In vitro amino acid absorption using hydrolysed sardine muscle or soybean meal at different intestinal regions of the Pacific bluefin tuna (Thunnus orientalis)

The amino acid (AA) absorption along the intestinal tract of the Pacific bluefin tuna (Thunnus orientalis) was evaluated using two hydrolysed protein sources (fresh sardine muscle and soybean meal) with the everted intestine technique. Pork pepsin and pancreatic enzyme extract from the bluefin tuna were used to hydrolyse the protein from fresh sardine (FSH) and soybean meal (SMH) under optimal bluefin tuna fish physiological conditions. Both of the hydrolysate solutions were tested within three intestinal sections from the bluefin tuna. The everted intestinal fractions immersed in the hydrolysate solutions were sampled at different times to analyse for AA and absorption rate calculations. Fresh sardine and SMH contained greater amounts of essential amino acids (EAA) than those of non‐essential amino acids (NEAA); however, the profiles of AA absorbed showed higher absorption of NEAA in both cases. Using a similar concentration solution, the absorption rates within the intestinal fractions showed a preferential absorption in the proximal and distal regions for Arg and His when FSH was used. However, the absorption rates for Lys resulted in a decreasing proximal‐to‐distal gradient between the different intestinal regions for FSH and SMH. The possibility of a catabolic role of certain AAs in the enterocytes being able to explain the differences in absorption is discussed.     

美济礁附近海域3种金枪鱼肌肉成分检测与营养评价

为分析中国南海美济礁附近海域大目金枪鱼(Thunnus obesus)、蓝鳍金枪鱼(T.thynnus)、黄鳍金枪鱼(T.albacares)的营养成分与营养价值,该研究采用国标法检测了3种金枪鱼的水分、蛋白质、灰分、脂肪酸和氨基酸成分及其结构,并进行营养评价.结果显示,3种金枪鱼中黄鳍金枪鱼蛋白质含量最高,蓝鳍金枪鱼...     

Intestinal absorption of amino acids in the Pacific bluefin tuna (Thunnus orientalis): in vitro lysine–arginine interaction using the everted intestine system

The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL^?1). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.     

Comparison of the lipid profiles from wild caught eggs and unfed larvae of two scombroid fish: northern bluefin tuna (<Emphasis Type="Italic">Thunnus thynnus</Emphasis> L., 1758) and Atlantic bonito (<Emphasis Type="Italic">Sarda sarda</Emphasis> Bloch, 1793)

Lipids and essential fatty acids are determinants of the reproductive process in marine fish, affecting fecundity, egg quality, hatching performance, pigmentation and larval malformation. We have analyzed and characterized the lipids of eggs and unfed larvae of two wild caught scombroid fish, the Atlantic northern bluefin tuna (Thunnus thynnus) and Atlantic bonito (Sarda sarda). Dry matter and total lipid contents, polar and neutral lipid classes and total lipid fatty acid contents were determined in the eggs of bluefin tuna and eggs and unfed larvae during the development of Atlantic bonito. Bluefin tuna eggs had slightly but significantly more dry mass than bonito eggs but very similar lipid content. However, bluefin tuna eggs presented a higher polar lipid content due to increased proportions of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). Bonito eggs and larvae showed increasing dry mass and decreasing lipid content with development. The proportion of polar lipids increased due to increased PE, PS and PI, whereas choline-containing polar lipids (phosphatidylcholine and sphingomyelin) remained relatively constant. Free cholesterol also increased, whereas the levels of other neutral lipids, especially triacylglycerol and steryl ester fractions, decreased, presumably due to utilization for energy to drive development. Bluefin tuna eggs had higher levels of n − 3 and n − 6 highly unsaturated fatty acids due to higher docosahexaenoic and arachidonic acid contents, respectively, than bonito eggs. The results are discussed in relation to the lipid and fatty acid requirements of larval scombroid fish in comparison to those of other larval marine finfish species under culture conditions.     

Biochemical Characterization and Comparison of Pancreatic Lipases from the Pacific Bluefin Tuna,Thunnus orientalis; Totoaba,Totoaba macdonaldi; and Striped Bass,Morone saxatilis

Lipolytic activity from pancreatic crude extracts from the Pacific bluefin tuna (PBT), Thunnus orientalis; totoaba, Totoaba macdonaldi; and striped bass, Morone saxatilis, were biochemically characterized to detect possible differences in the digestive capacity of each fish. The maximum activity was at temperatures ranging from 35 to 45 C and at pH 8.0; the activity decreased significantly at a pH lower than 7.0. Lipases from PBT and totoaba showed an increase in activity with the presence of sodium taurocholate (NaTC; 6 mM). However, no increase in activity could be observed when a mixture of bile salts or natural bile extracts was used in the reaction mixture, which was observed in the presence of NaTC. In contrast, the lipase activity from striped bass increased accordingly when NaCl was added at different levels (0–1.5 M). Calcium was not required in the reaction mixture for any of the fish species tested. We concluded that all three lipases have different physicochemical properties and ionic strength and emulsifier requirements to obtain their maximum activity. Striped bass was far too sensitive to NaCl, while totoaba and PBT were sensitive to NaTC. The differences in activity shown here suggest the possibility of studying the use of different additives on feed formulations to maximize lipase efficiency.     

Mortality processes of hatchery‐reared Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel) larvae in relation to their piscivory

In mass culture of Pacific bluefin tuna Thunnus orientalis, yolk‐sac larvae of other species are fed as a major prey item to tuna larvae from 7 to 8 mm in total length. Marked growth variations in tuna larvae are frequently observed after feeding of yolk‐sac larvae, and this variation in the growth of tuna larvae is subsequently a factor leading to the prevalence of cannibalistic attacks. To elucidate details of the mortality process of hatchery‐reared tuna larvae after the initiation of yolk‐sac larvae feeding, we compared the nutritional and growth histories of the surviving (live) tuna larvae to those of the dead fish, found dead on the bottom of the tank, as direct evidence of their mortality processes. Cause of mortality of tuna larvae 3 and 5 days after the initiation of feeding of yolk‐sac larvae was assessed from nitrogen stable isotope and otolith microstructure analyses. Stable isotope analysis revealed that the live fish rapidly utilized prey fish larvae, but the dead fish had depended more on rotifers relative to the live fish 3 and 5 days after the initiation of feeding of yolk‐sac larvae. The growth histories based on otolith increments were compared between the live and dead tuna larvae and indicated that the live fish showed significantly faster growth histories than dead fish. Our results suggest that fast‐growing larvae at the onset of piscivory could survive in the mass culture tank of Pacific bluefin tuna and were characterized by growth‐selective mortality.     

Molecular characterization of southern bluefin tuna myoglobin (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

The primary structure of southern bluefin tuna Thunnus maccoyii Mb has been elucidated by molecular cloning techniques. The cDNA of this tuna encoding Mb contained 776 nucleotides, with an open reading frame of 444 nucleotides encoding 147 amino acids. The nucleotide sequence of the coding region was identical to those of other bluefin tunas (T. thynnus and T. orientalis), thus giving the same amino acid sequences. Based on the deduced amino acid sequence, bioinformatic analysis was performed including phylogenic tree, hydropathy plot and homology modeling. In order to investigate the autoxidation profiles, the isolation of Mb was performed from the dark muscle. The water soluble fraction was subjected to ammonium sulfate fractionation (60–90 % saturation) followed by preparative gel electrophoresis. Autoxidation profiles of Mb were delineated at pH 5.6, 6.5 and 7.4 at temperature 37 °C. The autoxidation rate of tuna Mb was slightly higher than that of horse Mb at all pH examined. These results revealed that tuna myoglobin was unstable than that of horse Mb mainly at acidic pH.     

Horizontal and vertical movements of juvenile bluefin tuna (Thunnus orientalis) in relation to seasons and oceanographic conditions in the eastern Pacific Ocean

Electronically tagged juvenile Pacific bluefin, Thunnus orientalis, were released off Baja California in the summer of 2002. Time‐series data were analyzed for 18 fish that provided a record of 380 ± 120 days (mean ± SD) of ambient water and peritoneal cavity temperatures at 120 s intervals. Geolocations of tagged fish were estimated based on light‐based longitude and sea surface temperature‐based latitude algorithms. The horizontal and vertical movement patterns of Pacific bluefin were examined in relation to oceanographic conditions and the occurrence of feeding events inferred from thermal fluctuations in the peritoneal cavity. In summer, fish were located primarily in the Southern California Bight and over the continental shelf of Baja California, where juvenile Pacific bluefin use the top of the water column, undertaking occasional, brief forays to depths below the thermocline. In autumn, bluefin migrated north to the waters off the Central California coast when thermal fronts form as the result of weakened equatorward wind stress. An examination of ambient and peritoneal temperatures revealed that bluefin tuna fed during this period along the frontal boundaries. In mid‐winter, the bluefin returned to the Southern California Bight possibly because of strong downwelling and depletion of prey species off the Central California waters. The elevation of the mean peritoneal cavity temperature above the mean ambient water temperature increased as ambient water temperature decreased. The ability of juvenile bluefin tuna to maintain a thermal excess of 10°C occurred at ambient temperatures of 11–14°C when the fish were off the Central California coast. This suggests that the bluefin maintain peritoneal temperature by increasing heat conservation and possibly by increasing internal heat production when in cooler waters. For all of the Pacific bluefin tuna, there was a significant correlation between their mean nighttime depth and the visible disk area of the moon.     

养殖和野生亚东鲑机体成分比较分析

亚东鲑(Salmo trutta fario)是鲑属鱼类在青藏高原仅有的鱼类种群,是亚东地区全国农产品地理标志产品。本研究对西藏亚东地区野生和养殖亚东鲑的常规营养成分及各组织氨基酸和脂肪酸组成进行分析,旨在比较野生和养殖亚东鲑营养成分的异同,为养殖亚东鲑的品质评价和饲料配方的完善提供参考信息。实验采集亚东河中野生亚东鲑和亚东渔业产业园中使用配合饲料养殖的亚东鲑各10尾用于相关成分分析,每尾为一个独立样本。结果显示,野生组肥满度显著低于养殖组,而全鱼水分和灰分显著高于养殖组。全鱼粗蛋白、粗脂肪以及肝脏常规成分在野生组和养殖组间无显著差异。野生组肌肉粗脂肪显著低于养殖组,而水分含量显著高于养殖组。野生组全鱼必需氨基酸总量显著高于养殖组,且野生组肌肉中苏氨酸、缬氨酸、苯丙氨酸、赖氨酸和甘氨酸含量显著高于养殖组。野生组全鱼、肌肉和肝脏中饱和脂肪酸、n-3多不饱和脂肪酸(n-3 PUFA)总量、EPA、C20:4n-6以及肌肉中DHA含量高于养殖组,而全鱼和组织中单不饱和脂肪酸和n-6 PUFA总量低于养殖组。综上所述,目前养殖亚东鲑和野生亚东鲑在机体成分上存在较大差异。由于养殖鱼类体成分很大程度上反映了饲料组成,因此,亚东鲑养殖中饲料营养组成可能有待进一步优化。     

Universal PCR primers for ribosomal protein gene introns of fish

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.     

Partial characterization of the digestive enzymes of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> under culture conditions

The digestive enzyme activities of Pacific bluefin tuna Thunnus orientalis were evaluated for specific activity and characterized for pH and temperature optima in crude extracts of stomach, caecal mass, and proximal, middle and distal intestine. A higher level of alkaline proteolytic activity was detected in the caecal mass than in the proximal intestine. Total alkaline proteases, trypsin, chymotrypsin and leucine aminopeptidase (LAP) were tested. The temperature and pH analyses showed that proteolytic activity as well as lipase were maximal in the alkaline range, with a maximum at pH 9.0 and at temperatures between 35 and 60°C, except for the pepsin, which showed maximum activity at the same temperatures but in the acid range (pH 3.0). The α-amylase activity showed a broader range in activity, both for pH and temperature, with higher activity over the alkaline pH values and higher temperature. The lipase activity seems to be nondependent on bile salts under our assay conditions, resulting in a significant activity reduction in the presence of bile salts. This knowledge will allow the development of a gastrointestinal model (everted intestine) where food or feed will be hydrolysed with the fish’s own enzymes, a project that is being undertaken in our laboratory as a contribution to the development of novel diets for tuna fish.     

Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish

ABSTRACT:   In the present study, the cDNA encoding myoglobin (Mb) of bigeye tuna Thunnus obesus was cloned and its amino acid sequence deduced in order to investigate the relationship between the primary structure and thermostability of scombridae fish Mb. An open reading frame of bigeye tuna Mb cDNA contained 444 nucleotides encoding 147 amino acids. The primary structure of bigeye tuna Mb was highly conserved when compared with those of bluefin tuna and yellowfin tuna Mb, the sequence identity being 95.2–100.0%. It also showed relatively high identity (82.3–89.1%) with the counterparts of scombridae fish. Myoglobin was then isolated from the dark muscle of four scombridae fish including bigeye tuna. Differential scanning calorimetry and circular dichroism measurements on these Mb revealed that the thermostability of bigeye tuna Mb was lowest and that of skipjack Katsuwonus pelamis Mb highest among the scombridae fish Mb examined. The α-helical contents of scombridae fish Mb at 10°C were in the range of 39.8–44.8%, clearly lower than that of horse Mb (55.3%), suggesting instability of fish Mb. The melting temperatures of these Mb fell in the range of 75.7–79.9°C, lower than that of horse Mb (84.2°C). These results strongly suggest the instability of fish Mb.     

Purification and characterization of glutathione peroxidase 1 in the red muscle of Pacific bluefin tuna Thunnus orientalis

Glutathione peroxidase GPx1 was purified from the red muscle of Pacific bluefin tuna Thunnus orientalis and its enzymatic properties were characterized. The enzyme was optimally active at pH 7.4 with a specific activity for hydrogen peroxide and a K _m of 6.7 μM. cDNA was also isolated and it contained a predicted open reading frame (ORF) encoding a 188 amino acid protein. The phylogenetic tree shows that fish including Pacific bluefin tuna, pufferfish, and zebrafish, but not mammals, possess two genetically distinct types of GPx1, i.e., GPx1a and GPx1b. The purified enzyme was classified as a fish GPx-1b enzyme on the basis of the phylogenetic tree of the GPx1 family.     

Effect of water temperature on the feeding activity and the resultant mercury levels in the muscle of cultured bluefin tuna Thunnus orientalis (Temminck and Schlegel)

Tuna muscle often contains high levels of mercury, and fish samples with mercury concentrations ten times higher than the specified safety standards have been reported. Here, we report on the relationship between water temperature and the concentration of mercury in the tail muscle tissue of cultured bluefin tuna Thunnus orientalis. The fish used in this study were cultured at Fisheries Laboratory of Kinki University (Amami Experimental Station, Kagoshima, Japan). One hundred fish weighing 26.2–89.4 kg were selected for analysis between February 2007 and January 2008. Water temperature during rearing ranged from 21 to 29 °C. The total mercury levels were measured using the reduction vaporizing atomic absorption method after acid digestion. Body weight increased approximately 1.5 times that observed in a previous study, despite feeding activity either being the same or less than that observed previously. The average mercury concentration in white muscle was 0.353 mg kg^?1, remaining almost constant and independent of body growth. Unlike previous studies, seasonality was not observed in this study. Based on these findings, water temperatures within a certain range were considered to stabilize feeding activity and increase feeding efficiency. Consequently, water temperature is considered to have a moderating effect on seasonal fluctuations in muscle mercury concentrations in cultured bluefin tuna.     

Gustatory responses in Pacific bluefin tuna Thunnus orientalis (Temminck and Schlegel)

Gustatory neural responses of the Pacific bluefin tuna Thunnus orientalis (Temminck and Schlegel) to extracted compounds of prey organisms, such as amino acids, nucleotide‐related substances, organic acids and organic bases, were electrophysiologically recorded from the facial nerve supplying the anterior palate. Of the 17 amino acids tested, l ‐proline was the most potent amino acid at 10^?2 M, and its threshold was the lowest at around 10^?6 M. l ‐leucine, l ‐methionine, l ‐alanine, l ‐valine and l ‐isoleucine were also highly stimulatory at 10^?2 M; however, the other 11 amino acids examined were not as effective or were ineffective. Thus, the gustatory receptors for amino acids of the Pacific bluefin tuna show a narrowly tuned response profile. Among the seven nucleotide‐related substances tested, uridine‐5′‐monophosphate, inosine‐5′‐monophosphate and adenosine‐5′‐diphosphate were highly stimulatory, and their thresholds were 10^?4–10^?5 M. Inosine elicited a positive response at 10^?2 M but its response magnitude was not so high. Organic acids l ‐lactic and pyruvic acids were effective at 10^?2 M, but no response was elicited at 10^?3 M. Among organic bases, betaine was highly stimulatory, and its response magnitude at 10^?2 M is almost equal to that of l ‐proline at the same concentration. The threshold for betaine was determined to be at around 10^?5 M. Trimethylamine oxide and ammonium chloride were ineffective.     

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.     

Bluefin tuna (Thunnus thynnus) distribution in relation to sea surface temperature fronts in the Gulf of Maine (1994–96)

Fishery‐linked aerial surveys for bluefin tuna (Thunnus thynnus) were conducted in the Gulf of Maine (GOM) from July through October, 1994–96. Each year, from 507 to 890 surface schools were detected and their locations examined in relation to oceanographic conditions. Correlations between bluefin tuna presence and environmental variables were explored for sea surface temperature (SST), distance to a SST front, frontal density (relative density of all SST fronts seen in a given 1 km area for 2 weeks prior to each tuna sighting), and bottom depth and slope. Mean SST associated with bluefin schools was 18.1°C (±2.8). Schools were located at a mean distance of 19.7 km (±19.6) from SST fronts, and in water masses with an average frontal density of 28.2 m km^?2 (±35.7). Mean bottom depth of detected schools was 139.0 m (±70.3), and mean bottom slope was 0.7% rise (±0.7). A binomial generalized linear model fit to these variables indicated that bluefin are seen closer to fronts than locations in which no tuna were seen. Using simple and partial Mantel tests, we investigated the spatial correlation between bluefin tuna presence and the environmental variables, controlling for spatial autocorrelation. For each day that schools were sighted, we performed 24 Mantel tests, on a combination of response and predictor variables. The spatial relationship between bluefin tuna and SST fronts was inconsistent. Our analysis identified significant spatial structure in the bluefin school locations that had no significant correlation with any of the measured environmental features, suggesting that other untested features, such as prey density, may be important predictors of bluefin distribution in the GOM.     

Absorption of glucose, amino acids, and dipeptides by the intestines of Atlantic salmon (Salmo salar L.)

Rates of glucose, amino acid and dipeptide absorption by the intestine of Atlantic salmon (Salmo salar L.) were measured in vitro at 10 °C as functions of concentration and region using intact tissues (everted sleeve method). Salmon (weight range 300–1300 g) fed a commercial, extruded salmon feed, were kept at 12–13 °C in freshwater. Maximum rates (V _max) of glucose and dipeptide transport were low compared to most amino acid V _max values. There was a declining proximal-to-distal gradient of absorption along the post-gastric intestinal tract. A saturable component of absorption was not evident for proline and glycyl-proline in the distal intestine, and glycyl-sarcosine in any region. `Apparent diffusion', which may include low affinity, high capacity carrier systems and carrier-independent influx, appears to account for the majority of total uptake at higher concentrations of amino acids and dipeptides. There was competition between the dipeptides for transporter sites in the pyloric ceca and mid intestine, suggesting a common carrier. There was also indication of hydrolysis of these dipeptides by brush border membrane enzymes.