Effects of cold acclimation on serum biochemical parameters and metabolite profiles in Schizothorax prenanti.

BACKGROUND: Environmental temperature is critical in regulating biological functions in fish. S. prenanti is a kind of cold-water fish, but of which we have little knowledge about the metabolic adaptation and physiological responses to long-term cold acclimation. RESULTS: In this study, we determined the physiological responses of S. prenanti serum after 30 days of exposure to 6℃. Compared with the control group, the levels of TC, TG, and LDL-C in the serum were significantly (P < 0.05) increased, and the level of glucose was significantly (P < 0.05) decreased under cold acclimation. Cold acclimation had no effect on the gene expression of pro-inflammatory factors and anti-inflammatory factors of S. prenanti. Metabolomics analysis by LC-MS showed that a total of 60 differential expressed metabolites were identified after cold acclimation, which involved in biosynthesis of amino acids, biosynthesis of unsaturated fatty acids, steroid degradation, purine metabolism, and citrate cycle pathways. CONCLUSION: The results indicate that cold acclimation can alter serum metabolites and metabolic pathways to alter energy metabolism and provide insights for the physiological regulation of cold-water fish in response to cold acclimation.

Impact of Hygrophila auriculata supplementary diets on the growth, survival, biochemical and haematological parameters in fingerlings of freshwater fish Cirrhinus mrigala (Hamilton, 1822).

The present study investigates the effects of Supplementary diet Hygrophila auriculata on the growth, survival, biochemical and haematological parameters of Cirrhinus mrigala. The seaweed was administered to the fish possessing an initial average weight of 14.063 ± 1.828 g. Fish were fed with supplementary diet H. auriculata exhibited significant difference (P < 0.05) in the growth performance, haematological indices such as RBC count, haematocrit volume, haemoglobin, WBC, MCV, MCH and MCHC concentration in contrast to the control after a period of 8 weeks. Also, there were significant differences in biochemical parameters (P < 0.05), between the fish supplemented with dietary H. auriculata extract and the control group. These findings suggest that the administration of H. auriculata extract has a positive effect on the immunological indices and the immune system activity in Mrigal fish.

Establishment of a rare minnow (Gobiocypris rarus) model for evaluation of experimental vaccines against a disease induced by grass carp reovirus genotype II.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD.

Exogenous phytase and xylanase supplementation of formulated diets for rohu (Labeo rohita): impact on haematology, histology and IGF I gene expression.

The present study aims to delineate the effect of exogenous enzyme supplementation of fermented or non-fermented de-oiled rice bran (DORB) on haematology, histology and expression of IGF I gene expression of Labeo rohita. Four test diets, namely, T1 (DORB), T2 (fermented DORB), T3 (DORB+exogenous enzyme) and T4 (fermented DORB+exogenous enzyme) were formulated and fed to the L. rohita for a period of 60 days. The test diets T3 and T4 were supplemented with 0.1 g kg-1 xylanase (16,000 U kg-1) and 0.1 g kg-1 phytase (500 U kg-1) enzymes. A total of 120 juveniles of L. rohita (average weight 5.01 ± 0.02 g) were stocked in 12 rectangular tanks with 10 fish per tank in triplicates. At the end of the experiment, haematology, histology and IGF I gene expression of the different groups were analysed. The haemoglobin (Hb) content, RBC count and WBC count of L. rohita varied significantly (p < 0.05) among different groups. T3 diet-fed group had the highest Hb content, RBC count and WBC count as compared to the other groups. The MCV content was significantly (p < 0.05) lower in T3 group. The DORB type, exogenous enzyme supplementation and their interaction have no significant (p > 0.05) effect on MCH, MCHC and lymphocyte content of the cultured fish. The group which were fed T3 diet had significantly (p < 0.05) higher IGF-I gene expression as compared to other groups. The histological examination of liver revealed no pathological alteration of this organ. Similarly, there were no pathological changes observed in intestinal tissue in any dietary treatment group. Based on the findings of the present study, it is concluded that exogenous enzyme supplementation of DORB-based diets improves the physiological status and growth performances of Labeo rohita.

Histopathological, hematological, and biochemical changes in high-latitude fish Phoxinus lagowskii exposed to hypoxia.

Hypoxia is one of the most significant threats to biodiversity in aquatic systems. The ability of high-latitude fish to tolerate hypoxia with histological and physiological responses is mostly unknown. We address this knowledge gap by investigating the effects of exposures to different oxygen levels using Phoxinus lagowskii (a high-latitude, cold-water fish) as a model. Fish were exposed to different oxygen levels (0.5 mg/L and 3 mg/L) for 24 h. The loss of equilibrium (LOE), an indicator of acute hypoxia tolerance, was 0.21 ± 0.01 mg/L, revealing the ability of fish to tolerate low-oxygen conditions. We sought to determine if, in P. lagowskii, the histology of gills and liver, blood indicators, enzyme activities of carbohydrate and lipid metabolism, and antioxidants changed to relieve stress in response to acute hypoxia. Notably, changes in vigorous jumping behavior under low oxygen revealed the exceptional hypoxia acclimation response compared with other low-latitude fish. A decrease in blood parameters, including RBC, WBC, and Hb, as well as an increase in MCV was observed compared to the controls. The increased total area in lamella and decreased ILCM volume in P. lagowskii gills were detected in the present study. Our results also showed the size of vacuoles in the livers of the hypoxic fish shrunk. Interestingly, an increase in the enzyme activity of lipid metabolism but not glucose metabolism was observed in the groups exposed to hypoxia at 6 h and 24 h. After combining histology and physiology results, our findings provide evidence that lipid metabolism plays a crucial role in enhancing hypoxia acclimation in P. lagowskii. Additionally, SOD activity significantly increased during hypoxia, suggesting the presence of an antioxidant response of P. lagowskii during hypoxia. High expression levels of lipogenesis and lipolysis-related genes were detected in the 6 h 3 mg/L and 24 h 3 mg/L hypoxia group. Enhanced expression of lipid-metabolism genes (ALS4, PGC-1, and FASN) was detected during hypoxia exposure. Together, these data suggest that P. lagowskii's ability to tolerate hypoxic events is likely mediated by a comprehensive strategy.

Effect of photoperiod and temperature on indicators of immunity and wellbeing of endangered golden mahseer (Tor putitora) broodstock.

Recently, we reported the possibility of successfully inducing captive maturation and spawning in golden mahseer through photothermal manipulation. Subsequently, we felt that it was imperative to understand the impact of these environmental manipulations on immunity, stress response, antioxidant potential, and general well-being of adult mahseer to develop a healthy broodstock. For this purpose, two experiments were carried out with changes in photoperiod (experiment I) and temperature (experiment II). In experiment I, random groups of adult female and male Tor putitora were subjected to three photoperiods (8L:16D, 12L:12D and 16L:8D) for 100 days. Decreasing levels of plasma melatonin with increasing photoperiod confirmed the physiological significance of different light-dark conditions in mahseer brooders. In terms of stress, plasma cortisol levels showed a linear increase with decreasing light duration in both males and females. Similarly, the level of thiobarbituric acid reactive substances was also significantly higher in males kept at 8L:16D. Plasma concentration of total immunoglobulins was found reduced in female brooders at 8L:16D, but this was not evident in males. In females, total antioxidants were found significantly elevated at 12L:12D. On the contrary, superoxide dismutase activity was lower at 12L:12D in females. The photoperiod has substantially influenced the plasma total protein and albumin levels in males. In experiment II, random groups of adult T. putitora were reared at ambient (21.2 ± 1.4 °C) or elevated temperature (23.7 ± 1.3 °C) groups for 121 days. The higher temperature was found to significantly decrease lysozyme, myeloperoxidase, and anti-protease activities in female mahseer brooders. However, total immunoglobulin levels were reduced significantly at elevated temperature both in males and females. No other temperature-related significant changes were observed in antioxidant potential, anti-oxidative enzymes or well-being related indices.

Effects of dietary α-linolenic acid/linoleic acid ratio on growth performance, tissue fatty acid profile, serum metabolites and Δ6 fad and elovl5 gene expression in silver barb (Puntius gonionotus).

BACKGROUND: Silver barb (Puntius gonionotus) is a medium-sized carp that is promising for freshwater aquaculture in Asia. This study's aim was to investigate the ideal dietary α-linolenic acid (ALA): linoleic acid (LA) ratio for maximizing long-chain polyunsaturated fatty acid (LC-PUFA) synthesis and their deposition in the muscle of silver barb, as that of fish oil based control diet. RESULT: Fish (with an initial body weight of 11.07 ± 0.12 g) were fed for 60 days with five experimental iso-proteinous, iso-lipidic, and iso-caloric diets, supplemented with linseed oil and peanut oil at varying levels to obtain ALA:LA ratios of 0.35, 0.51, 0.91, 2.04, 2.66. A control diet was prepared by supplementing fish oil. The dietary ALA:LA ratio did not influence the growth performance of fish. With increased dietary ALA:LA ratios, LA content decreased and ALA content increased in the muscle and liver of silver barb. The n-3 LC-PUFA level in muscle and liver was not influenced by feeding different ratios of ALA:LA, whereas n-6 LC-PUFA was decreased in the muscle and increased in the liver with increased dietary ALA:LA ratios. Increasing dietary ALA:LA ratio increased the Δ6fad and elovl5mRNA expression in the liver, muscle, brain, and intestinal tissues of silver barbs. CONCLUSION: Silver barb possess the ability to elongate and desaturate ALA and LA to their end products EPA and DHA. The highest level expression of Δ6 fad and elovl5 mRNA at the dietary ALA:LA ratio of 2.66 suggests greater affinity of these enzymes towards ALA than LA in silver barb. © 2019 Society of Chemical Industry.

Hematological analysis of Ctenopharyngodon idella, Megalobrama amblycephala and Pelteobagrus fulvidraco: Morphology, ultrastructure, cytochemistry and quantification of peripheral blood cells.

The grass carp (Ctenopharyngodon idella), blunt snout bream (Megalobrama amblycephala) and yellow catfish (Pelteobagrus fulvidraco) are economically important fishes in China. Fish hematological features, especially the type and number of peripheral blood cells, are crucial for the evaluation of fish health and the diagnosis of fish diseases. Since the automatic blood cell count equipment for human is not suitable for fishes, the manual method is critical in the quantification of fish blood cells. To make sense of the comparison and interpretation of the blood cell count studies in different articles, the standardization of blood cell classification is necessary. In this study, erythrocytes (red blood cell, RBC), thrombocytes (TC) and leucocytes (i.e. white blood cells, WBC, including lymphocytes, neutrophils and monocytes) were well distinguished in blood smears with Giemsa staining and confirmed by transmission electron microscopy. RBC, TC and WBC were directly counted with an improved Neubauer counting chamber in a modified diluting solution. The differential leucocyte count (DLC) was carried out in blood smears. In view of the labeling characteristics of peroxidase (PO) positivity in neutrophils and non-specific esterase (α-ANAE) positivity in monocytes, PO positive cell percentage and α-ANAE positive cell percentage were also determined in cytochemistry staining smears. No difference was found for the percentages of neutrophils and monocytes between Giemsa staining and cytochemistry staining. The standardized classification, normal count ranges and sizes of the peripheral blood cells by the present systemic studies will provide useful references for monitoring the health status of grass carp, blunt snout bream and yellow catfish.

Effects of dietary fenugreek seed extracts on growth performance, plasma biochemical parameters, lipid metabolism, Nrf2 antioxidant capacity and immune response of juvenile blunt snout bream (Megalobrama amblycephala).

Dietary administration of some plant-derived substances have been proved of great economic value in aquaculture. In order to investigate the effects of dietary fenugreek seed extracts (FSE) on juvenile blunt snout bream (Megalobrama amblycephala), a feeding trial was conducted for 8 weeks. The results showed that final weight (FW), weight gain (WG), feed conversion ratio (FCR) and specific growth rate (SGR) were not significantly affected by dietary FSE levels. The whole body lipid contents of fish fed with 0.04%, 0.08% and 0.16% FSE diets were significantly lowered compared to the control group. Dietary FSE diets significantly affected plasma complement component 3 (C3), immunoglobulin M (IgM), albumin (ALB) and total protein (TP). The relative expressions of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP1) mRNA in the liver of fish decreased significantly with increasing dietary FSE levels from 0% up to 0.04%. FSE supplementation diets lowered the liver pro-inflammatory genes expressions by regulating tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8) mRNA levels and increased anti-inflammatory genes expression by regulating transforming growth factor (TGF-ß) and interleukin 10 (IL-10). FSE diets increased growth factor-1 (IGF-1) and target of rapamycin (TOR) mRNA levels from 0% up to 0.04%, 0.04% FSE diets significantly increased growth factor-1 (IGF-1) mRNA levels and S6 kinase-polypeptide 1 (S6K1) mRNA levels compared to the control group. 0.04% FSE diets significantly increased superoxide dismutase (SOD) activities and 0.08% FSE diets significantly increased catalase (CAT) and glutathione peroxidase (GPx) activities, 0.16% FSE diets significantly increased total antioxidant capacity (T-AOC) activities compared to the control group. Additionally, compared to the control group, 0.04% dietary FSE significantly up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels and glutathione peroxidase-1 (GPx1) mRNA levels, at the same time, 0.02%, 0.04%, 0.08%, 0.16% FSE diets significantly down-regulated kelch-like ECH-associated protein 1 (Keap1) mRNA levels. However, no significant effects were observed on copper zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD). Our study indicated that dietary FSE could improve plasma biochemical parameters, regulate lipid metabolism related genes, promote Nrf2 antioxidant capacity and enhance immune response of juvenile blunt snout bream.

Serum biochemistry, liver histology and transcriptome profiling of bighead carp Aristichthys nobilis following different dietary protein levels.

Dietary protein plays a major role in determining the rate of fish growth and overall health. Given that the liver is an important organ for metabolism and detoxification, we hypothesized that optimal dietary protein levels may benefit liver function. Herein, we investigated the effects of dietary protein level on serum biochemistry, liver histology and transcriptome profiling of juvenile bighead carp Aristichthys nobilis fed for 8 weeks on a diet supplemented with high protein (HP, 40%), low protein (LP, 24%) or optimal protein (OP, 32%; controls). The results revealed a significant change in liver morphology in LP and HP groups compared with the OP group, coupled with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. RNA sequencing (RNA-Seq) analysis of the liver transcriptome yielded 47 million high-quality reads using an Illumina platform, which were de novo assembled into 80,777 unique transcript fragments (unigenes) with an average length of 1021 bp. Subsequent bioinformatics analysis identified 878 and 733 differentially expressed unigenes (DEGs) in liver in response to LP and HP diets, respectively. KEGG enrichment analysis of DEGs identified immune and metabolism-related pathways, including Toll-like receptor signaling, PI3K-Akt signaling, NF-κB signaling, complement and coagulation, peroxisome, nitrogen metabolism, PPAR signaling, and glycolysis and gluconeogenesis pathways. Transcriptome profiling results were validated by quantitative real-time PCR for 16 selected DEGs. The findings expand our understanding of the molecular mechanisms underlying the effects of dietary protein level on liver function in bighead carp.

Iron mediated hematological, oxidative and histological alterations in freshwater fish Labeo rohita.

Iron is an essential element for many physiological functions of several organisms but in excess it causes toxicity. High iron content in water bodies of mountainous states is considered as one of the major factor, responsible for low productivity in aquaculture systems. But, till date comprehensive reports on the adverse effect of iron overload in aquatic organisms, especially cultured fishes are scanty. Therefore, the present study was undertaken to investigate the adverse effects of iron overload in economically important aquaculture fish species Labeo rohita. Three sub-lethal test concentration of iron (ferrous) viz., 1/16th, 1/8th and 1/4th of LC50 (post 96 h) i.e. 8.25, 16.51 and 33.01 mg L-1, respectively, were used for in vivo exposure. Blood cells and tissue samples of the control & exposed specimens were sampled at intervals of 24, 48, 72 and 96 h to assess alterations in hematological, oxidative stress and histological parameters. Significant changes in erythrocyte and leukocyte counts, hemoglobin, lipid peroxidation, antioxidant enzyme activity (super oxide dismutase and catalase) and tissue iron accumulation were observed in the exposed fish. Significant increase in lipid peroxidation, coupled with significant reduction in free radicals scavengers like super oxide dismutase and catalase revealed a compromised anti-oxidative defense mechanism in the fishes exposed to iron overload. Histological examination of gills and liver showed severe tissue injury and histological alternations. Severity was found to increase in time and concentration dependent manner. Perl's staining revealed accumulation of excess iron in liver of the exposed fish. The observed patho-physiological changes in the present study provide the most comprehensive insight of iron overload stress in L. rohita.

Pharmacokinetics of florfenicol in blunt-snout bream (Megalobrama amblycephala) at two water temperatures with single-dose oral administration.

The pharmacokinetics and residue elimination of florfenicol (FFC) and its metabolite florfenicol amine (FFA) were studied in healthy blunt-snout bream (Megalobrama amblycephala, 50 ± 10 g). The study was conducted with a single-dose (25 mg/kg) oral administration at a water temperature of 18 or 28°C, while in the residue elimination study, fish were administered at 25 mg/kg daily for three consecutive days by oral gavage to determine the withdrawal period (WDT) at 28°C. The FFC and FFA levels in plasma and tissues (liver, kidneys and muscle) were analysed using high-performance liquid chromatography (HPLC). A no-compartment model was used to analyse the concentration versus time data of M. amblycephala. In the two groups at 18 and 28°C, the maximum plasma concentration (Cmax ) of FFC was 5.89 and 6.21 µg/ml, while the time to reach Cmax (Tmax ) was 5.97 and 2.84 hr, respectively. These suggested that higher temperature absorbed more drug and more quickly at M. amblycephala. And the elimination half-life (T1/2 kß ) of FFC was calculated as 26.75 and 16.14 hr, while the total body clearance (CL) was 0.09 and 0.15 L kg-1  hr-1 , and the areas under the concentration-time curves (AUCs) were 265.87 and 163.31 µg hr/ml, respectively. The difference demonstrated that the elimination rate of FFC in M. amblycephala at 28°C was more quickly than that at 18°C. The results of FFA showed the same trend in tissues of M. amblycephala. After multiple oral doses (25 mg/kg daily for 3 days), the k (eliminate rate constant) of FFA in M. amblycephala muscle was 0.017, the C0 (initial concentration) was 3.07 mg/kg, and the WDT was 10 days (water temperature 28°C).

Effects of dietary synbiotic on innate immunity, antioxidant activity and disease resistance of Cirrhinus mrigala juveniles.

The dietary supplementation of synbiotic in Cirrhinus mrigala juvenile (with initial body weight ranging from 2.87 ±â€¯0.01 g to 3.26 ±â€¯0.05 g) was evaluated in terms of changes in innate immunity, antioxidant activity and disease resistance against Aeromonas hydrophilla infection. One hundred eighty acclimatized juveniles of mrigal were randomly distributed in the three replicates of each of four experimental groups i.e. control (without Probiotic and Prebiotic), T1 (High Probiotic + Low Prebiotic), T2 (Low Probiotic + High Prebiotic) and T3 (High Probiotic + High Prebiotic), using completely randomized design (CRD). At the end of the feeding trial for 60 days, fish were challenged by Aeromonas hydrophila and survival rate was recorded for the next 15 days. Bacillus subtilis used as a probiotic source and MOS used as a prebiotic source in the experiment. Results showed that innate immunity was comparatively improved in T3 group. Lysozyme activity and respiratory burst activity (NBT) were significantly (P < 0.05) affected in T3 group. Highest activities of antioxidant enzymes (P < 0.05) were reported in T3 group. Cumulative mortality % was found to be lower in the fish fed dietary synbiotic on T3 group after challenging with Aeromonas hydrophilla infection. The results of this study showed that under the experimental conditions, dietary supplementation of synbiotic had a synergestic effect on enhancing innate immunity and disease resistance of Cirrhinus mrigala (P < 0.05).

Modulation of immune response and protective efficacy of recombinant outer-membrane protein F (rOmpF) of Aeromonas hydrophila in Labeo rohita.

The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1ß (IL-1ß), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.

Bisphenol A induced abnormal DNA methylation of ovarian steroidogenic genes in rare minnow Gobiocypris rarus.

Bisphenol A (BPA), an ubiquitous environmental endocrine disruptor chemical, disturbs the mRNA expressions of steroidogenic genes and subsequently steroid hormone synthesis in mammals and aquatic species. However, the underlying regulation mechanisms are barely understood, especially in fish. To explore the regulation mechanism, we exposed female rare minnow Gobiocypris rarus (G. rarus) to BPA at a nominal concentration of 15 µg/L for 7 and 14 days in the present study. Results showed significant increase of gonad somatic index (GSI) and serum estradiol (E2) levels in response to BPA at day 14. The 7-day BPA exposure notably repressed the expression of two ovarian steroidogenic genes (star and hsd11b2) and suppressed their capacity of estrogen response elements (ERE) to recruit estrogen receptor (ER), while the 14-day BPA treatment remarkably induced transcript of hsd3b and enhanced the capacity of ERE to recruitment ER in ovaries. Furthermore, the 7-day BPA exposure caused DNA hypermethylation of star (CpGs: -742 bp and -719 bp) and hsd11b2 (CpG: -1788 bp). However, 14-day BPA exposure resulted in DNA hypomethylation of hsd3b (CpG: -181 bp). Correlation analysis revealed that the DNA methylation levels at specific CpGs in star, hsd3b and hsd11b2 were significantly correlated to their mRNA levels and ER-EREs interactions. These findings suggest that the disturbed steroidogenesis and the transcripts of ovarian steroidogenic genes might attribute to the altered DNA methylation status of these ovarian steroidogenic genes in response to BPA.

Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens.

A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.

Genotoxic and hematological effects of chlorpyrifos exposure on freshwater fish Labeo rohita.

Chlorpyrifos is a commonly used organophosphate insecticide that causes toxicological effects in aquatic organisms especially in fish. This study determined the effects of chlorpyrifos on the genotoxic and hematological parameters of freshwater fish, Labeo rohita. The genotoxic effects of different sublethal concentrations of chlorpyrifos were investigated in the erythrocytes of Labeo rohita (commonly known as Rohu) using the Micronucleus test. Effects of chlorpyrifos on the hematological parameters of the fish were also observed. Fish specimens were exposed to three sublethal concentrations of chlorpyrifos viz., sublethal I (SL-I, 1/6th of LC50 = ∼73.8 µg/L), sublethal II (SL-II, 1/4th of LC50 = ∼110.7 µg/L) and sublethal III (SL-III, 1/2nd of LC50 = ∼221.4 µg/L) for 96 h. Blood samples were collected at every 24 h and were subjected to the Micronucleus assay. The observed micronucleus frequencies were concentration and time-dependent. The MN induction was significantly highest (p < 0.01) at all the concentrations on 96-h exposure. During the experimental period, hematological parameters like total erythrocytes count (TEC), hemoglobin (Hb) and packed cell volume (PCV) decreased, whereas total leukocytes count (TLC) increased. This study indicated that the Micronucleus assay is a useful tool to detect genotoxic potential of chlorpyrifos in fish.

Evaluation of dietary Hybanthus enneaspermus (Linn F. Muell.) as a growth and haemato-immunological modulator in Labeo rohita.

This study evaluated the effects of Hybanthus enneaspermus aqueous extract (HEE) as a feed supplement on growth, immune responses, haematological, and biochemical parameters of Labeo rohita and its susceptibility to Aeromonas hydrophila infection. Diets containing five variable concentrations of HEE (g kg-1) (0 [basal diet], 1.0 [H1], 2.0 [H2], 3.0 [H3], and 4.0 [H4]) were fed to fish (average weight: 17.2 ± 0.21 g) for 6 weeks. Growth parameters, immune parameters, and haemato-biochemical parameters were examined 6 weeks post-feeding. The results showed that growth parameters, such as final weight (39.47 ± 3.1 g) and specific growth rate (1.92 ± 0.2), were significantly higher in the H3 group than in the control. Immunological parameters such as serum lysozyme (24.73 ± 2.16 U mg-1), phagocytic activity (19.14± 0.26%), and respiratory burst activity (0.307 ± 0.005) were significantly higher in the H3 group, compared with the control. Among the haematological parameters examined, red blood cells, white blood cells, neutrophils, total serum protein, and albumin levels were significantly higher in the H3 group. However, HEE administration had no significant effect on haematocrit, haemoglobin, monocytes, or eosinophils levels. Blood biochemical parameters, including superoxide dismutase and alkaline phosphatase activities, were higher (P < 0.05) in the H3 group than in the control; however, the opposite result was found for aspartate transaminase, alanine transaminase, glucose, and cholesterol. Supplementation with 3 g kg-1 HEE increased the activities of intestinal digestive enzyme (amylase, protease, and lipase) in fish. Moreover, fish fed diets supplemented with 3 g kg-1 HEE exhibited the highest disease resistance against A. hydrophila infection. These results suggest that dietary administration of HEE (at 3 g kg-1) has several positive effects on growth, immune and haematological responses in L. rohita.

Dietary supplementation with selenium yeast and tea polyphenols improve growth performance and nitrite tolerance of Wuchang bream (Megalobrama amblycephala).

In order to explore the effects of dietary selenium yeast, tea polyphenols and their combination on growth of Wuchang bream (Megalobrama amblycephala) and its resistance to nitrite stress, 360 healthy Wuchang bream with initial body weight of (55.90 ± 2.60) g were randomly divided into four groups: a control group fed with basal diet and three treated groups fed with basal diets supplemented with 0.50 mg/kg selenium yeast, 50 mg/kg tea polyphenols, and the combination of 0.50 mg/kg selenium yeast and 50 mg/kg tea polyphenols, respectively. After 60 d of feeding, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to nitrite stress of 15.0 mg/L. The serum stress hormones, liver histology and hepatic antioxidant responses were evaluated before nitrite exposure (0 h) and at 6, 12, 24, 48 and 96 h after exposure. The results showed that before nitrite exposure, compared with the control, the weight gain, specific growth rate, liver total antioxidant capacity, the activities and transcriptional levels of hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) in the selenium yeast and combination groups were significantly increased, while feed conversion rate was decreased significantly, which suggested that the combined use of selenium yeast and tea polyphenols as well as the single selenium yeast supplementation improved growth performance and enhanced antioxidant capacity in fish. After nitrite exposure, compared with the control, liver total antioxidant capacity as well as the activities and transcription levels of catalase superoxide dismutase and glutathione peroxidase in three treatment groups were significantly increased in varying degrees whereas serum cortisol contents and liver malondialdehyde levels were decreased significantly. By contrast, the combined use of selenium yeast and tea polyphenols was more effective than the single supplementation with selenium yeast or tea polyphenols. In consistent with this, alterations of the liver histostructure in three treatment groups were slower and less severe than in the control group after nitrite exposure. In conclusion, a basal diet supplemented with the combination of 0.50 mg/kg selenium yeast and 50 mg/kg tea polyphenols could effectively improve growth performance and nitrite resistance in Wuchang bream.

Effects of fluconazole based medicated feed on haemato-immunological responses and resistance of Labeo rohita against Saprolegnia parasitica.

Fluconazole (FLZ) is a new azole antifungal drug having no earlier record of its utilization for the treatment of fish diseases. A 55-days experiment was carried out to delineate the role of FLZ based medicated feed on haemato-immunological responses and prevention of fungal infection in Labeo rohita fingerlings. Three hundred and sixty fingerlings were randomly distributed into four experimental groups in triplicates. Four iso-caloric and iso-nitrogenous purified medicated feeds were prepared with 0, 100, 200 and 300 mg FLZ 100 g-1 feed. Haemato-immunological parameters like erythrocyte counts (EC), haemoglobulin (Hb), leucocyte counts (LC), packed cell volume (PCV), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), nitro-blue tetrazolium (NBT), albumin, globulin, total plasma protein were studied. The present study revealed that the continuous feeding of FLZ based feed for 15-30 days significantly (P < 0.05) increases the erythropoiesis, heme-synthesis, as well as the leucocytosis in rohu. However, all the doses exhibited equal protection from the infectious S. parasitica after 45 days of continuous feeding.