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.     

湘华鲮仔、稚鱼消化系统发育组织学观察

采用连续石蜡切片、苏木精-伊红染色技术, 对湘华鲮[Sinilabeo decorus tungting (Nichols)]出膜后 2~80 d 的仔稚鱼消化系统发生进行研究。结果表明, 2 日龄的仔鱼口、咽腔、肠腔、肛门形成, 但口咽腔未贯通; 肝细胞紧紧围绕着前肠周围, 形成肝细胞索。4 日龄, 消化道上皮细胞开始功能性分化; 肝细胞团增多, 填充于卵黄囊与消化道间, 胞质着色较浅初现肝血窦; 消化道贯通, 仔鱼由内源性营养向混合性营养转变。6 日龄口裂加深, 呈“＞” 形, 吻部初具形状, 初现黏液细胞; 咽喉胼胝垫出现角质化, 鳃弓黏膜上皮增厚, 初现黏液细胞和味蕾; 食道肌层变厚, 环形肌为主; 肝细胞间形成大量空泡, 肝细胞变小; 肠表皮出现皱褶和纹状缘, 前肠到后肠皱褶逐渐变短, 杯状细胞增多。9 日龄咽后部的胼胝垫处肌层发达, 胼胝垫对侧的黏膜层开始凹陷形成皱褶; 食管环肌发达, 纵肌不明显; 肠道均分布有杯状细胞, 后肠段最多。10 日龄, 唇部细胞出现角质化趋势, 胼胝垫表面形成梳状突起。13 日龄, 卵黄囊完全消失, 混合性营养转变为外源性营养。37 日龄, 稚鱼肝胰脏、食管组织结构均似成鱼, 发育基本完成。43 日龄, 口咽腔、鳃与肠道组织发育基本完成。本研究将湘华鲮仔稚鱼消化系统的发育分为 3 个阶段, 即卵黄期、混合性营养及外源性营养阶段。研究发现, 湘华鲮消化器官组织结构的发育和变化, 始终是与食性的转变相适应的。湘华鲮其仔稚鱼消化系统组织学研究可为湘华鲮苗种培育中饵料管理提供理论依据, 对提高湘华鲮人工养殖的存活率和生长性能具有重要意义。     

Organogenesis of the digestive system in Pacific red snapper (Lutjanus peru) larvae

This study reports the ontogenetic development of the digestive system of larval Pacific red snapper (Lutjanus peru), an important candidate species for aquaculture on the Pacific coast of Mexico. Histological sections of larvae were cut and dyed using the haematoxylin–eosin technique. The development of the digestive tract of Pacific red snapper larvae follows a general pattern of differentiation that can be divided into three stages. Stage I lasted from 1–3 days post hatching (DPH) and included the endogenous nutrition period; it was characterized by the initial differentiation of the digestive tract in preparation for the onset of exogenous feeding (3 DPH). At this time, the digestive tract was differentiated into buccopharynx, oesophagus, stomach anlage, anterior intestine, posterior intestine and a short rectum. The liver, pancreas and kidney were also present. The mouth and anus were open. Stage II occurred after first feeding, lasted for 16 days (4–23 DPH) and included both preflexion and flexion larvae. The main changes that occurred during this stage reflected the adaptation to exogenous feeding and the concomitant growth. Stage III (24–30 DPH) included post‐flexion larvae and started with the appearance of the gastric glands and pyloric caeca. The presence of the gastric glands suggests that early weaning during culture trials of the Pacific red snapper larvae may be possible at this early age.     

Effect of L‐tryptophan supplemented diets on serotonergic system and plasma cortisol in Totoaba macdonaldi (Gilbert, 1890) juvenile exposed to acute stress by handling and hypoxia

Brain serotonin and cortisol play a central role in integrating the neuroendocrine response to stress. Previous studies in aquatic species show that dietary supplementation with the 5‐HT precursor L‐tryptophan amino acid is able to modulate this response. The aim of this study was to evaluate the effect of dietary tryptophan (Trp) on the serotonergic system and acute stress response in Totoaba macdonaldi. Four groups of fish were fed with one of four isonitrogenous and isocaloric diets formulated with different Trp content: 0.5% (1X TRP, control diet), 1.1% (2X TRP), 1.7% (3X TRP) and 2.3% (4X TRP). After a feeding period of 21 days, groups were exposed to acute stress by handling with a net or hypoxia. Feeding with Trp‐enriched diets resulted in increased 5‐HT content in telencephalon of undisturbed fish. Handling increased the 5‐hydroxyindole acetic acid (5‐HIAA) and serotonin turnover (5‐HIAA/5‐HT) in telencephalon of fish fed with 1X TRP and 2X TRP but was not significant for the fish fed 3X TRP and 4X TRP, and no significant increase was observed by hypoxia in both telencephalon and hypothalamus in any of the diets. An inverted U‐shaped response was observed in plasma cortisol increasing with 2X TRP after both handling and hypoxia and decreasing with diets rich in Trp, 3X TRP and 4X TRP. A higher increase was observed in haematocrit, plasma glucose and lactate in hypoxia than the handling groups. Our results indicate that Trp supplementation modulated cortisol response and mediated negative feedback of 5‐HT system in telencephalon after acute stress.     

Ontogenetic development of digestive tract and digestive enzymatic activities in Squaliobarbus curriculus larvae

Squaliobarbus curriculus is an economically important freshwater fish. The ontogenetic development of the digestive system of S. curriculus larvae was studied histologically and enzymatically from hatching to 30 days posthatching (DPH). Amylase, lipase, alkaline phosphatase and pepsin activities were detected from the hatching stage, indicating that these enzymes were genetically preprogrammed. Marked increases in intestinal amylase, trypsin and alkaline phosphatase activities between 10 and 20 DPH corresponded to feed acquisition and transformation. Larval development in S. curriculus could be divided into three phases: phase I (endotrophic period): 1–3 DPH; phase II (endo‐exotrophic period): 4–5 DPH; and phase III (exclusively exotrophic period): from 6 DPH onward. At hatching, the digestive tract of the larvae was an undifferentiated straight tube. On 3 DPH, the digestive tract differentiated into the mouth cavity, oesophagus and intestine. On 6 DPH, feeding was totally exotrophic and the yolk sac was completely exhausted. During the growth of S. curriculus larvae, the intestinal mucosa formed and the number of goblet cells and microvilli increased, demonstrating maturation of the digestive system. The study about the digestive development of S. curriculus larvae will contribute to better larval‐rearing strategies.     

Replacement of fishmeal by soya protein concentrate in the diets of Totoaba macdonaldi (Gilbert, 1890) juveniles: effect on the growth performance,in vitro digestibility,digestive enzymes and the haematological and biochemistry parameters

The potential of soya protein concentrate (SPC) as an alternative protein source in diets for Totoaba macdonaldi juveniles was evaluated. Seven isonitrogenous and isolipidic diets were formulated containing 15–100% SPC (SPC15, SPC30, SPC45, SPC60, SPC75, SPC90 and SPC100) to replace fishmeal (FM‐protein), and a FM‐protein‐based diet without SPC was used as a reference diet (RD). Each diet was randomly assigned to triplicate groups of 20 totoaba (50 ± 1.0 g) and was fed twice daily to apparent satiation. After 60 days of experiment, effects on totoaba were evaluated. Growth performance in fish fed RD to SPC45 was similar. The maximum FM‐protein replacement for weight gain (g kg ABW^?1 day^?1) was estimated to be 34.17% using a broken‐line model. In vitro digestibility of fish fed RD, SPC15 and SPC30 was similar. Trypsin activity was higher in fish fed the RD (9.38 ± 0.52 mU × 10^?3 mg protein^?1. Chymotrypsin activity was similar in fish fed RD, SPC15 and SPC30. Activity of alkaline protease and phosphatase was similar in fish fed RD, SPC15 and SPC30. Red blood cells (RBC) were the highest in fish fed RD to SPC60. Fish fed the RD, SPC15 and SPC30 present the highest haematocrit (HT) and haemoglobin (HB) content. Diets SPC90 and SPC100 presented similar MCV and MCHC. Total protein (TP) profile overall decreased in fish fed SPC90 and SPC100, pointing to nutritional hypoproteinemia due to deficient digestion and absorption. These results indicate that SPC‐based diets could be used efficiently by totoaba with FM‐protein replacement of less than 45%.     

中华倒刺鲃仔稚鱼期消化酶及碱性磷酸酶活性变化的研究

本试验选取了中华倒刺鲃从初孵仔鱼到30日龄稚鱼,分析测定了中华倒刺鲃的特定生长率、胰蛋白酶、淀粉酶、脂肪酶和碱性磷酸酶的活性。结果表明：在整个试验期间,中华倒刺鲃的SGR为26.43%。在初孵仔鱼体内就能检测到胰蛋白酶、淀粉酶、脂肪酶和碱性磷酸酶的活性。胰蛋白酶在内源营养阶段具有较高的活性,在4日龄开口时其比活力降到一较低值,随后呈现增加的趋势,在18日龄达到最低值后又缓慢增加。淀粉酶在试验期间表现出较高的活性,比活力一直呈增加的趋势。脂肪酶在18日龄前其比活力一直增加,并且在7-18日龄表现出较高的比活力水平,18日龄后快速下降。碱性磷酸酶活性则呈现出一直增加的趋势。     

Activities of digestive enzymes and histology of digestive system during larval development of devil stinger (Inimicus japonicus)

Devil stinger is a valuable demersal scorpaenid fish while the rearing of stinger larvae still relies on live prey. This study was conducted to illustrate the development of the main digestive enzymes and digestive system during larval development of this species to provide evidence for the application of artificial feeds. Enzymatic and histological assays were conducted from 1 day post hatching (dph) to 36 dph in larvae. The result showed that the selected digestive enzyme activities increased significantly after 15 dph. Specifically, the total trypsin activities increased significantly from 18 dph to 33 dph. The total pepsin and amylase activities increased significantly first and thereafter decreased significantly. The lipase activities followed the similar pattern with trypsin. With regard to the histological study, the stinger larvae open their mouth to first feeding at 3 dph and turned into totally exogenous nutritional stage at 6 dph. In addition, mucous membrane, rich in goblet cells, was widely distributed in oesophagus epithelium at 18 dph. The height and amounts of gastric gland in cardia and main body of the stomach increased gradually with the development of stinger larvae after 15 dph. The intestine length of stinger larvae was short, and goblet cell was abundant in anterior intestine after 12 dph, not the posterior intestine. The ontogeny of liver and pancreas started from newly hatched stage, and the differentiation of liver was prior to pancreas. The above findings would provide evidence for the use of artificial feeds from the larval stage of stinger larvae (at least from 21 dph).     

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.     

圆斑星鲽仔鱼变态前消化系统发生的形态学和组织学研究

对初始孵化到孵化后26d（变态前）的圆斑星鲽（Verasper vazriegatus Temminck et Schlegel）仔鱼的消化系统发生进行形态学和组织学研究。研究表明，在水温为12．5～15．5℃时，初始孵化仔鱼消化道细而直，两端封闭，位于卵黄囊背方。随着仔鱼发育，消化道延长，肝胰脏出现。孵化后6d，仔鱼开口，消化道贯通，开始摄食。孵化后10d，消化道明显分为口咽腔、食道、胃、小肠、直肠，黏膜上皮褶皱增多。孵化后14d，卵黄囊消失，仔鱼完全依靠外源性营养。孵化后26d，胃壁较厚处出现胃腺，但不发达，幽门盲囊尚未出现。由结果可见，孵化后6d仔鱼开口，可开始投喂轮虫；孵化后26d，消化系统的结构和功能逐渐完善，消化吸收能力增强，但由于胃的结构和功能尚不完善，此阶段还应以活体饵料为主，一方面可逐渐加大轮虫的投喂量，另一方面可适当投喂较大的卤虫无节幼体等。本研究旨为圆斑星鲽人工育苗中饵料种类及投喂时间的选择提供理论依据。     

灰鲳仔稚鱼消化系统的发育

对出膜后1~35 d灰鲳仔稚鱼的消化系统进行了形态学和组织学观察。初孵仔鱼具有很大的卵黄囊,消化管为一简单的直形盲管。2日龄仔鱼消化系统中肠区首先开始分化,内面可见纹状缘。3日龄仔鱼胃区膨大,卵黄消失,肠道分化出小肠和直肠。侧囊在4日龄仔鱼时出现分化。5日龄仔鱼观察到幽门盲囊的初始结构,油球消失,从内源性营养向外源性营养过渡基本完成。6日龄仔鱼摄食主动。14日龄稚鱼肝脏分为两叶,体积增大,胰脏体积增大,胃弯曲呈"U"形,幽门盲囊指状分支已增加到几十个。22日龄稚鱼肠道弯曲更多,在胸腹腔中肠道盘曲程度已接近幼鱼。28日龄稚鱼侧囊背腹形成一纵隔,出现角质剌。35日龄稚鱼胃腺已十分丰富,侧囊结构也已完善,标志着灰鲳消化系统更趋完善。     

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.     

三疣梭子蟹幼体消化酶活力及氨基酸组成的研究

在三疣梭子蟹幼体发育过程中，五种消化酶活力表现出三种变化模式，其中胃蛋白酶和类胰蛋白酶活力呈上升趋势，而且在蚤状幼体Ⅰ期时就较高，淀粉酶活力逐渐减小，纤维素酶和脂肪酶活力极微。氨基酸含量随幼体发育逐渐增加。必需氨基酸中以亮氨酸含量最高，色氨酸含量最低；非必需所基酸中含量最高者为谷氨酸，最低者为胱氨酸。同时单个必需氨基酸含量与必需氨基酸总量的比值（Ａ／Ｅ）在幼体不同发展阶段略有差异，但基本趋于一致。     

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.