Interactive effects of dietary leucine and isoleucine on growth, blood parameters, and amino acid profile of Japanese flounder Paralichthys olivaceus.

A 60-day feeding trial was conducted to assess the interactions of dietary leucine (Leu) and isoleucine (Ile) on Japanese flounder. Fish of 2.69 ± 0.04 g were fed experimental diets containing two levels of Leu (2.58 and 5.08% of diet) combined with three levels of Ile (1.44, 2.21, and 4.44% of diet), respectively. After the feeding trial, growth, proximate composition, muscle total amino acid profile, blood parameters, mucus lysozyme activity, and stress tolerance to freshwater were measured. Statistically significant (P < 0.05) interactive effects of Leu and Ile were found on growth parameters (final body weight, body weight gain, and special growth rate) of Japanese flounder. Antagonism was discovered in high dietary Leu groups, while stimulatory effects were obtained for increased dietary Ile in low Leu groups. Interactive effects of these two branched-chain amino acids were also found on hepatosomatic index of test fish. In addition, crude lipid content of fish whole body was significantly altered by various diets, with antagonism observed in low dietary Leu groups. Interactive effects also existed in muscle amino acid profiles for low fish meal diets, but no interactive impacts were observed on blood parameters. Furthermore, lysozyme activities and freshwater stress were significantly affected by different diets. And antagonism was found on lysozyme activities in low Leu groups. Moreover, high Leu and high Ile levels of diet significantly altered freshwater stress tolerance of Japanese flounder. These findings suggested that dietary Leu and Ile can effect interactively, and fish fed with diets containing 2.58% Leu with 4.44% Ile and 5.08% Leu with 1.44% Ile showed better growth performance.

Molecular characterisation and expression of parathyroid hormone-related protein in the caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

Parathyroid hormone-related protein (PTHrP) is a hypercalcemic factor in fish, but the source of circulating PTHrP remains unclear. In this study investigation of the caudal neurosecretory system (CNSS), considered one of major sources of PTHrP in fish, provided valuable insights into this regulatory system. We report pthrpa and pthrpb gene cloning, characterization, expression, and responses to low salinity and hypocalcemia challenge in flounder. The pthrpa and pthrpb precursors, isolated from a European flounder CNSS library, consist of 166 and 192 amino acid residues, respectively, with an overall homology of approximately 59.2%. Both precursors contain a signal peptide and a mature peptide with cleavage and amidation sites. The flounder PTHrPA and PTHrPB peptides share only 41% sequence identity with human PTHrPA. Quantitative PCR analysis demonstrated that the bone and bladder, are respectively major sites of pthrpa and pthrpb expression in flounder. Urophysectomy confirmed the CNSS as a likely contributor to circulating PTHrP peptides. There were no significant differences in CNSS pthrpa and pthrpb mRNA expression or plasma PTHrP levels between seawater (SW) and freshwater (FW)-adapted fish, though plasma total calcium concentrations were higher in FW animals. The intraperitonial administration of EGTA rapidly induced hypocalcemia and concomitant elevation in plasma PTHrP accompanied by increases in both pthrpa and pthrpb expression in the CNSS. Together, these findings support an evolutionary conserved role for PTHrP in the endocrine regulation of calcium.

Comparison of the effects of dietary single and multi-probiotics on growth, non-specific immune responses and disease resistance in starry flounder, Platichthys stellatus.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP1, 2 × 109 CFU kg-1 diet), single-probiotics 2 (Bacillus licheniformis; SP2, 2 × 109 CFU kg-1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP1, 2 × 109 CFU kg-1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP2, 2 × 109 CFU kg-1 diet) and oxytetracycline (OTC) at 5 g OTC kg-1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1 and MP2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1, MP2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder.

Effect of dietary supplementation with Suaeda maritima on blood physiology, innate immune response, and disease resistance in olive flounder against Miamiensis avidus.

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.

Probiotics and herbal mixtures enhance the growth, blood constituents, and nonspecific immune response in Paralichthys olivaceus against Streptococcus parauberis.

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 µl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 107 CFU ml⁻¹). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis.

Physiological responses and expression of metallothionein (MT) and superoxide dismutase (SOD) mRNAs in olive flounder, Paralichthys olivaceus exposed to benzo[a]pyrene.

We cloned complementary DNA (cDNA) encoding metallothionein (MT) and superoxide dismutase (SOD) from the liver of olive flounder, Paralichthys olivaceus. The full-length MT cDNA consists of 183 base pairs (bp) and encodes a protein of 60 amino acids; partial SOD cDNA consists of 326 bp and encodes a protein of 109 amino acids. We investigated the dose- and time-related effects of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) on MT and SOD mRNA using quantitative polymerase chain reaction (QPCR). The expression levels of MT mRNA were highest at 24 h (about five times) in 10 microg/L BaP, and at 6 h (about twelve times) in 30 microg/L BaP. The expression levels of SOD mRNA were highest at 12 h (about three times) in 10 microg/L BaP, and at 6 h (about six times) in 30 microg/L BaP, and then decreased toward the end of the experiment. We also measured plasma glucose and cortisol, all of which increased with BaP exposure. These results suggest that MT and SOD play an important role in the detoxification of reactive oxygen species (ROS) caused by BaP exposure, and thus may be indicators of oxidative stress responses.