to avoid the overestimation, particularly of alkaline phosphatase (AP), because it is present in other tissues; iii) the use of the proper reaction conditions at the normal range of values in terms of ions, temperature and intestinal alkalinity for the species of interest, and AP unit calculation. The implementation of these recommendations will promote the standardization of actual analytical procedures, as well as improve the reliability of comparative studies between different fish species or rearing procedures.
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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.
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The South American continent is known for its high production and exports in fisheries and aquaculture, but has not reached its full potential in fish farming. The latest data on fish production in Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Peru, Uruguay and Venezuela were explored in this review. Aspects of biology, production, market and health of the species most produced in South America are described in detail. These species include the round fish (Colossoma spp., Piaractus spp. and hybrids) and catfish (Pseudoplatystoma spp. and hybrids), in addition to the promising pirarucu Arapaima gigas, yellowtail tetra Astyanax altiparanae and silver catfish Rhamdia quelen. Among the countries mentioned, Chile and Brazil are two of the largest intensive fish producers in the world. Chile relies primarily on marine fish, whereas Brazil is prominent for continental production. Special emphasis is given to the black pacu Colossoma macropomum because it is a commonly farmed fish in South American countries and offers several desirable productive characteristics (domesticated, omnivorous and easy to reproduce). Furthermore, this fish has the greatest potential to compete economically with tilapia production in South America. The production of native fish is currently overtaking the production of exotic species in some countries, which is considered a milestone for South American aquaculture. Regarding diseases, the main pathogens are similar to those observed throughout the world, such as Ichthyophthirius multifiliis, different species of monogeneans and trichodinids, and the bacteria Aeromonas hydrophila. Local pathogens, such as the parasites Perulernaea gamitanae and Goezia spinulosa, are also concerning.
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Aquaculture is the main vector for introduction of non-native species in Brazil and around the world. Despite the potentially serious and irreversible ecological impacts caused by non-native species, they continue to be in many cases the preferred option in aquaculture farms, of which the recent plans of aquaculture expansion promoted by the Brazilian Government are an emblematic example. In this study, we present a survey of publicly available information on aquaculture parks to be installed across the Brazilian territory, with emphasis on species status as native or non-native, and discuss the implications for the conservation of aquatic biodiversity. One hundred and thirty-nine aquaculture parks (APs), with a total of 1556 sites covering 941.38 hectares, have been called for bids. Among these, 122 APs will contain at least one non-native species, and 68 APs will be based exclusively on their cultivation. A predictable consequence is the enhancement of propagule pressure in surrounding aquatic ecosystems, increasing the risk of non-native species establishment or persistence, which will likely intensify the environmental impacts already in course in four major river basins and along the Brazilian coast. These impacts will add up to more direct effects of aquaculture farming – for example elevated input of nutrients and organic matter – and include changes in habitat and water quality, spread of diseases, biotic homogenization, loss of population viability resulting from hybridization and outbreeding depression, and the local extirpation of native species. © 2016 Wiley Publishing Asia Pty Ltd
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The aim of the study was to evaluate the influence of different dietary protein and lipid levels and their ratios on larval growth, survival and the incidence of cannibalism in Pseudoplatystoma punctifer. Larvae were raised in a recirculation system from 3 to 26 days post-fertilization (dpf) (2–25 days post hatching, dph) at an initial density of 40 larvae L−1, 27.8 ± 0.65°C and 0L : 24D photoperiod. Larvae were fed from 4 to 12 dpf with Artemia nauplii and weaned onto four different compound diets from 13 dpf within 3 days, then fed exclusively with these diets until 26 dpf. These diets contained 30 : 15, 30 : 10, 45 : 15 or 45 : 10 protein : lipid (P : L) (in % of dry matter) levels. A control group was fed Artemia nauplii until 17 dpf and weaned thereafter with the 45P : 10L compound diet. The experiment was carried out in triplicate. Results showed higher growth and survival rates and lower incidence of cannibalism in the group fed the 45P : 15L diet than in the other treatments. Differences in larval survival and growth performance were associated with the higher protein and lipid content rather than the protein : lipid ratio of this diet. When comparing diets with the same protein level, the increase in dietary lipid led to an improvement in growth, suggesting that energy from lipids spares protein for growth in P. punctifer fingerlings. An Artemia feeding period longer than 12 dpf did not improve larval growth or survival.
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This study aimed at describing the normal bony skeleton of Pseudoplatystoma punctifer juveniles to use as a reference when assessing the adequacy of nutritional and environmental conditions in experimental rearing during the early developmental stages and to provide a baseline for characterizing skeletal anomalies that might appear in rearing trials with this species. Fertilized eggs and newly hatched P. punctifer larvae were incubated at 27.8 ± 0.4°C in two 60-L tanks (50-L water volume) connected to a clear water recirculating system. At 3 days post fertilization – dpf (2 days post hatching – dph) larvae were reared in three 40-L tanks (30-L water volume; initial n = 2700 larvae per tank; 28.3 ± 0.4°C, pH 6.9 ± 0.2, dissolved oxygen 8.2 ± 0.5 mg L−1, N–NO2 0.04 ± 0.02 mg L−1, N–NH4 0.14 ± 0.05 mg L−1; 0L:24D photoperiod) and fed as follows: non-enriched Artemia spp. nauplii from 4 to 21 dpf (3–20 dph) and a commercial compound diet from 18 dpf onwards. Pseudoplatystoma punctifer juveniles (23.2 ± 5.5 mm standard length, SL, n = 58) were stained with alizarin red and their skeletal structures analysed and identified under stereoscope. Pseudoplatystoma punctifer presents an osseous skeleton typical of catfishes, consisting of a broad and depressed skull containing small eyes, 43–44 vertebrae (44 being the most frequent), a caudal fin complex composed of one epural, five hypurals, one parhypural and two hypurapophyses, dorsal and pectoral fins with spines and anal and adipose fins. The observed occurrence of several skeletal anomalies indicates that the rearing conditions might have been suboptimal.
Article
Fish larvae are astonishing organisms. In spite of being considered the smallest free-living vertebrates that hatch with a very simple digestive system, they show a remarkable efficiency with regards to vital processes like feeding behavior (prey detection, capture and ingestion) and food digestion (enzymatic digestion and neuroendocrine control of the digestive system). Thus, some fish larvae can achieve growth rates close to 100% per day. Knowledge of the histological and morphological differentiation of the digestive system coupled with functional studies, including the digestion and absorption of nutrients and their neuronal and hormonal regulation is essential for understanding the digestive and nutritional physiology of fish larvae. The basic mechanisms of sensorial and digestive organ development are similar among teleosts, although the timings of ontogenetic development can vary considerably. The time of organ development and its associated physiologicalfunctions are affected by the life history pattern of each species (precocial vs. altricial) and by anumber of abiotic and biotic factors, such as water temperature, foodavailability and composition during early life stages. These factors determine the nutritional and physiological performances of fish larvae and therefore, their ability to deal with challenges during their subsequent life. The enzymes required for digestion of the main nutrients appear early in the development of most fish species, being proteases the most diversified and active ones during the initial stages. As a general trend, enzyme activities increase with age, with the exception of those involved in the intestinal absorption of macromolecules (i.e. cytosolic proteases). The activity of digestive enzymes is affected by a number of factors, but most greatly by variations in the amount and composition of food, as well as by the feeding pattern. Compared to digestion, much less is known on the mechanisms that regulate feeding in fish larvae and how these change during ontogeny. Studies performed mostly on adult teleosts appear to indicate the involvement of similar and well conserved central and peripheral neuropeptides and hormone-like peptides potentially regulating feeding and digestive processes but functional studies on such complex systems are still practically inexistent. This chapter aims to review recent literature on fish larvae nutritional physiology concerning the morphological structures, biochemical, physiological and neuro-hormonal mechanisms that regulate food capture and ingestion and digestion in fish larvae, in order to identify gaps in scientific knowledge that should be bridged for a better understanding of the feeding and digestive processes taking place in fish larvae.
Article
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Article
The histological development of the digestive tract and accessory organs of Nieuhofii's walking catfish, Clarias nieuhofii from hatching until 46 dah (day after hatching) was described using light microscopy. The mucosubstances and the liver glycogen were histochemical studied. At hatching, the digestive tract of C. nieuhofii was composed of a straight tube lining with the simple low columnar epithelium. Undifferentiated liver and pancreas cells were found just after hatching for 36 and 48 hrs, respectively and the glycogen in hepatocytes were observed at 3 dah. The opening of mouth and anus occurred at 2 dah, with distinct structure of esophagus, stomach, and intestine. By 3 dah, the buccopharygeal tooth bud was found coinciding with the stratification of esophageal epithelium with acid mucosubstance. At 5 dah, the yolk sac was not morphologically noticeable and yolk completely depleted by dah 7, indicating the end of endogenous feeding, while the onset of endo-exogenous feeding of C. nieuhofii was between 4-7 dah. By 4 dah, the first gastric gland appeared in mucosa of stomach and from this moment onwards several rapid histological changes of the digestive tract occurred in term of increasing in size of organ and number of cell structure with fish age to the end of investigation (46 dah). This study provides a basis for the optimization of formulated feeding during larval culture of this species. Background The Nieuhofii's walking catfish, Clarias nieuhofii is the freshwater catfish in the family Clariidae, order Siluriformes. Due to its tender flesh and good flavor, C. nieuhofii has been targeted to be a commercially important fishery as human food, as well as to increase production of C. nieuhofii fingerling for stocking to natural waters through aquaculture as the population of this catfish in nature is being decline. Basically, rearing of fish larvae is an extremely important issue from the commercial point of view, since it is the only way of successful reproduction of fish. The process of yolk sac resorption and exogenous initial feeding is a critical period as from this moment onwards, coinciding with the morphological changes of digestive tract, this transition is important as a source of larval growth and survival [1]. Histological analysis is essential for the accurate determination of the functional relationship between feeding and absorption of nutrients at the time of start exogenous feeding through fish larval life [2]. As the basic knowledge of development in digestive system of C. nieuhofii is limited, and with the marked diversity in morphology and function of digestive tract in fishes, thus the purpose of this study was to describe the ontogenic development of the digestive tract in this species for the use of improving management in rearing conditions and to provide a basis for the optimization of formulated feeding during larval culture.
Article
Despite considerable progress in recent years, many questions regarding fish larval nutrition remain largely unanswered, and several research avenues remain open. A holistic understanding of the supply line of nutrients is important for developing diets for use in larval culture and for the adaptation of rearing conditions that meet the larval requirements for the optimal presentation of food organisms and/or microdiets. The aim of the present review is to revise the state of the art and to pinpoint the gaps in knowledge regarding larval nutritional requirements, the nutritional value of live feeds and challenges and opportunities in the development of formulated larval diets.
Article
The digestive physiology of butter catfish was studied by assessing the activity of different pancreatic (trypsin, chymotrypsin, alpha-amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase) enzymes from hatching until the juvenile stage (30 dph). Larvae were reared at 27 degrees C and fed with Artemia nauplii from 2 days post hatching (dph) until 10 dph, from 7-10 dph with Artemia nauplii and zooplankton (Cyclopoida) and from 10 dph onwards only with zooplankton. The assessment of the activity of digestive enzymes showed that enzymes involved in the digestion of proteins, lipids and carbohydrates were present in butter catfish larvae before mouth opening and increased after the onset of exogenous feeding, coinciding with the histological organization of the exocrine pancreas. The specific activity of most of the pancreatic enzymes increased until 15 dph and decreased thereafter coinciding with the increase of pepsin. A progressive shift in activity from alkaline (trypsin and chymotrypsin) to acid (pepsin) proteases indicated a change in the digestive physiology of the specimen, as alkaline proteases were no longer the main digestive enzymes involved in protein digestion after the onset of acidic digestion between 15 and 21 dph. The maturation of the intestine and the achievement of a juvenile-like mode of digestion were demonstrated by changes in enzyme activities from the exocrine pancreas and stomach that coincided with alterations in enzyme production occurring in the intestine (e. g. alkaline phosphatase). Considering the ontogenetic development of the digestive enzymes from the pancreas, stomach and intestine, butter catfish larvae might be weaned between 15 and 21 dph, as larvae have achieved the complete maturation of their digestive capacities. These results contradict previous recommendations, which were based solely on the histological organization of the gastric gland and histochemical properties of mucous cells from the stomach, to wean butter catfish larvae at earlier ages. These findings on the functional development of the digestive system in butter catfish would be useful to improve the actual larval rearing techniques for this promising catfish species from the Indian sub-continent.
Article
Fish hybrids provide genetically manipulated products of excellent value for the commercial aquaculture industry. However, if handled or marketed incorrectly, they can cause great financial loss to producers as well as threaten the native species. Herein, molecular markers are established to identify hybrid lineages of pimelodids and characterize them in relation to their parental species, Pseudoplatystoma corruscans, Pseudoplatystoma reticulatum, Phractocephalus hemioliopterus and Leiarius marmoratus. The results show that the mitochondrial genes are useful for identification of the cross-direction through the characterization of the maternal lineage. The nuclear genes allow identification of the interspecific hybrids. Use of genetic markers can avoid misidentification of hybrids that occur in simple morphological analysis. Thus, the present results allow the routine monitoring of pimelodid hybrids for their correct management and trade in aquaculture.
Article
The feeding process is composed of nine stereotyped movement patterns (particulate intake, gulping, rinsing, spitting, selective retention of food, transport, crushing, grinding, and deglutition). The sequence and frequency of these movements are adjusted to the type, size, and texture of food. Better understandings of food intake and mechanisms of food processing reveal intraspecies plasticity and interspecies trophic interactions. This knowledge is essential to manage multispecies communities and maximize productivity of polyculture systems. Feeding periodicity has been observed in marine fishes feeding on algae at the time of peak algal energy due to early afternoon photosynthesis. Morphological features of the digestive system are of great consequence in respect to the type of diet larval/juvenile fish are able to utilize, especially at the highest growth rates during early ontogenetic development. Intestinal transepithelial transport of nutrients reflects a general tendency for fish species to consume diets containing either more carbohydrates (herbivores and omnivores) or more protein/amino acids (carnivores). Seasonality, feeding migrations, and ontogeny result in dietary modulations that are reflected in the intestinal nutrient transport=uptake as well. The mechanistic basis for changes in nutrient absorption can be analyzed using brush border membrane vesicles (BBMV), intact intestinal tissue preparations (in vitro), and the “whole animal” approach where nutrients acquired are measured along the digestive tract.
Article
The growth, survival and trypsin, lipase and amylase activities of red drum larvae were measured in two experiments. For the first trial, a group was fed live prey only (L) and another group was fed a combination of a microparticulate diet (MPD) and live food (L-MP). For the second growth trial a group fed the MPD only (MP) and a starvation group (ST) were examined in addition to the L and L-MP treatments. Enzyme activities of live prey were measured to estimate their possible contribution to larval digestion. No significant (P > 0.05) differences in final size and survival were observed between treatments L and L-MP. Larvae subjected to starvation or fed the MPD diet alone were smaller than treatments fed live prey and did not survive past days 5 and 14, respectively. Trypsin, lipase and amylase activities were detectable at hatching. No significant differences (P > 0.05) in total enzyme activities among treatments were observed before day 14. Specific activity of trypsin, lipase and amylase peaked on day 3 (prior to first feeding) and subsequently decreased. For trypsin, the percentage of enzyme activity potentially attributable to ingested prey increased with age to a maximum of 17%. For lipase and amylase this fraction was less than 5% throughout the study, except on day 8 (12% and 24%, respectively). The lack of significant differences observed in the activity of digestive enzymes among treatments suggests that dietary regime, availability of prey and possible effects of exogenous enzymes did not significantly influence enzyme activity. Therefore, the lower growth rate observed in the L-MP, MP and starved treatments cannot be attributed to low digestive enzyme production of the enzymes measured. It is more likely that the MPD failed to supply the required nutrients for adequate development.
Article
The silver catfish, Rhamdia quelen, is endemic to North, Central and South America with high aquaculture potential and wide acceptance in the market. Breeder fish were subjected to induced reproduction through hypophysation using a crude common carp pituitary extract. Egg characteristics, oocyte surface ultrastructure and histology of larval ontogenesis until whole yolk resorption were described for the first time for this species. Oocytes and semen were obtained by manual extrusion, and fertilization was conducted using the dry method. After fertilization, eggs were kept in incubators at 24 °C. The embryonic development was monitored using a stereomicroscope every 10 min until hatching. To analyse the larval development, larvae samples were collected from incubators daily until the fifth day, fixed in Bouin's fluid and subjected to routine histological techniques. The oocyte extrusion occurred 8 h after the second hormone dose at 26 °C. The oocytes were spherical, non-adhesive and yellow, with a diameter of 1471.75±47.63 μm. Scanning electron microscopy revealed a thin jelly coat covering the zona radiata in the animal pole around the micropyle. The blastopore closure occurred within 8 h after fertilization, and the fertilization rate was 79.9±5.2% at 24 °C. Embryonic development was completed within 25 h 30 min after fertilization. The complete resorption of the yolk and the formation of the digestive system organs and the mouth opening occurred on the fifth day, indicating a need for exogenous feeding. The results of this study provide information important for improvement in R. quelen culture and management.
Article
The sea bass Dicentrarchus labrax is a pelagic egg spawner; sinking eggs are unable to develop into embryos, and this is a limitation in the controlled reproduction of this species. The eggs were divided into good and poor quality, by virtue of their ability to float or sink in seawater. High levels of cathepsins B, D, and L were detected in the eggs, whereas no cathepsin A, C, and E activity was detected. Cathepsin D was found at significantly higher levels in sinking eggs, whereas cathepsin L was more abundant in floating eggs. Since degradation of yolk proteins is essential for the early development of the embryo, the levels of cathepsins A, B, C, D, E, and L were tested in different stages of embryo development. Cathepsin A activity was detectable from the morula stage at which time cathepsin B activity reached its maximal level. Cathepsins A and L reached maximal activity during segmentation, and this corresponded with major changes in the electrophoretic pattern of yolk proteins during embryogenesis suggesting their involvement in yolk protein mobilization at this time. Cathepsin D reached its maximal activity during hatching.
Article
A rapid one-step assay for intestinal peptide hydrolase activity in mucosal homogenates, and in cytosol and particulate fractions prepared from homogenates is described. Peptide hydrolysis, and estimation of liberated l-amino acids by use of l-amino acid oxidase, peroxidase, and a chromogen are achieved by a single incubation step. The most suitable peptide substrates are dipeptides composed of one amino acid which reacts strongly and rapidly with the l-amino acid oxidase reagent and one which does not react. Brush border peptide hydrolase activity may also be estimated with this assay by performing the incubation in the presence of p-hydroxymercuribenzoate.
Article
Brooders of Surubí (Pseudoplatystoma fasciatum) were caught in the Ichillo River (Bolivian Amazon) and adapted to captivity conditions for 1 year in the facilities of the experimental aquaculture station of ‘El Prado’ (Santa Cruz de la Sierra) under natural temperature and photoperiod conditions. Induced reproduction was obtained by means of Ovaprim® (Syndel, Canada) injections and artificial fertilization. Sperm and ova were obtained by gentle stripping of male and female brooders. Fertilized eggs were incubated in 60 L Zug jars. A mean hatching rate of 73.7±19.0% was obtained after 24 h at 26.5 °C. For larval rearing, several protocols were tested with different settings of photoperiod, light intensity, food type and period of distribution, and stocking density. The best survival rates were obtained with Artemia nauplii feeding in total darkness. A high level of aggressiveness between larvae and precocious appearance of jumpers was observed, but these can be controlled with appropriate rearing conditions.
Article
Proteinases (EC 3.4.21-24) in langostilla extract and crayfish hepatopancreas were classified using site specific effectors or chelators for either serine, cysteine, aspartic or metallo classes. Azocasein hydrolysis by langostilla and crayfish preparations was inhibited 50% and 40%, respectively, when assayed in the presence of serine proteinase inhibitor, phenylmethylsulfonyl fluoride. A similar degree of inhibition was observed with a trypsin inhibitor. However, no inhibition occurred with a chymotrypsin inhibitor. Less inhibition of azocasein hydrolysis, i.e., 20% and 14%, respectively, resulted with 1,10 phenanthroline. Inhibitors for cysteine and aspartic proteinases did not reduce the azocasein hydrolysis activities significantly. The amidase activity, with N-benzoyl-DL-Arg-p-nitroanilide as substrate was more sensitive, in both extracts, to serine proteinase inhibitors than the azocasein hydrolase activity. Results showed the presence of serine proteinases, i.e., trypsin but not chymotrypsin, as the major component and some minor activity of metallo dependent proteinases in the decapods extracts. Zymograms obtained after SDS-PAGE showed a dozen proteinases in each extract. Some of them were inhibited by a serine proteinase inhibitor and two to three were inhibited by 1,10 phenanthroline, supporting the results of the test tube proteinase activity assays. Furthermore, the reported technique for zymograms allowed direct comparison between extracts and provided information about their composition and the molecular weight of the targeted enzymes.
Article
We examined allometric growth of larval green sturgeon ( Acipenser medirostris) reared from hatching to metamorphosis. Three different phases were detected in allometric growth in weight. From hatch to 6 dph ( days post hatch) growth was negatively allometric ( b = 0.76), reflecting utilization of yolk for morphogenesis, growth, and metabolic energy. The growth coefficient increased in late lecitotrophic phase ( b = 2.35; 6-14 dph) and the growth was positively allometric during exogenous feeding phase ( b = 3.38; 15-50 dph). The head and eye growth was isometric throughout development. Green sturgeon was similar with Siberian sturgeon in the allometric growth of mouth, pectoral fin, and tail. The differences between two species were attributed to the larval size, larval behaviour after hatching, and some heterochrony in development. The allometric growth of green sturgeon matched developmental and behavioural events observed in early ontogeny of this threatened species.
Sea bass (Dicentrarchus labrax) larvae were weaned with a microparticulated diet at various times after hatching: 10, 15, 20 and 25 days; a control group was fed live prey (Artemia salina). The earlier the weaning, the lower the larval growth obtained. The amylase activity in the pancreatic segment increased swiftly after the weaning in all groups. This increase was the result of an extensive synthesis induced by the starch content (12%) of the compound diet. The enhancement of specific activities of intestinal peptidases, leucine aminopeptidase and γ-glutamyl transpeptidase after weaning was the result of compensatory adaptation, as was described in the case of malnutrition. The depressed activities of alkaline phosphatase, in the brush border membrane fraction, indicated malnutrition in weaned groups. Weaning before day 20 stops or delays larval development, particularly maturation of some digestive processes in larvae, such as the onset of pancreas secretory functions.
Article
The digestive physiology of common dentex was studied by assessing the activity of different pancreatic (trypsin, chymotrypsin, amylase and lipase), intestinal (alkaline phosphatase, aminopeptidase N, maltase and leuncine–alanine peptidase) and gastric (pepsin) enzymes from hatching until the juvenile stage (50 dph at 19 °C). Enzymes involved in the digestion of protein, lipid and carbohydrate were present in common dentex larvae at hatching and before the onset of exogenous feeding. The specific activity of trypsin and chymotrypsin in newly hatched larvae was higher than that of amylase and lipase, indicating the importance of these enzymes in the cleavage of yolk proteins and hatching, while after hatching, the activity of these serine proteases dramatically decreased, whereas lipase showed the opposite trend. A progressive shift in activity from alkaline to acid proteases was observed during larval development, reflecting that alkaline proteases were not longer the main digestive enzymes involved in protein digestion after the development of gastric glands and onset of acidic digestion. Lipase total activity in common dentex peaked at 35 dph and decreased after weaning. Diet change due to weaning might partially explain the decrease in lipase activity, although this change might be also indicative of a change in the nutritional requirements of this species, since the juveniles prefer diets with high protein levels than those with lower protein and higher lipid content. Regarding intestinal enzymes, leucine–alanine peptidase and alkaline phosphatase and aminopeptidase N were found in newly hatched larvae, while maltase was detected after the onset of exogenous feeding. The achievement of an efficient brush border membrane digestion takes place much earlier (6–12 dph) than in any of the studied species which might reflect the different metabolic profile and rapid growth of this species.
Article
The study on histological and ultrastructural characteristics of the digestive tract of yellow catfish (Pelteobagrus fulvidraco) was carried out from hatching (0 day after hatching, DAH) until 35 DAH. Larvae for this study were maintained in the laboratory conditions (water temperature ranged from 23 °C to 25 °C). They were fed with zooplankton from 3 DAH to 17 DAH, with zoobenthos added from 10 DAH, and only zoobenthos from 18 DAH to 35 DAH. Development of the digestive tract in P. fulvidraco followed the general pattern described for other fish species with some peculiar findings. At hatching, it consisted of an undifferentiated straight tube laying over the yolk sac. The digestive tract was differentiated into buccopharynx, esophagus, primary stomach and intestine by 2 DAH. The liver and pancreas also appeared at this time. The intestine became differentiated into anterior and posterior regions separated by the intestine bend at 3 DAH. Gastric gland appeared in cardiac stomach at 3 DAH, the earliest appearance time among fishes studied to date. Oxynticopeptic cell contained pepsinogenic granules and abundant tubulovesicular systems at 3 DAH. As larvae grew, more pepsinogenic granules but less tubulovesicular systems were found in oxynticopeptic cell. The abundant visible tubulovesicular systems suggested that oxynticopeptic cell was still in rest phase with little hydrogen chloride (HCl) secreted at the first appearance time. The ultrastructure of oxynticopeptic cell indicated the asynchronous development of acid-secreting and pepsinogen-secreting function. The epithelial absorptive cell of the anterior and posterior intestinal segments showed electron-opaque lipid droplets and heavy pinocytosis, respectively at 3 DAH. Heavy pinocytosis could be observed in the posterior intestine until 25 DAH. Lipid vacuole accumulation appeared in liver at 13 DAH, the same time as the storage of abundant glycogen. These results suggested that the development of the digestive tract of P. fulvidraco larvae was functional rapidly, however it was still incomplete at 3 DAH. The functions of digestive tract and accessory glands were developed gradually until 25 DAH.