Dietary Lipid Levels and Bile Acid Supplementation: Effects on Growth, Metabolism and Fatty Acid Composition in Asian Seabass (Lates calcarifer)

Impact of dietary starch on extrahepatic tissue lipid metabolism in farmed European (Dicentrarchus labrax) and Asian seabass (Lates calcarifer)

Osmoregulation is necessary for euryhaline teleosts in maintaining internal ion and water balance in response to environmental salinity changes. In the epithelia of teleostean gills, the ionocytes (i.e. mitochondrion‐rich cells) are thought to be the primary site of osmo/ionoregulation. In the ionocytes, the Na+, K+‐ATPase (NKA) plays a crucial role in triggering ion‐transporting systems. Asian sea bass (Lates calcarifer) is a marine euryhaline teleost and an important economic species in Taiwan, Southeast Asia, and Australia that can be cultured in environments from seawater (SW) to fresh water (FW). However, the modulatory mechanisms of osmoregulation in gills of euryhaline Asian sea bass were not clear to date. Hence the purposes of the present study were to investigate changes in physiological parameters, morphology of apical openings of gill ionoctyes, as well as expression and localization of branchial NKA in the SW‐ and FW‐acclimated Asian sea bass. The results showed that the sea bass was able to maintain their internal physiological parameters including plasma osmolality and muscle water content in a tolerated range. Observed by the scanning electron microscope, different types of apical surfaces of ionocytes with distinct features were found in the afferent epithelia of gills in FW and SW milkfish. On the other hand, the specific activity of branchial NKA was lower in the FW milkfish compared to the SW individuals. The immunohistological observation further revealed that most NKA‐immunoreactive cells in gills of the FW‐ and SW‐acclimated Asian sea bass were distributed in the afferent and interlamellar regions of the filaments. Taken together, these results illustrated that the Asian sea bass was an efficient osmoregulator with these salinity‐dependent alterations to maintain homeostasis of ions and water in environments of different salinities. These findings contributed to the knowledge of phenotypic plasticity of ionocytes and changes in activity of NKA, the crucial enzyme for ionoregulation, in gills of the Asian sea bass upon salinity challenge.Support or Funding InformationThis study was supported by a grant to THL from the Ministry of Science the Technology (MOST), Taiwan (105‐2313‐B‐005‐027‐MY3).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Supplementation exogenous bile acid improved growth and intestinal immune function associated with NF-κB and TOR signalling pathways in on-growing grass carp (Ctenopharyngodon idella): Enhancement the effect of protein-sparing by dietary lipid

Malaysian Lates calcarifer (Asian seabass) is readily adapted to the freshwater environment and is suggested to have different long-chain polyunsaturated fatty acids biosynthetic activities compared to other marine fish. Additionally, fatty acid profiling of L. calcarifer byproducts are not yet studied. Hence, in this study, fatty acid compositions of L. calcarifer byproducts (head, liver and intestine) were analyzed. The byproducts were excised and the lipid was extracted using modified Bligh and Dyer method and converted into fatty acid methyl ester before being analyzed by gas chromatography coupled with mass spectrometry. In all byproducts analyzed, major class of fatty acid was saturated fatty acids with the range of 48.25 ± 0.99% – 64.31 ± 0.79%, followed by monounsaturated fatty acids within the range 18.94 ± 1.47% – 40.19 ± 1.00% and the least was polyunsaturated fatty acids with the range between 6.08 ± 0.47% – 21.23 ± 1.20%. The C16:0, C18:1n9(c), C20:5n3(c), C22:6n3(c) and C18:2n6(c) were found to be predominant fatty acids in byproducts. Interestingly, L. calcarifer head has the highest and lowest content of omega-3 fatty acids and omega-6 fatty acids, respectively, with the amount of 16.46 ± 1.06% and 4.77 ± 0.40%. In contrast, omega-3 fatty acids were not detected from intestine, with omega-6 fatty acids appeared in greatest amount of 11.56 ± 0.11%. Such kind of useful fatty acids can be utilized to increase the nutritional value of animal feeds upon refinery.

LC-MS-based lipidomics analyses of alterations in lipid profiles of Asian sea bass (Lates calcarifer) induced by plasma-activated water treatment

Immunostimulatory Effects of Polysaccharide Compound from Seaweed Kappaphycus alvarezii on Asian seabass (Lates calcarifer) and it’s Resistance against Vibrio parahaemolyticus Kappaphycus alvarezii is a red seaweed rich in polysaccharides. In the present study an attempt was made to isolate polysaccharide from K. alvarezii and also tested for immunostimulatory effects on Asian seabass (Lates calcarifer) using Vibrio parahaemolyticus as a test pathogen. Two sets of experiments were designed as mixed with fed diets and intraperitoneal (IP) injection of the crude extracts. Every five days of interval, the non specific immune responses were analysed by the parameters like WBCs count, Lysozyme activity and NBT assay. After 15 days of treatment, the animals were challenged against V. parahaemolytcius and cumulative survival rates were recorded. The total white blood cells were decreased in intraperitoneal injection of polysaccharides extract after 5th day. But in fed diet cells count were significantly increased from 1st to 15th day. Fish fed diet with polysaccharide extract improved the lysozyme level than IP injection, which reduced simultaneously over the period compared with control. In the present study, the respiratory burst activity significantly enhanced in the fed diet polysaccharide extract in Asian seabass (Lates calcarifer) by oral administration and the highest activity reached in 0.5% fed diet on 15th day. After challenge with V. parahaemolyticus, all treated groups showed a reduced mortality compared to the control group. The best survival rate was observed in the group polysaccharide extract treated with 0.5% by oral administration. Among the other groups, fish treated by intraperitoneal injection had a lower survival rate than the fish treated by oral administration. However, the differences between the two sets of experiment (oral administration or intraperitoneal injection) were only 10%. UV, FT-IR, 1H and 13C NMR spectra were carried out to characterize the polysaccharide extract. The UV spectrum of the crude extract compound was showed a major peak at 282 nm. In FT-IR spectrum, major peak was observed at 1000-1100 cm-1 supports the presence of ring vibrations C-O, C-O-C (polysaccharides region) and minor peak of 1647.93 cm-1, which supports amide group. And based on the 1H and 13C NMR spectra, the presence of carbonyl carbons in the purified compound was confirmed. Findings of the present study evidenced the excellent immunostimulatory activity of polysaccharide from the seaweed Kappaphycus alvarezii. Further complete purification and characterization of the active polysaccharide and its field evaluation is needed to bring this seaweed polysaccharide as a promising candidate for the development of good immunostimulatory agent in the field of aquaculture.

Australasia has a large fish industry, and fish skin by-products from the processing industry could be used for the commercial production of fish collagen. The aim of this study was to characterize collagen extracted from the Asian sea bass (Australian barramundi) (Lates calcarifer) and snapper (Pagrus auratus) skin as an alternative to mammalian-derived collagen in gelatin products. The acid-soluble fractions of collagen from Asian sea bass and snapper skin were extracted and yielded about 8 and 7.5 % collagen (on a dry weight basis), respectively. The electrophoretic and chromatography patterns indicated that both collagens comprise of α1, α2, α3, and β chains, corresponding to the properties of calf skin collagen type I. Amino acid analysis and peptide mapping of digested collagen suggested differences in their amino acid sequences and collagen primary structure. Fourier transform infrared spectroscopy demonstrated that the helical structure of collagen was completely maintained in Asian sea bass and partially in snapper. Transition temperatures for the completion of the melting process in the two collagen networks were confirmed with differential scanning calorimetry and dynamic oscillatory rheology to be about 29 °C. Zeta potential analysis identified the isoelectric points (pI values) of collagen from Asian sea bass and snapper skin at pH 6.90 and 7.75, respectively. Thus, Asian sea bass and snapper skin could be an important alternative source of collagen to replace mammalian collagen for industrial applications.

In the present study, metallothionein (MT) proteins and cDNA were isolated from Asian sea bass (Lates calcarifer) livers. Immunochemical protocols (i.e. an enzyme-linked immunosorbent assay (ELISA) and Western blot) were developed for quantification of MT protein levels in Asian sea bass. These were applied to analyze MT protein levels in 15 different feral fish species from two different areas, one urban area Angsila and one industrial area Map Ta Phut in the Gulf of Thailand. An MT protein band was detected in Shrimp scad (Alepes djadaba) and Indian ilisha (Ilisha melastoma) from both areas. Higher MT protein expressions were seen in shrimp scad from cadmium-contaminated areas near shore as well as off shore. These results suggest that induction of hepatic MT immunoreactive proteins in the shrimp scad may be a good early warning signal for heavy metal exposure in environmental monitoring programmes in Thailand waters. Protocol for quantification of MT mRNA levels in Asian sea bass was developed using quantitative PCR. Asian sea bass were exposed in the lab to different doses of CdNO $_3$ and sampled at different times after injection. There appears to be a bi-phasic dose-response pattern with highest MT mRNA levels in fish injected with 4 mg CdNO $_3$ /kg. In these fish, the highest expression was seen after one day and lowest induction after three days. These results suggest that induction of MT mRNA levels in Asian sea bass liver can be used as a sensitive early warning signal for cadmium exposure in tropical waters.

Bile Acid Supplementation Reduced Hepatic Lipid Deposition and Modulated Bile Acid Metabolism and the Gut Microbiota in Weaned Piglets.

An essential function carried out by bile acids (BA) is to aid in lipid absorption, as they emulsify lipids and combine with them to form chylomicrons. While BA have been utilized as lipid emulsifiers in monogastric and aquatic animals, their impact on ruminants remains relatively unexplored. Two experiments were conducted to investigate the effects of BA on the lipid absorption of lactating dairy goats. In the Exp. 1, twelve lactating goats with similar days in milk (42 ± 2 d), body weight (46.2 ± 5.1 kg), and milk yield (1.62 ± 0.33 kg) were administered four BA doses (0, 1, 2, or 4 g/d) to explore the effects of BA supplementation. The results observed a positive correlation between the BA dose and plasma total bile acid concentration (P < 0.001). Notably, when goats received BA, there was a trend of increase in the proportion of preformed fatty acids in milk (P = 0.079). In the Exp. 2, eighteen lactating goats with similar days in milk (100 ± 6 d), body weight (45.0 ± 3.7 kg), and milk yield (1.60 ± 0.31 kg) were divided into two groups (HF and HFB): HF group received no BA, while HFB group received BA (4 g/d). Both the groups were fed diets with fat powder supplementation (1% of dry matter) for 5 weeks. BA supplementation led to a significant increase in milk yield (P = 0.024) and fat concentration (P = 0.045). Meanwhile, there was a significant rise of total bile acid concentration in plasma (P < 0.001), rumen (P < 0.001) and feces (P < 0.001). Additionally, BA supplementation had a significant impact on blood metabolism, up-regulating 241 metabolites and down-regulating 208 metabolites (P < 0.05). BA supplementation also altered the correlations among various plasma lipid components (P < 0.05). In fatty acid (FA) analysis, 26 different FA were detected, out of which 16 showed significant down-regulation upon BA supplementation (P < 0.05). In conclusion, BA supplementation positively influenced lactating dairy goats, particularly when fed diets with fat powder supplementation. BA supplementation enhanced milk yield, improved milk fat concentration, and significantly altered lipid-related blood metabolism. These findings suggest that BA supplementation could be a valuable strategy for enhancing dairy production and lipid metabolism in dairy goats, offering potential benefits for the dairy industry.

Simple SummaryAs a proinflammatory cytokine, interleukin-8 (IL-8) plays a crucial function in inflammatory responses by recruiting and regulating monocytes and lymphocytes during the early stages of inflammation. In this study, a cDNA encoding the Asian seabass (Lates calcarifer) IL-8 gene was cloned and referred to as LcIL-8. Moreover, the expression levels of LcIL-8 in various tissues of normal and diseased fish were analyzed by means of qRT–PCR. Additionally, the recombinant LcIL-8 protein (rLcIL-8) was overexpressed in the Escherichia coli system, and its biological functions under various conditions were investigated. LcIL-8 transcripts were expressed in all the tested tissues of normal Asian seabass. The constitutive mRNA expression of LcIL-8 was quantified in six tissues of fish infected with virulent Streptococcus iniae and Flavobacterium covae at three different concentrations. The minimum inhibitory concentration (MIC) and therapeutic effects of the rLcIL-8 protein against S. iniae were also thoroughly investigated. The obtained findings could be used to further develop prophylactic or therapeutic strategies applicable to the Asian seabass farming industry.In this research, the proinflammatory cytokine interleukin-8 (IL-8) was shown to play a key role in inflammatory responses in fish. This study involved the cloning of the gene that encodes IL-8 in Asian seabass (Lates calcarifer) as well as analyses of its expression and function in this fish. The expression levels of LcIL-8 indicated that it was broadly expressed in most analyzed tissues, with the most predominant expression in the whole blood 6 to 24 h after infection with S. iniae at concentrations of 105 colony-forming units (CFU)/fish (p < 0.05). After fish were immersed in F. covae, the LcIL-8 transcript was upregulated in the gills, liver and intestine, and the highest expression level was observed in the gills. However, LcIL-8 was downregulated in all the tested tissues at 48 and 96 h after infection with the two pathogenic strains, indicating that Lc-IL8 has a short half-life during the early immune responses to pathogens. Moreover, the MIC of the rLcIL-8 protein against S. iniae was 10.42 ± 3.61 µg/mL. Furthermore, functional analyses clearly demonstrated that 10 and 100 µg of the rLcIL-8 protein efficiently enhanced the phagocytic activity of Asian seabass phagocytes in vitro (p < 0.05). Additionally, in vivo injection of S. iniae following the rLcIL-8 protein indicated that 50 and 100 µg of rLc-IL-8 were highly effective in protecting fish from this pathogen (p < 0.001). The obtained results demonstrate that rLcIL-8 possesses a biological function in the defense against bacterial infections in Asian seabass.

Aims: EPS extracted from marine bacteria, which associated with Asian sea bass has potential antimicrobial activities. Methodology and Results: Two marine Bacteria were isolated from Asian sea bass (Lates calcarifer) obtained from aquaculture farm, located at Johor bahru Malaysia. 16S rRNA analysis for bacteria identity revealed that bacteria ors1 had 99 % identity to Bacillus cereus and ors2 had 96 % identity with Brachybacterium sp. All bacteria shared many similarities and variation in terms of biochemical reactions and microscopic observation. Exopolysaccharides (EPSs) were extracted and purified from bacteria as they produced mucous colonies. Average analysis of EPS components showed 50 % carbohydrates, 26 % protein and 24 % fatty acids. The FTIR analysis confirmed the functional groups of

The Asian seabass is of importance both as a farmed and wild animal. With the emergence of infectious diseases, there is a need to understand and characterize the immune system. In humans, the highly polymorphic MHC class I (MHC-I) molecules play an important role in antigen presentation for the adaptive immune system. In the present study, we characterized a single MHC-I gene in Asian seabass (Lates calcarifer) by amplifying and sequencing the MHC-I alpha 1 and alpha 2 domains, followed by multi-sequence alignment analyses. The results indicated that the Asian seabass MHC-I α1 and α2 domain sequences showed an overall similarity within Asian seabass and retained the majority of the conserved binding residues of human leukocyte antigen-A2 (HLA-A2). Phylogenetic tree analysis revealed that the sequences belonged to the U lineage. Mapping the conserved binding residue positions on human HLA-A2 and grass carp crystal structure showed a high degree of similarity. In conclusion, the availability of MHC-I α1 and α2 sequences enhances the quality of MHC class I genetic information in Asian seabass, providing new tools to analyze fish immune responses to pathogen infections, and will be applicable in the study of the phylogeny and the evolution of antigen-specific receptors.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019-2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 μg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

BackgroundFor Asian seabass (Lates calcarifer, Bloch 1790) cultured at sea cages various aquatic pathogens, complex environmental and stress factors are considered as leading causes of disease, causing tens of millions of dollars of annual economic losses. Over the years, we conducted farm-based challenges by exposing Asian seabass juveniles to complex natural environmental conditions. In one of these challenges, we collected a total of 1,250 fish classified as either ‘sensitive’ or ‘robust’ individuals during the 28-day observation period.ResultsWe constructed a high-resolution linkage map with 3,089 SNPs for Asian seabass using the double digest Restriction-site Associated DNA (ddRAD) technology and a performed a search for Quantitative Trait Loci (QTL) associated with robustness. The search detected a major genome-wide significant QTL for increased robustness in pathogen-infected marine environment on linkage group 11 (ASB_LG11; 88.9 cM to 93.6 cM) with phenotypic variation explained of 81.0%. The QTL was positioned within a > 800 kb genomic region located at the tip of chromosome ASB_LG11 with two Single Nucleotide Polymorphism markers, R1-38468 and R1-61252, located near to the two ends of the QTL. When the R1-61252 marker was validated experimentally in a different mass cross population, it showed a statistically significant association with increased robustness. The majority of thirty-six potential candidate genes located within the QTL have known functions related to innate immunity, stress response or disease. By utilizing this ddRAD-based map, we detected five mis-assemblies corresponding to four chromosomes, namely ASB_LG8, ASB_LG9, ASB_LG15 and ASB_LG20, in the current Asian seabass reference genome assembly.ConclusionAccording to our knowledge, the QTL associated with increased robustness is the first such finding from a tropical fish species. Depending on further validation in other stocks and populations, it might be potentially useful for selecting robust Asian seabass lines in selection programs.