Amino acid composition of Arctic charr, Salvelinus alpinus (L.) and the prediction of dietary requirements for essential amino acids

Embryo somatic tissues, non‐somatic yolk‐sac materials, and whole, individual fingerlings (age 0+) of Arctic charr, Salvelinus alpinus (L.), as well as a commercial trout diet, were analysed for a wide spectrum of amino acids. Analytical material consisted of prefeeding swim‐up fry that were separated into discrete yolk sac and somatic embryo tissue samples. Amino acid concentrations in fry somatic tissue and whole fingerlings were generally very similar to each other, but were lower than those measured in yolk materials. Higher correlations were observed between the majority of specific amino acid concentrations in the trout diet when compared with fingerling data (r² = 0.91) and fry somatic tissue data (r² = 0.89), than when correlated with fry yolk sac material (r² = 0.76). These results indicate that the essential amino acid profiles of fry somatic tissue and whole fingerlings are closer to that of a commercial feed than they are to the endogenous profiles found in the embryonic yolk sac material itself. The dietary ratios of individual essential amino acids were also compared with the total essential amino acid concentrations (A/E ratios) in whole fingerling tissues, and these ratios could be used to accurately estimate the apparent essential amino acid requirements of Arctic charr. The rationale for using carcass amino acid composition data to estimate the dietary essential amino acid requirements of Arctic charr is discussed.     

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.     

Effect of the lipid droplet adherence on growth and survival of the European hake (Merluccius merluccius) larvae

The effect of the adherence of the lipid droplet in the yolk‐sac on the growth and survival of the European hake (Merluccius merluccius) larvae was studied under starved and feeding conditions. Recently hatched hake larvae with adhered and non‐adhered lipid droplet (ALD and non‐ALD) were maintained during 8 days under starvation conditions. The adherence of the lipid droplet positively affected larval survival during starvation. At the end of the yolk consumption period (7–8 days after hatching), 90% of the surviving larvae had an ALD, while 10% of the survivors had a non‐ALD and showed a higher mortality rate. In a month feeding experiment, results show that the presence of non‐ALD in newly hatched hake larvae negatively affected their growth and survival. Dry weight and length (TL) of 20‐days‐old larvae with ALD were significantly higher than those with non‐ALD. Furthermore, lifespan of those larvae with non‐adhered lipid droplet was shorter, with most of them dying during the first 15–20 days of culture. As conclusion, the percentage of the lipid droplet adherence at hatching could be used as an indicator of ‘quality’ for European hake larvae: the greater the number of larvae with adhered lipid droplet, the better larval growth and survival.     

Early development of Lamprey Lampetra japonica (Martens, 1868)

Studies of lamprey early development have produced major insights into primitive developmental patterns and the evolution of vertebrates. In this study, the effectiveness of the morphological and histological characteristics of Lampetra japonica during embryological, yolk‐sac larval and ammocoete stages were examined. Based on the external and histological morphology, the embryological phase was divided into eight stages, including the zygote, cleavage, blastula, gastrula, neural plate and groove, head protrusion, prehatching and hatching stages. A set of key morphological and histological transformations occurred through the yolk‐sac larval stage, reflecting the larva‐ammocoete transition. In the ammocoete stage, all organ systems were differentiated. Except for the melanophores, the lamprey did not develop any further morphological complexity beyond the ammocoete stage. The total length and body weight of larva and ammocoete generally increased over time. We hope that this study might form the basis for future studies of the phylogenetic development of this interesting group of animals.     

Free Amino Acids in Pacu,Piaractus mesopotamicus,Eggs and Larvae

Free amino acids (FAA) are used principally as substrate in protein synthesis and the source of energy in aerobic catabolism. In marine fish, embryo and larvae FAA are used to maintain body fluid osmolality during fish early development. However, there is essentially no information about FAA concentrations in early ontogeny of freshwater neotropical species in comparison to marine fishes. Therefore, the aim of this study was to evaluate the FAA concentrations in pacu, Piaractus mesopotamicus, eggs and larvae. Broodstock fish were induced to spawn and ovulated females were stripped of their eggs and immediately sampled for analysis. Larvae were sampled right after hatching (HL) and after the completion of the yolk‐sac absorption (YSA). The wet weight of the HL and YSA larvae amounted to 0.5 ± 0.1 mg and 1.1 ± 0.3 mg, respectively. HL larvae showed higher levels of most of the indispensable amino acids (IAA) in comparison to eggs and YSA larvae. Exceptions were observed with His and Trp that showed higher or similar levels, respectively, in YSA larvae. The FAA Orn, Tau, Glu, Gln, Gly, and Tyr increased concentrations in both larval stages while that of Tau was found in higher concentration in all analyzed stages. Also, the concentrations of Asn, Ala, Pro, Ser, and Asp were higher in HL larvae. Both larval stages displayed a rise in total free IAA/total free DAA (dispensable amino acids) ratio. The authors conclude that the highest level of FAA in HL pacu larvae is indicative of active proteolysis of yolk reserves and a probable catabolism regulation of some FAA through spare‐effect. In addition, Tau is one of the major FAA occurring during pacu ontogeny and may be performing regulation on body fluid osmolality regulation .     

Development of digestive system and swim bladder of larval nase (Chondrostoma nasus L.)

Digestive tract and swim bladder differentiation during nase (Chondrostoma nasus L.) larval development was evaluated using histological methods. The mouth, pharynx and oesophagus were impervious at the time of hatching. The straight tubular intestine consisted of undifferentiated cells and was situated over the yolk sac. The intestine, lined with a single layer of columnar epithelial cells, opened on 2 day posthatching (dph). First evidence of protein digestion and absorption by the enterocytes was observed on the sixth day, while lipid digestion and absorption began on the ninth day of larval development. Intestinal mucous cells developed and became active by the fourth day. The posterior chamber of the swim bladder inflated on the third day, while the anterior one filled 12 dph. Complete yolk sac resorption took place on the ninth day.     

Effects of embryonic exposure to α‐lipoic acid or ascorbic acid on hatching rate and development of zebrafish (Danio rerio)

This study investigates the effects of embryonic exposure to two different antioxidants on growth and development in fish. Zebrafish (Danio rerio) embryos (100 per group) were exposed to lipoic acid (LA, 6–12 μM) or ascorbic acid (AA, 100–200 μM) and the hatching rate, standard lengths (SL) at hatching, development and growth post‐hatching monitored. The SLs at hatching were increased (P<0.05) in both antioxidant‐exposed groups relative to the controls, with no effect on yolk reserves. This enhanced development persisted up to 15 days post hatching. At hatching, cell proliferation rates (P<0.0005) and basic fibroblast growth factor (P<0.001), were greater in the antioxidant‐exposed fish than in the controls (0 μM antioxidant); no oxidative DNA damage was detected (P>0.05). Activity of the endogenous antioxidant enzyme superoxide dismutase was greater (P<0.001) in LA‐treated fish than in the controls. The results suggest that embryonic treatment of zebrafish with LA or AA during embryogenesis enhanced cell proliferation, leading to increased somatic growth in the larval stages, persisting into the juvenile stage. The findings support the treatment of embryonic fish with antioxidants for enhanced results in aquaculture.     

Compartmental changes in the contents of total lipid, lipid classes and their associated fatty acids in developing yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

Total lipid, lipid classes and their associated fatty acids have been measured in whole halibut, Hippoglossus hippoglossus (L.) larvae and in dissected animals separated into yolk and body compartments. At hatching the larval body contained 17 μg ind^?1 of lipid (11% of larval body dry weight), while the yolk contained 190 μg ind^?1. Phosphatidylcholine (PC) accounted for 57% of total yolk lipids while phosphatidylethanolamine (PE), triacylglycerol (TAG), cholesterol and sterol ester (SE) accounted for 12%, 12%, 9% and 6% respectively. The main fatty acids in the PC fraction were 22:6n-3 (25.6 μg ind^?1), 16:0 (19.2 μg ind^?1) and 20:5n-3 (12.6 μg ind^?1). Between hatch and 200 day-degrees post hatch (D°PH) a net decline in total lipids of 29% was seen. There seemed to be some, but relatively minor, changes in the relative composition of lipids in the yolk throughout development, which are indicative of a non-selective endocytotic bulk uptake of lipids from the yolk. Towards first-feeding there was a selective catabolism of PC and a net synthesis of PE in the developing body, resulting in a shift in the lipid class composition in the body compared with that of the yolk. The fatty acids released from lipid hydrolysis were mainly used as energy substrates by the growing halibut larvae; 22:6n-3 was quantitatively one of the most important fatty acid fuel in energy metabolism. At the same time 38% and 23% of the 22:6n-3 released from PC was retained by the PE and neutral lipids in the growing larval body respectively. Except for 20:5n-3 (2%, 14%) no similar retention was seen in any of the other fatty acids. The observed net synthesis of PE in developing yolk-sac larvae of Atlantic halibut and the preferential retention of 22:6n-3 into it, increasing from 28% at hatching to 45% at 200 D°PH, may point to a high biological value of this compound.     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

黄颡鱼早期发育阶段受精卵和鱼体脂肪酸组成变化

研究了黄颡鱼受精卵孵化期间和仔鱼发育阶段脂肪含量和脂肪酸的组成变化规律.采用常规化学分析方法和气相色谱法对黄颡鱼从鱼卵受精开始至仔鱼孵化后未投饵的7 d内的脂肪含量和脂肪酸组成进行测定.结果表明,受精卵在整个孵化期间脂肪含量有下降趋势.受精卵中不饱和脂肪酸含量大于饱和脂肪酸含量.受精卵在整个孵化期间各种脂肪酸含量无明显变化.仔鱼孵化后,鱼体总脂肪含量急剧下降,总脂含量从0日龄的4.57%降低到7日龄的0.75%.仔鱼在饥饿期间鱼体脂肪酸组成发生明显变化,单不饱和脂肪酸含量下降最为明显,尤其是C18:1.仔鱼在饥饿期间,脂肪酸按n-9>n-6>n-3顺序被先后利用,黄颡鱼仔鱼发育阶段主要以单不饱和脂肪酸作为能量代谢基质,而C20:4n6(AA)和C22:6n3(DHA)优先于C20:5n3(EPA)被保存下来.     

The effects of dietary supplements of polyunsaturated fatty acid on pearl oyster,Pinctada margaritifera L., gonad composition and reproductive output

Black‐lip pearl oyster, Pinctada margaritifera broodstock was collected from the wild. Egg production, hatching rate and larval development were compared between oysters induced to spawn within 2 days after collection in the wild (T1), oysters fed a pure microalgae diet during 24 days before spawning (T2) and oysters fed the same microalgal diet in which 10% of the algae were replaced with 2 μm polyunsaturated fatty acid (PUFA)‐rich microspheres (T3). Administration of lipid microspheres resulted in larger sized eggs, a higher percentage of D‐larvae and larger sized 48‐h‐old larvae (P<0.05). The total and neutral lipid contents of the gonad increased after oysters were fed with microalgae only or with supplementary diet. The major neutral and polar fractions of saturated fatty acid (SFA) were 16C and 18C fatty acids, and not influenced by the diet (P>0.05). The gonads of oysters fed supplementary PUFA contained more docosahexaenoic acid (DHA) and less monounsaturated fatty acids. Higher level of DHA in gonads of T3 was associated with oogenesis and embryogenesis success. The n‐3/n‐6 ratio in the neutral lipid fraction provides a good indication of the spawning condition and predicting egg size and hatching rate.     

雅砻江鲈鲤的人工繁殖、胚胎及卵黄囊仔鱼发育

采用不同配伍的外源激素对培育成熟的野生鲈鲤进行人工催产,并对其胚胎和1-10日龄卵黄囊仔鱼的发育进程进行详细的观察和描述。注射促黄体素释放激素类似物A2、地欧酮和绒毛膜促性腺激素的鲈鲤亲鱼组的催产效果明显好于注射鲤鱼脑垂体和促黄体素释放激素类似物A2的亲鱼组,效应时间为58h,平均受精率和平均孵化率分别为87.33%和90.75%。在水温(16±2)℃条件下,鲈鲤胚胎发育历时117.83h,所需积温为1855.33℃·h,出膜10d后仔鱼开始平游。鲈鲤胚胎发育分为受精卵、卵裂期、囊胚期、原肠胚期、神经胚期、器官形成期和出膜期7个阶段,共26个时期。分析发现,在人工催产配伍激素中添加适量的绒毛膜促性腺激素,有助于鲈鲤亲本顺利排卵;鲈鲤卵黄囊仔鱼在孵化出膜后的前5d处于内营养阶段,第6d起转入混合营养阶段,为仔鱼培育的第一危险时期。     

Fatty acid utilization during the early larval stages of Florida pompano (Trachinotus carolinus) and Common snook (Centropomus undecimalis)

The pattern of conservation and loss of fatty acids from the yolk sac during the endogenous feeding period and subsequent starvation was studied in pompano and snook larvae. Fundamental information on the early fatty acid dynamic and mobilization of pompano and snook larvae was collected. In both species, fatty acids were utilized as an energy source after hatching. Mono‐unsaturated fatty acids were catabolized, while saturated and poly‐unsaturated fatty acids were conserved. High levels of arachidonic acid (ARA) in pompano and snook eggs, as well as selective retention in the unfed larvae suggest a high dietary requirement for this fatty acid during the early stages of larval development. The effect of an ARA supplementation was therefore investigated in snook larvae at the rotifer feeding stage. The fatty acid profile of the larvae was successfully influenced to match that of wild eggs; however, no significant improvement in growth or survival was observed. Future research should be carried out over a longer period of time and include factors related to stress resistance.     

The effects of constant and oscillating temperature on embryonic development and early larval morphology in longfin yellowtail (Seriola rivoliana Valenciennes)

Constant and oscillating egg incubation temperatures on embryonic development and early larval morphology were studied in longfin yellowtail (Seriola rivoliana Valenciennes). We investigated the effects of constant temperatures from 16 to 32°C on embryo development and larval morphology at hatch, and whether oscillating temperature during embryogenesis could lead to larval morphological variations. After hatching, larval morphology and development during yolk sac (YS) utilization were examined in larvae at constant temperatures and larvae at 25°C that had oscillating temperature during egg incubation. Hatching rates were > 75%, only decreasing to ~ 50% at 30°C. At constant temperatures, the largest larvae occurred at 22 and 24°C. The oscillating temperature did not affect the timing of embryo development but resulted in larger and smaller larvae with a smaller and bigger YS, respectively, with a similar hatching time. Therefore, a growth response occurred in embryos during a window of development before hatching, depending on the adaptive response to temperature (spawn‐specific). After hatching, most of the YS was absorbed within 24 hr in all treatments, and the growth of the larval head was a priority with an optimal development at 26°C. There was compensatory growth in smaller larvae resulting in similar sizes after YS utilization, but larvae showed variations in body structure that could be important in further aquaculture research.     

Is batch variability in hatching time related to size heterogeneity and cannibalism in pikeperch (Sander lucioperca)?

Larval size heterogeneity is known to induce cannibalism, and procedures to avoid larval size differences are consequently implemented already during egg incubation and hatching. We investigated the relation between larval development variability, size heterogeneity and cannibalism in pikeperch. Larvae were sorted into five groups according to the time of hatching during a 65‐h period. The larvae with different times of hatch were then reared separately or together during an 18‐day period. Late hatched larvae were longer (P=0.003) and had less yolk remaining (P<0.001) than early hatched individuals at the time of hatching. However, on 11 days post fertilization, the late‐hatching larvae tended to have larger yolk reserves than earlier hatched individuals (P=0.07). Furthermore, the next day, a lower proportion in the late fraction had switched to exogenous feeding (P=0.024). That larvae with a late hatching time developed slower suggests a positive relationship between the hatching time and the embryonic developmental rate. However, differences in the length and available yolk reserves at hatching between larval fractions with different hatching times suggest that hatching is not strictly associated with a specific developmental stage, and that factors other than the development rate of the embryo are involved in the timing of hatching.     

Utilization of free amino acids, yolk proteins and lipids in developing eggs and yolk-sac larvae of walleye pollock Theragra chalcogramma

ABSTRACT:   To elucidate the utilization of the major yolk nutrient stocks in eggs and larvae of walleye pollock Theragra chalcogramma , the contents of free amino acids (FAA), the major yolk protein (180 kDa lipovitellin originated from vitellogenin B in ovulated eggs: oLv B), and lipids were measured. Most eggs hatched 18 days after fertilization at 5°C, and all larvae absorbed almost all their yolk mass by 28 days. The total FAA content showed no change during the first 6 days, and then decreased to 28% of the initial level by 18 days. The oLv B contents, measured by an enzyme-linked immunosorbent assay using a specific antiserum against oLv B, gradually decreased from 6 to 18 days, followed by a rapid decline. The content of phospholipids (PL) and triacylglycerols (TG) showed no marked change until hatching, and then decreased until disappearance of yolk sac. From these results, it is proposed that there are two main periods for nutrient utilization in embryos and larvae of walleye pollock. In the first period, FAA was mainly utilized until 18 days after fertilization. Active utilization of oLv B and lipids (PL and TG) instead of FAA occurred during the second period from 18 to 28 days.     

Annual variation in otolith increment widths of walleye pollock (Gadus chalcogrammus) larvae in Funka Bay,Hokkaido, Japan

To clarify relationships between year‐class strength and larval growth of walleye pollock (Gadus chalcogrammus), and oceanographic conditions in the Pacific stock off Hokkaido and Tohoku, Japan, we undertook conductivity/temperature/depth (CTD) observations and investigated larval densities, larval otolith increment widths and larval prey densities (of copepod nauplii) of the 2008, 2009, 2010 and 2011 yr classes in Funka Bay. Oyashio Coastal Water (OCW) flowed into the bay in late February in 2008, 2010 and 2011, and the mean water temperatures decreased to 1.9–3.1 °C in March. OCW was not observed in 2009, and it was warm in late February (≥3.4 °C). Increment widths of lapillar otoliths during the yolk‐sac stage were wide in 2009 and 2011, medium in 2010 and narrow in 2008. Increment widths during the first‐feeding stage tended to become wider as the hatch month progressed, and the annual variation during the first‐feeding stage was larger than that of the yolk‐sac stage. The densities of the primary food for the larvae were high in 2008 when larval increment widths were narrowest, so the effect of prey abundance on larval growth appeared to be small. The ranking of the larval abundance in March was nearly coincident with that of the increment width during the larval stage. We, therefore, suggest that the larval growth rate is associated with the mortality rate and that the growth–mortality hypothesis may be applicable to walleye pollock in Funka Bay. Feeding success under warm water conditions may be an important factor that contributes towards high growth rates.     

Digestibility of nutrients and amino acids of different protein sources in practical diets by largemouth bass Micropterus salmoides (Lacepéde, 1802)

This study evaluated the apparent digestibility coefficients (ADCs) of dry matter, protein, lipid, energy, calcium, phosphorus, and essential and non‐essential amino acid availability of animal and plant protein sources in practical diets for the carnivorous fish, largemouth bass Micropterus salmoides (8.0±0.5 g; 10.0±0.3 cm). Fish were conditioned to accept artificial feed and those stocked in plastic cages were fed pelleted test diets. Diets contained 30% of the ingredient tested mixed with 70% of a reference ration (RD) containing 40 g 100 g^?1 of crude protein, 19.85 kJ g^?1 crude energy, and 0.5% of chromic oxide. Cages were then transferred to cylindrical, conical‐bottomed aquaria (200 L) where the faeces were collected by sedimentation in a refrigerated container. All the tested ingredients had high ADCs for protein and lipid; ADCs of amino acids of varying protein sources showed significant differences (P<0.01). Plant protein sources did not significantly influence the ADCs of diets. The results allow the inference that the availability of amino acids in plant and animal protein sources varies considerably.     

红螯螯虾胚胎发育过程中的生化成分变化

采用生物化学方法对红螯螯虾胚胎发育过程中主要生化成分(糖类、蛋白质及氨基酸、脂类及脂肪酸)进行了测定和分析,以期了解胚胎形态发生与营养需求的关系.结果显示:红螯螯虾胚胎发育过程中糖类总体含量很低.总蛋白与总氨基酸(TAA)的含量在胚胎发育过程中均呈下降趋势.在必需氨基酸(EAA)中亮氨酸(Leu)和精氨酸(Arg)的含量较高,而在非必需氨基酸中谷氨酸(Glu)和天冬氨酸(Asp)的含量较高.总脂在胚胎发育过程中的含量总体呈下降趋势,其中性脂和磷脂的主要脂肪酸均为C16:0、C18:0 、C18:1ω9和C18:3ω3.研究认为:在红螯螯虾胚胎发育过程中,糖类主要参与多种特异复合物的合成以及甲壳构建,这些特异复合物在细胞分化中起着重要的信号传递作用.蛋白质和脂类都是红螯螯虾胚胎发育过程中主要的结构和能源物质,蛋白质主要在早期被利用而脂类主要在晚期起作用.饱和脂肪酸(SFA)和单不饱和脂肪酸(MUFA)(C16:0,C18:0,C16:1ω7 和 C18:1ω9)主要作为能源物质被消耗而高不饱和脂肪酸(PUFA)(20:5ω3和22:6ω3)在胚胎发育过程中对于构建细胞膜以及调节中枢神经系统的发育起重要作用.     

Biochemical and technical observations supporting the use of copepods as live feed organisms in marine larviculture

The use of live prey is still necessary for a large number of raised fish species. Small sized rotifers are usually used as live preys during the first days of feeding in small mouth fish. An alternative to this is the use of copepods as prey for first feeding. In this study, the sizes, weight and biochemical contents of two copepods and one rotifer species raised on similar algal food conditions were compared. Rotifers contained a higher proportion of essential amino acids in the free amino acid (FAA) fraction (43%) than copepods (30–32%). However, rotifers had lower levels of important fatty acids like DHA (7% compared with 23–32% in copepods) and their DHA/EPA ratio was lower than that in copepods (0.54 compared with 1.35–1.63 in copepods). The FAA pattern of the preys tended to be species‐specific and its implications from an aquaculture point of view is discussed. In contrast, the‐protein bound amino acids tended to be very conservative among the studied organisms. The second part of the work is focused on ‘the price’ of hatching in Acartia tonsa eggs before or after cold storage at 3°C. The fatty acid contents in A. tonsa tended to decrease with the storage time. It also decreased with hatching of the nauplii, but its proportion compared with the dry weight remained constant.