Effects of Dietary Bovine Lactoferrin on Growth Performance and Immuno-physiological Responses of Asian Sea Bass (Lates calcarifer) Fingerlings

Probiotics and Antimicrobial Proteins - The aim of this study was to evaluate the effects of lactoferrin (Lf) on growth and feeding performance, biochemical and immune parameters in Asian sea bass...     

Novel Microsatellites from Asian Sea Bass (Lates Calcarifer) and Their Application to Broodstock Analysis

We isolated novel dinucleotide, trinucleotide, and tetranucleotide microsatellites from the genome of Asian sea bass (Lates calcarifer). Two genomic DNA libraries were established, one was enriched for (CA)n repeats, while the other for (GATA)n, (GACA)n, and (AAC)n repeats. Sixty clones containing an insert between 250 and 1000 bp in size were sequenced from each library; altogether 50 (43%) of them contained microsatellites. Forty microsatellites were characterized in 16 unrelated Asian sea bass individuals. Twenty-eight of them (70%) showed specific amplification and polymorphism. The allele number per loci varied between 2 and 20 with an average of 5.3, while expected heterozygosity ranged from 0.31 to 0.95 with an average of 0.64. At some loci allele sizes spread over a wide range (>100 bp). No significant correlation (r = 0.23, df = 31, P > 0.05) was found between the repeat number and the number of alleles. A whole broodstock containing 170 individuals was analyzed by using 8 selected polymorphic microsatellites. The average number of alleles per locus was 11.8 (range, 4–21). The expected heterozygosity ranged from 0.57 to 0.90 with an average of 0.75, while the fixation index was 0.02. Genetic similarity between individuals ranged from 0 to 0.72. Comparison of allele frequencies between the broodstock and the 24 nonrelated individuals revealed some unique alleles.     

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer)

Two separate experiments were conducted to determine the dietary requirements of juvenile Asian sea bass Lates calcarifer Bloch for lysine and arginine. Fish (average initial weight: lysine experiment, 13.12 ± 0.12 g; arginine experiment, 2.56 ± 0.13 g) were given amino acid test diets for 12 weeks containing fish meal, zein, squid meal, and crystalline amino acids. Each set of isonitrogenous and isocaloric test diets contained graded levels of L ‐lysine or L ‐arginine. The feeding rate in the lysine experiment was at 4–2.5% of the body weight day^?1, while in the arginine experiment it was at 10–4% of the body weight day^?1. The fish (20 per tank, lysine experiment; 15 per tank, arginine experiment) were reared in 500‐L fibreglass tanks with continuous flowthrough sea water at 27 °C and salinity of 31 ppt in the lysine experiment and at 29 °C and salinity of 29 ppt in the arginine experiment. The experiments were in a completely randomized design with two replicates per treatment. Survival was high in fish given adequate lysine or arginine. Mean percentage weight gains were significantly different in fish fed varying levels of lysine or arginine. Fish fed high levels of L ‐arginine suffered high mortalities. No significant differences were obtained in the feed efficiency ratios (FER, g gain g^?1 feed) of fish fed graded lysine, although the values tended to increase as the dietary lysine level was increased up to the requirement level. In contrast, in the arginine experiment, significant differences in FER of fish among treatments were obtained; the highest FER was observed in fish fed the diet containing an optimum arginine level. On the basis of the growth response, survival, and FER, the lysine and arginine requirements of juvenile Asian sea bass were estimated to be 20.6 g kg^?1 dry diet (4.5% protein) and 18.2 g kg^?1 dry diet (3.8% protein), respectively. These data will be useful in the further refinement of practical diet formulations for the Asian sea bass.     

Chilled storage of Asian seabass,Lates calcarifer Bloch semen: Effects of ions,extenders and storage periods on sperm quality and fertilization ability

The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non-ionic solutions, extender types (Ringer's solution, Ca-F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled-storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non-ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl₂ (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl₂, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled-stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non-ionicity effects on the motility of Asian seabass sperm.     

Bioactivity of stored luteinizing hormone-releasing hormone analogue (LHRHa) in sea bass, Lates calcarifer Bloch

The spawning induction activity of dissolved and pelleted (D-Ala⁶, Pro⁹ N ethylamide) luteinizing hormone-releasing hormone analogue (LHRHa) stored for various periods was assessed in mature female sea bass. The spawning response of mature fish was reduced significantly after injection of dissolved LHRHa (20 μg kg⁻¹) stored for more than 90 days in a refrigerator (4–10°C) or for more than 30 days at room temperature (28–30°C). Similar to fish administered fresh preparations of LHRHa, fish spawned successfully after injection of a solution of LHRHa previously frozen, subjected to alternate freezing and thawing, exposed to sunlight or implanted pelleted LHRHa (50 ng kg⁻¹) stored at room temperature for 30–120 days. Loss of hormone bioactivity after prolonged storage may have been due to bacterial growth in solubilized preparations. Injection or implantation of stored LHRHa did not influence egg production among treated sea bass. These results demonstrated the relatively prolonged shelf life of stored LHRHa.     

Cloning and characterization of the calreticulin gene in Asian seabass (Lates calcarifer)

Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. We cloned and characterized the CRT gene in an important marine food fish species Asian seabass (Lates calcarifer). The full-length DNA of the CRT gene was 2194 bp, including a complete open reading frame encoding 420 amino acid residues, a 113 bp 5'-untranslated region and an 818 bp 3'-untranslated region. The CRT gene contained nine exons and eight introns covering a total of 2772 bp genomic DNA from the start to stop codon. Ten single nucleotide polymorphisms (SNPs) were detected in introns and an exon in six individuals collected from five different locations. The CRT gene was assigned to linkage group 4 of the linkage map of Asian seabass. Quantitative real-time PCR revealed that the CRT gene was highly expressed in liver at the age of 1, 3 and 7 months under normal conditions, whereas its expression in liver reduced sharply after 0.5 to 2 h cold challenge at 16°C, and then increased slowly. A preliminary association analysis showed a significant (P < 0.001) association between the SNP6 in the CRT gene and the mortality after cold challenge at 16°C. Our results suggest that the CRT gene is associated with cold tolerance of Asian seabass and further investigation will be necessary to illustrate the underlying mechanisms.     

The use of sectioned otoliths to age barramundi (Lates calcarifer) (Bloch, 1790) [Centropomidae]

The relationship between the number of rings present in sagittal otoliths and the age of barramundi, Lates calcarifer (Bloch, 1790) [Centropomidae], was investigated by examining cross sectioned otoliths of 37 tagged fish of known age between 1 and 5 years from the Johnstone River, north Queensland. Concentric rings were clearly visible in all otolith sections and were validated as annual marks. The technique was then used to estimate the age and calculate von Bertalanffy growth parameters for 70 barramundi from the Fitzroy River, central Queensland. Growth appeared to be rapid but variable in the first year; the von Bertalanffy growth parameters for length versus age were L =690 mm, K=0.53, t ₀= 0.003 years. October 1 was designated as the birth date. Whole otolith length, width and thickness were also approximated well by the von Bertalanffy equation. We suggest that examination of otoliths is a useful technique for ageing barramundi but note that further validation of the ageing method is still needed for fish older than six years.     

Methodology in the study of seabass (Lates calcarifer Bloch): nutritional requirements in Singapore

This paper describes the effort of the Marine Aquaculture Section of the Primary Production Department in Singapore and the Institut Francais de Recherche pour l'Exploitation de la Mer in developing adequate facilities for fish nutritional requirement studies in Singapore, and in introducing appropriate nutritional methodology so that results are reliable and accurate. One of the first achievements was the demonstration of homogeneous fish (tropical seabass, Lates calcarifer Bloch) growth response in a newly established tank system before any nutritional experiments were initiated. The application of statistical procedures that take into consideration the power of a test and number of replications required has allowed for more accurate interpretation of results. This can be seen in the results of the first nutritional requirement experiment on the optimal dietary protein level of the seabass at constant energy.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

Ontogenetic eye development and related behavioural changes in larvae and juveniles of barramundi Lates calcarifer (Bloch)

Larvae and juveniles of barramundi Lates calcarifer (Bloch) were examined for the development of the retina, occurrence of the retinomotor response and retinal tapetum and change in eye size with age in days. The barramundi hatched with unpigmented non-functional eyes in which retinal cells had not yet differentiated into the various elements. Soon it was followed by rapid changes in the histology of the retina. Two-day-old larvae had a well-pigmented retina with area temporalis which would allow acute vision and prey attack in the nasal direction. At 10 days, rod cells and the retinal tapetum first appeared in the retina and the retinomotor response first occurred; these would allow feeding in dim light. The retinal tapetum moved in unison with the cones and the pigment epithelium during the retinomotor response. At 26 days, the horizontal cells were divided into two layers and the twin cones appeared. These changes in the eyes occurred concurrently or in anticipation of behavioural changes, such as the onset of the first feeding at 2 days, the shift of habitat from coastal waters to swamps at the notochord flexion stage at 7–15 days, the abrupt change in feeding behaviour from roving zooplanktivore to lurking predator at 25–30 days and a later shift of habitat from turbid swamps to open coastal or lake areas at the early juvenile stage.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

DNA barcoding reveals a likely second species of Asian sea bass (barramundi) (Lates calcarifer)

DNA barcoding – the sequencing of a c. 650 base pair region of the mitochondrial cytochrome c oxidase I gene – strongly suggests that barramundi ( Lates calcarifer ) from Australia and from Myanmar are different species (Kimura 2 parameter distance of c. 9·5%). Cytochrome b sequence data support this conclusion (distance c. 11·3%). Further examination, both genetic and morphological, of L. calcarifer throughout its range is recommended.     

An inexpensive tag for short-term studies in milkfish (Cbanos chanos Forsskal) and in seabass (Lates calcarifer Bloch)

An opercular tag for marking adult milkfish ( Chanos chanos Forsskal) and seabass ( Lates calcarifer Bloch) is described. High tag retention and relatively low mortality rates were observed in adult fish handled two to ten times during 14-to 60-day tests. The features and advantages of the tag for marking large-sized fish in short-term studies are discussed.

Zusammenfassung

Eine preiswerte Markierung für Kurzzeitstudien des Milchfisches (Chanos chanos Forsskal) und der Centropomidae (Lates calcarifer Bloch)
Eine Kiemendeckel-Markierung für adulte Milchfische (Chanos chanos Forsskal) und Centropomidae (Lates calcacifer Bloch) wird beschrieben. Sie zeichnet sich durch gute Haltbarkeit aus und verursacht relativ geringe Mortalität bei adulten Fischen, die in Versuchen von 14 bis 60 Tage Dauer 2-bis 10mal untersucht wurden. Die Eigenschaften und Vorteile dieser Markierung für große Fische in Kurzzeitstudien werden diskutiert.

Résumé

Un marquage économique pour des études de courte durée du chanidé (Chanos chanos Forsskal) et du centropomidé (Lates calcarifer Bloch)
Un marquage d'opercule pour les chanidés (Chanos chanos Forsskal) et les centropomidés (Lates calcarifer Bloch) adultes est décrit. Une bonne conservation et une mortalité relativement basse ont été observées chez des poissons adultes examinés 2 à 10 fois pendant des expériences d'une durée de 14 à 60 jours. Les caractéristiques et les avantages du marquage de poissons de grande taille pendant des expériences d'une courte durée sont discutés.     

Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species. Received April 10, 2000; accepted July 13, 2000.     

Assessment of growth characteristics,the survival rate and body composition of Asian sea bass Lates calcarifer (Bloch, 1790) under different feeding rates in closed aquaculture system

One of the essential factors to be addressed in the development of aquaculture is the feeding regime. This study was investigated to assess the effects of feeding rate on growth performance, feed utilization, chemical body composition survival rate, cannibalism and morphological indices of Asian Seabass, Lates calcarifer. Intended for the trial, one hundred forty sea bass individuals with an average weight 5.47 ± 0.11 g were randomly distributed in 4 concrete tanks (914 cm × 183 cm 122 cm) each; length × width × depth) and volume 18,399 L, for total 68 days. The fishes were fed with a pelleted diet containing 46% crude protein for different feeding groups designated as (T1, T2, T3 and T4). The feed was supplied with a rate of (T1) 3%, (T2) 4%, (T3) 6%, and (T4) 9% of fish biomass per day and feeding frequency were maintained three times per day to all the groups. At the end of the trial water physicochemical parameters was in acceptable range for Asian sea bass growth. The average daily weight gain (g), weight gain (g) and specific growth rate (%) was significantly higher (p < 0.05) in T3 and T4 as compared to T1 and T2 group. The poorest feed conversion ratio was recorded in T1 group with 3% biomass per day. The cannibalism rate was significantly (p < 0.05) higher in T1 (3%) compared to T3 and T4 treatment. The morphological indices, condition factors (CF), viscerosomatic index and hepatosomatic index (HSI) was significantly higher in T4 group as compared to other treatments. The protein, moisture and ash contents of the whole biomass of the Asian sea bass were not significantly influenced by feeding rate. The fat levels in the fish bodies increased significantly (p < 0.05) with increasing feeding ratio. The phenomenal regression indicates that 6.5% feeding rate per day is optimum for best growth performance, survival and minimum cannibalism rate for Asian sea bass in captivity. The outcome of the finding will help in promotion for not only the coastal aquaculture in Pakistan abut also elsewhere.     

Lunar synchronization of spawning in sea bass, Lates calcarifer (Bloch): effect of luteinizing hormone-releasing hormone analogue (LHRHa) treatment

Based on egg collection records, spontaneous spawning activity of sea bass, Lates calcarifer (Bloch), reared in floating net cages followed a semilunar cycle. The peak of multiple spawnings coincided with declining spring tides of quarter moon periods. Maximum diameter of intra-ovarian, ripe oocytes (0.51–0.55 mm) occurred in synchrony with the quarter moon periods. Smaller oocytes (0.44–0.47 mm) were sampled during the new and full moon periods. Two structural analogues of luteinizing hormone-releasing hormone (LHRHa) (des-Gly¹⁰, d -Ala⁶-LHRH ethylamide and d -Ala⁶, Pro⁹-Nethylamide-LHRH), in pelleted or dissolved form, induced mature female sea bass with a mean egg diameter of at least 0.40 mm to spawn at any day during the lunar cycle. The onset of spontaneous and LHRHa-induced spawnings occurred during low tides in the evening until dawn (from 19.00 to 05.00 hours). These results demonstrate that LHRHa can effectively induce mature sea bass to spawn independent of the highly predictable semilunar spawning rhythm. In addition, the occurrence of both spontaneous and hormone-induced spawnings at a precise period of the day suggest a tidal and diurnal cue entraining spawning of mature female sea bass.     

Variable growth rates of the tropical estuarine fish barramundi Lates calcarifer (Bloch) under different freshwater flow conditions

Relationships between freshwater flows and growth rates of the opportunistic predatory finfish barramundi Lates calcarifer in a dry tropical estuary were examined using data from a long-term tag-recapture programme. Lagged effects were not investigated. After accounting for length at release, time at liberty and seasonal variation ( e.g. winter, spring, summer and autumn), growth rates were significantly and positively related to fresh water flowing to the estuary. Effects were present at relatively low levels of freshwater flow ( i.e. 2·15 m³ s⁻¹, the 5th percentile of the mean flow rate experienced by fish in the study during time at liberty). The analysis, although correlative, provides quantitative evidence to support the hypothesis that freshwater flows are important in driving the productivity of estuaries and can improve growth of species high in the trophic chain.