Comparative intestinal absorption of amino acids in rainbow trout (Oncorhynchus mykiss), totoaba (Totoaba macdonaldi) and Pacific bluefin tuna (Thunnus orientalis)

The kinetics of amino acid absorption in the proximal section of three fish species were studied using an everted intestine technique and a pancreatic digest of casein (Tryptone), as the model amino acid mixture. Fresh intestine of rainbow trout (Oncorhynchus mykiss) was used to set up the experimental system, and results were compared with those obtained using intestines from totoaba (Totoaba macdonaldi) and bluefin tuna fish (Thunnus orientalis). The kinetics of amino acid absorption was sigmoidal for all amino acids and for each species of fish tested. In general, specific absorptions of essential amino acids were higher than those of non‐essential ones. No correlation between the concentration of amino acids in the Tryptone solution and the corresponding absorptions was found. The maximum specific absorption rates of all amino acids for trout were 10 times higher than those determined for totoaba and bluefin tuna. The relative amounts of the different amino acids preferentially absorbed in all three species were different. Results obtained from the everted technique may be applicable to the design of formulated diets for large fish species with commercial value.     

Ontogeny and distribution of alkaline and acid phosphatases in the digestive system of California halibut larvae (Paralichthys californicus)

Studies aimed to assess the digestive physiology of marine fish larvae under culture conditions are important to further understand the functional characteristics and digestive capacities of the developing larvae. Most studies to date concentrate on intestinal lumen digestion and little attention to the absorption process. Thus, the objectives of this study were to histochemically detect and quantify some of the enzymes responsible for absorption and intracellular digestion of nutrients in the anterior and posterior intestine of California halibut larvae. Alkaline and acid phosphatases were detected from the first days post-hatch (dph). Alkaline phosphatase maintained a high level of activity during the first 20 dph in both intestinal regions. Thereafter, a clear intestinal regionalization of the activity was observed with the highest levels occurring in the anterior intestine. Acid phosphatase activity gradually increased in both intestinal regions during development, and a regionalization of the activity was not observed until late in development, once the ocular migration began. Highest levels were observed in the anterior intestine at the end of metamorphosis concomitant with the stomach development. The results from this study show some morphological and physiological changes are occurring during larval development and a clear regionalization of the absorption process as the larvae develops. These ontological changes must be considered in the elaboration of diets according to the digestive capacity of the larvae.     

Comparison of growth,digestive system maturation and skeletal development in sea bass larvae reared in an intensive or a mesocosm system

The quality of development in intensive or mesocosm hatchery‐reared Dicentrarchus labrax larvae was investigated using physiological indicators assessing ontogeny. Larvae were reared in intensive (120 L tanks) and in mesocosm systems (20 m³ enclosures) with the same feeding sequence, excluding the wild zooplankton naturally available in mesocosms. Faster growth was recorded since early development [16 day after hatching (DAH)] in the mesocosm. Maturation of the digestive system also occurred earlier as indicated by the higher amylase secretion ratios, the intestinal maturation index (alkaline phosphatase/leucine–alanine peptidase and aminopeptidase‐N/leucine–alanine peptidase ratios) and the more developed intestinal epithelium at 23 DAH. Nevertheless, the delay in digestive maturation in the intensive system seemed retrieved within few days. In both the groups, the number of vertebrae ranged between 24 and 26, with the dominant class being 25 vertebrae. However, the distributions differed between treatments for meristic characteristics, ossification stages and incidence of malformation types. Loss of a vertebra was more frequent in the intensive system, while the appearance of an additional vertebra was more frequent in the mesocosm. Ossification at 37 DAH was also more advanced in the mesocosm in addition to a lesser rate and severity of skeletal malformations. It is suggested that the early nutritional contribution of mesocosm wild zooplankton, yet at densities of 0.2–0.7 prey mL^?1, had key effects on larvae development since the early stages.     

Digestive protease characterization, localization and adaptation in blacklip abalone (Haliotis rubra Leach)

Digestive protease in blacklip abalone (Haliotis rubra Leach) from whole gut extracts was found to have dual pH optima at pH 3 and 10 and an optimum temperature of 45 °C. Over the biologically relevant range of pH (5–8), protease activity dropped to a minimum at pH 5 (53% of the maximal rate at pH 3) rising gradually and continuously up to (and beyond) pH 8. Over the biologically relevant range of temperatures (9–24 °C), protease activity increased continuously with activity at 24 °C being 75% higher than activity at 9 °C. Protease digestion was relatively uniform in gut extracts from sections containing gut contents. Digestion thus appears to be significantly extracellular (in the lumen of the gut). Analysis of gut sections washed free of food matter suggests the anatomical origin of protease from activity of gut regions in the order: digestive gland » salivary gland ≈ stomach > crop > intestine > upper oesophagus. Whole gut protease levels did not alter in response to a high protein artificial diet.     

Precocious development of the digestive system in relation to early appearance of piscivory in striped bonito Sarda orientalis larvae

ABSTRACT: Striped bonito Sarda orientalis larvae and juveniles were reared in a laboratory from hatching to 14 days post hatching, and the development of their digestive system was investigated histologically. On day 2 (first feeding day), the larval-type digestive system was differentiated. On day 3, a blind sac with densely distributed gastric glands developed and pyloric ceca appeared, suggesting that the digestive system was attaining adult-type structure. The yolk was completely absorbed on day 4. Bonito larvae preyed on rotifers and/or Artemia nauplii on day 2, but almost the whole gut was occupied by fish larvae from day 4. In the striped bonito, the adult-type digestive system was established right after first feeding, and this precocious development was well synchronized with the appearance of piscivory.     

Evaluation of weaning performance of California halibut (Paralichthys californicus) larvae using growth,survival and digestive proteolytic activity

This study evaluated weaning success of California halibut, Paralichthys californicus, larvae onto a microdiet at various stages of development utilizing growth, survival and digestive enzyme activity. Weaning onto a microdiet was evaluated at 16, 26, 36 and 46 days posthatch (dph). Alkaline and acid proteases and leucine aminopeptidase activities were measured after weaning and compared between the weaned treatment and Artemia‐fed controls. Survival was significantly lower in the microdiet‐fed treatments compared to the control groups. Growth was significantly reduced in all weaning treatments compared to the control, except for the 46 dph group. No differences in enzyme activities were detected between treatment and diet at 16 and 26 dph; however, activities were higher for the microdiet‐fed larvae at 36 and 46 dph. This study demonstrates that California halibut larvae possess a differentiated and effective digestive system early in development and can be weaned with relative success (>40% survival) before completion of the metamorphosis (i.e., 36 dph). The lack of weaning success at an early date cannot be entirely because of the absence of a functional stomach but could be related to, among other factors, the low‐microdiet ingestion rates observed and higher leaching of smaller microdiets.     

Ontogeny of the digestive tract in butter catfish Ompok bimaculatus (Bloch) larvae

The ontogeny of the digestive tract was studied histologically in butter catfish Ompok bimaculatus from hatching to 30?days post-hatching (dph). At hatching, the digestive tract of butter catfish consisted of a straight tube with a smooth lumen dorsally attached to the yolk sac. Between 1 and 2 dph, the mouth opened, oral valves were visible and canine-like teeth and taste buds were detected. During this period, intestine was differentiated into the anterior and posterior intestine, and the digestive accessory glands were also developed. Exogenous feeding started at 2?dph, and there was a 2-day mixed endogenous–exogenous feeding period. Most of the yolk sac reserves were consumed between 2 and 3 dph, and by 5?dph, the yolk sac was completely depleted and no longer visible in histological sections. Between 3 and 4 dph, several vacuoles (neutral lipids) were observed in the intestine and also in hepatocytes, indicating a functional absorption of nutrients from food. At 8 dph, differentiation of gastric glands was noticed, and by 9–11?dph, there were abundant gastric tubular glands arranged along numerous longitudinal folds. During the same period, pyloric sphincter appeared as an epithelial fold that separated the stomach from the anterior intestine. From 12 dph to the end of the study at 30?dph, no noticeable histological modifications were observed. The development of gastric glands is considered as the last major events in digestive tract development and their presence designates the end of larval period and the onset of the juvenile period. Hence, it is suggested that, butter catfish larvae have a morphologically complete digestive tract by 12?dph. These findings on the development of the digestive system in butter catfish may lead to a better understanding of the ontogeny and would be useful to improve the larval rearing techniques of this promising catfish species for freshwater aquaculture diversification.     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

The proteolytic digestive activity and growth during ontogeny of Parachromis dovii larvae (Pisces: Cichlidae) using two feeding protocols

The proteolytic digestive activity and growth of Parachromis dovii larvae during the ontogeny were evaluated in a recirculation system using two feeding strategies during a 28-day period. Larvae were reared using two feeding protocols (three replicates each): (A) Artemia nauplii (at satiation), fed from exogenous feeding [8 days after hatching (DAH)] until 15 DAH followed by nauplii substitution by formulated feed (20 % day^?1) until 20 DAH and then formulated feed until 28 DAH; (B) formulated feed (100 % BW daily) from exogenous feeding until 28 DAH. Levels of acid (pepsin type) and alkaline digestive proteases as well as growth and survival of larvae were measured along the feeding period. Survival was high and similar between treatments: 98.9 ± 0.0 for Artemia, 97.3 ± 0.0 % for formulated feed. The specific growth rate for length and weight was higher in larvae fed with Artemia nauplii than in larvae reared with formulated feed: 3.4 ± 0.1 versus 1.8 ± 0.1 % day^?1 for body length (P = 0.009) and 12.2 ± 0.1 versus 6.5 ± 0.3 % day^?1 for body weight (P = 0.002). The acid and alkaline proteolytic activity was detected, in both treatments, from the beginning of the experiment, at 8 DAH. The total enzymatic activity (U larva^?1) for acid and alkaline proteases was higher in larvae reared with Artemia after 12 DAH, whereas the specific enzymatic activity was similar for both enzyme types in the two treatments. The results suggest that P. dovii larvae were capable to digest formulated diets from the beginning of exogenous feeding and that they could be reared with formulated feeds. However, the formulated feed used should be nutritionally improved because of the poor growth obtained in this research.     

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

Changes in lipid class and fatty acid composition during development in pike (Esox lucius L) eggs and larvae

To establish the changes which occur during embryogenesis and early larval development, eggs, yolk sac larvae and swim-up larvae of pike were examined for lipid class and fatty acid composition. At a water temperature of 15.5°C, the embryonic phase was short (6 days) and characterized by a 41.3% decline in the lipid content of eggs, accompanied by large reductions in the amount of phosphatidylcholine (41.4% decrease), sterol esters and triacylglycerols (respectively a 41.2% decrease and a 58.1% decrease), but not phosphatidylethanolamine which increased markedly (35.6%). By the time of yolk sac absorption (7 to 11 days after fertilization) the larvae remained inactive and a limited utilization of lipids was observed. Yolk sac phosphatidylcholine was selectively incorporated into larval bodies while the levels of other lipid classes remained unchanged in the yolk. When the swim bladder was filled and the swimming stage was reached (11 days to 13 days af), the yolk was completely depleted and yolk phosphatidylcholine together with yolk triacylglycerols were catabolised. Yolk phosphatidylethanolamine and yolk sterol esters were partly incorporated into the body lipids. In the subsequent swim-up larval stage (13 to 15 days af), a steady decrease in lipids was observed (41.6%). Fluctuations in the levels of polyunsaturated fatty acids, monounsaturated fatty acids or saturated fatty acids examined from eggs or larvae were consistent with changes in lipid classes during pike development. During yolk sac absorption, pike incorporated yolk PUFA released on hydrolysis of phosphatidylcholine into the larval body. The results are discussed with reference to water temperature and in relation to the ontogenic and ecological context of pike development. This revised version was published online in July 2006 with corrections to the Cover Date.     

胭脂鱼仔鱼的生长和消化酶活力变化

对胭脂鱼(Myxocyprinus asiaticus)孵化后1～37 d仔鱼的体长、体重、RNA/DNA比率、胰蛋白酶、脂肪酶和淀粉酶的酶活力变化进行了测定。结果显示:胭脂鱼仔鱼1～37 d体长稳定增长。体重增加呈阶段性,在1～21 d体重增长缓慢,21～29 d体重增加较快,29～37 d体重快速增加。仔鱼从7 d开始摄食,1～6 d,RNA/DNA比率变化很小,蛋白含量逐渐降低;7～21 d,RNA/DNA比率波动较大,蛋白含量增长缓慢;21～37 d,RNA/DNA比率和蛋白质均增长迅速。生长率(GR)与RNA/DNA比率的回归方程为:GR=1.01RNA/DNA+1.6077(R2=0.8516)。从2、5和7 d开始分别检测到明显的胰蛋白酶、脂肪酶和淀粉酶活力,之后,消化酶活力随着发育先快速地上升然后波动性地下降到一个稳定水平。结果表明:胭脂鱼仔鱼从内源性到外源性营养转化过程中,不同的消化酶能够检测到明显酶活力的时间不同,到仔鱼开始摄食时已经具备消化蛋白质、脂肪和淀粉的能力;RNA/DNA比率可以作为判定仔鱼生长率的可靠指标。     

Ontogeny of the digestive tract and enzymes in rock bream Oplegnathus fasciatus (Temminck et Schlegel 1844) larvae

Histological development of the digestive tract and specific activities of three digestive enzymes (trypsin, alkaline phosphatase, and pepsin) were studied in rock bream Oplegnathus fasciatus from hatching to 50 days after hatching (DAH). At hatching, the digestive tract appeared as an undifferentiated straight tube and differentiated into the buccopharynx, esophagus, stomach, intestine, and rectum at mouth opening by 3 DAH. The taste bud and mandibular teeth were present in the buccopharyx at 8 DAH. The goblet cells appeared in the esophagus at 8 DAH and in the buccopharyx at 9 DAH. The stomach anlage was formed at 2 DAH and developed into cardia, fundus, and pylorus at 14 DAH. The gastric glands were visible at 16 DAH, and the pepsin was firstly detected on 22 DAH. At 2 DAH, the intestinal valve appeared and divided the intestine into anterior intestine (AI) and posterior intestine (PI). The rectum was differentiated from the PI at 3 DAH. The supranuclear vacuoles were visible in the rectum by 6 DAH, and the lipid inclusions were present in the AI at 8 DAH. The alkaline phosphatase was detected at 1 DAH, and the increase in its activity indicated the maturation of the intestine after 40 DAH. The hepatocytes and pancreatic cells were differentiated from the blast cells at 2 DAH, and the acidophilic zymogen granules in the exocrine pancreas were observed simultaneously. The trypsin was detected by 1 DAH and increased to the maximum at 19 DAH, followed by a decrease as the stomach became functional.     

Development of digestive enzyme activity in spotted rose snapper,Lutjanus guttatus (Steindachner, 1869) larvae

We describe digestive enzyme activity during the larval development of spotted rose snapper, Lutjanus guttatus. Trypsin, chymotrypsin, leucine aminopeptidase, pepsin, amylase, lipase, and acid and alkaline phosphatase activities were evaluated using spectrophotometric techniques from hatching through 30 days. The spotted rose snapper larvae present the same pattern of digestive enzyme activity previously reported for other species in which pancreatic (i.e., trypsin, chymotrypsin, amylase, and lipase) and intestinal (i.e., acid and alkaline phosphatases and leucine aminopeptidase) enzymatic activities are present from hatching allowing the larvae to digest and absorb nutrients in the yolk-sac and live prey by the time of first feeding. The digestive and absorption capacity of the spotted rose snapper increases during the larval development. A significant increase in individual activity of all enzymes occurs at 20 DAH, and around 25 DAH, the juvenile-type of digestion is observed with the appearance of pepsin secreted by the stomach, suggesting that maturation of the digestive function occurs around 20–25 DAH. Our results are in agreement with a previous suggestion that early weaning may be possible from 20 DAH. However, the patterns of enzymatic activities reported in our study should be considered during the formulation of an artificial diet for early weaning of the spotted rose snapper.     

Ontogenetic development of digestive functionality in golden pompano Trachinotus ovatus (Linnaeus 1758)

Ontogenetic development of the digestive system in golden pompano (Trachinotus ovatus, Linnaeus 1758) larvae was histologically and enzymatically studied from hatch to 32 day post-hatch (DPH). The development of digestive system in golden pompano can be divided into three phases: phase I starting from hatching and ending at the onset of exogenous feeding; phase II starting from first feeding (3 DPH) and finishing at the formation of gastric glands; and phase III starting from the appearance of gastric glands on 15 DPH and continuing onward. The specific activities of trypsin, amylase, and lipase increased sharply from the onset of first feeding to 5–7 DPH, followed by irregular fluctuations. Toward the end of this study, the specific activities of trypsin and amylase showed a declining trend, while the lipase activity remained at similar levels as it was at 5 DPH. The specific activity of pepsin was first detected on 15 DPH and increased with fish age. The dynamics of digestive enzymes corresponded to the structural development of the digestive system. The enzyme activities tend to be stable after the formation of the gastric glands in fish stomach on 15 DPH. The composition of digestive enzymes in larval pompano indicates that fish are able to digest protein, lipid and carbohydrate at early developmental stages. Weaning of larval pompano is recommended from 15 DPH onward. Results of the present study lead to a better understanding of the ontogeny of golden pompano during the larval stage and provide a guide to feeding and weaning of this economically important fish in hatcheries.     

泥鳅消化道指数及消化酶活性分布的研究

研究了泥鳅消化道指数及消化酶活性在肠道不同部位(前肠、中肠及后肠)和肝胰脏的分布.试验结果表明,泥鳅比肠长为0.335±0.006,比肠质量为0.021±0.002,比肝质量为0.015±0.001,其特征偏肉食性.泥鳅肝胰脏淀粉酶、蛋白酶和脂肪酶的活性均极显著高于其他部位,分别为(45.464±2.442)、(580.515±12.282)、(28.889±3.849) U/ml.     

摄食对大口黑鲈消化器官蛋白酶和淀粉酶活力的影响

试验从摄食方面研究食物对大口黑鲈主要消化器官蛋白酶和淀粉酶分泌的影响,对摄食后主要消化器官蛋白酶和淀粉酶活力进行了研究。结果表明,摄食对蛋白酶活力影响:摄食后1h胃蛋白酶降低,至摄食后4 h升高到平稳水平(P>0.05);幽门盲囊蛋白酶活力摄食后开始降低,而从摄食后4 h开始升高(P<0.05);肠道蛋白酶活力摄食后1 h出现升高,摄食后4 h降到最低,10 h又升高到较大活力。摄食对淀粉酶活力影响:胃淀粉酶活力摄食后开始降低,摄食后4h又升高到最大(P<0.05);肠道淀粉酶活力摄食后开始升高,至摄食后4 h达到稳定(P>0.05);幽门盲囊淀粉酶活力在摄食后开始降低,到摄食后4h升高到较高活力,并保持到摄食后10 h;摄食后1 h肝脏淀粉酶活力便保持在较高水平(P>0.05)。     

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea)

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine–alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine–alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.     

Development of testis and digestive tract in longnose gar (Lepisosteus osseus) at the onset of exogenous feeding of larvae and in juveniles

The aim of this study was to describe the ontogenetic development of the testis and the alimentary tract in longnose gar (Lepisosteus osseus) related to fish size and age at the onset of exogenous feeding and late ontogenesis. Using light microscopy, testes were first detected histologically by the appearance of primordial germ cells 9 days after the first exogenous feeding [31–31.5 mm total body length(TL)] and presumptive seminiferous tubules (maleness characteristic) in fish of 107 mm TL. The present histological studies indicated that the alimentary tract of lepisosteids is completely functional at the beginning of exogenous feeding, several days before the completion of yolk absorption. Based on these results, we have concluded that garfish larvae/juveniles can be effectively trained to consume formulated diets at early stages, after an initial feeding of live food for 2–3 days (23.5 mm TL). Our findings provide evidence of the first controlled rearing of longnose gar using live and formulated diets, providing the possibility of experimental work with this non‐teleost fish.