Impact of dietary leaf extracts of Black pepper Piper nigrum L on the growth, hematological and immunological parameters of Labeo rohita (Hamilton, 1822) cultured in glass aquaria / Impacto de extratos de folhas da dieta de pimenta-do-reino Piper nigrum L sobre o crescimento, parâmetros hematológicos e imunológicos de Labeo rohita (Hamilton, 1822) cultivado em aquários de vidro

A study was conducted to evaluate the effect of Piper nigrum (black pepper) leaf extract on on the growth performance, proximate composition, hematological parameters, and immune response of Labeo rohita fingerlings with an average weight of 22.14 ± 0.98g. After acclimation for two weeks, fish (n=25) were randomly selected and placed in four glass aquaria (T0, T1, T2 and T3) at constant water temperature (30.0 ± 1.0 °C), pH (7.50 ± 0.5) and total hardness (200 ± 2.0 mgL,-¹) for a period of 12 weeks, with three replicates each. Fish were fed with P. nigrum leaf extract supplemented feed @ 0.0%, 1.0%, 2.0% and 3.0% in T0, T1, T2 and T3, respectively. At the end of experiment, five fish were randomly selected from each aquaria for proximate composition, gut and skin microbial load, hematological parameters. Total proteins, albumins, and globulins were also recorded to evaluate immunological memory. The result revealed that fish in T2 showed better growth performance with an average weight gain of 56.11 ± 0.51 g. Thus, it had been concluded that Piper nigrum, a medicinal plant, can also be used to enhance the growth performance and immune response of Labeo rohita as attractive alternatives against antibiotics and vaccines and has shown no negative side effects on fish health as well as on its environment.

Um estudo foi conduzido para avaliar o efeito do extrato da folha de Piper nigrum (pimenta-do-reino) sobre o desempenho de crescimento, composição centesimal, parâmetros hematológicos e resposta imune de alevinos de Labeo rohita com peso médio de 22,14 ± 0,98g. Após aclimatação por duas semanas, os peixes (n = 25) foram selecionados aleatoriamente e colocados em quatro aquários de vidro (T0, T1, T2 e T3) em temperatura constante da água (30,0 ± 1,0 °C), pH (7,50 ± 0,5) e dureza total (200 ± 2,0 mgL-¹) por um período de 12 semanas, com três repetições cada. Os peixes foram alimentados com ração suplementada com extrato de folha de P. nigrum @ 0,0%, 1,0%, 2,0% e 3,0% em T0, T1, T2 e T3, respectivamente. Ao final do experimento, cinco peixes foram selecionados aleatoriamente de cada aquário para composição centesimal, carga microbiana intestinal e cutânea e parâmetros hematológicos. Proteínas totais, albuminas e globulinas também foram registradas para avaliar a memória imunológica. O resultado revelou que os peixes em T2 apresentaram melhor desempenho de crescimento com ganho de peso médio de 56,11 ± 0,51 g. Assim, concluiu-se que Piper nigrum, uma planta medicinal, também pode ser usado para melhorar o desempenho de crescimento e resposta imunológica de Labeo rohita como alternativas atraentes contra antibióticos e vacinas e não mostrou efeitos colaterais negativos na saúde dos peixes, bem como sobre seu ambiente.

Use of multi-strain probiotics in linseed meal based diet for Labeo rohita fingerlings / Uso de probióticos multi-cepas na dieta à base de farinha de linhaça para alevinos de Labeo rohita

Unavailability of probiotics in fish digestive system fingerlings is unable to digest and absorb their food properly. The current research was conducted to investigate the influence of probiotics added Linseed meal based (LMB) diet on hematology and carcass composition of Labeo rohita juveniles. Hematological parameters are essential diagnostics used to estimate the health status of fish. The usage of probiotics for fish health improvement is becoming common due to the higher demand for environment-friendly culture system in water. Linseed meal was used as a test ingredient to prepare six experimental test diets by adding probiotics (0, 1, 2, 3, 4 and 5 g/kg) and 1% indigestible chromic oxide for seventy days. According to their live wet weight, five percent feed was given to fingerlings twice a day. Fish blood and carcass samples (Whole body) were taken for hematological and carcass analysis at the end of the experiment. The highest carcass composition (crude protein; 18.72%, crude fat; 8.80% and gross energy; 2.31 kcal/g) was observed in fish fed with test diet II supplemented with probiotics (2 g/kg). Moreover, maximum RBCs number (2.62× 106mm-3), WBCs (7.84×103mm-3), PCV (24.61), platelets (63.85) and hemoglobin (7.87) had also been reported in the fish fingerlings fed on 2 g/kg of probiotics supplemented diet. Results indicated that probiotics supplementation has a critical role in improvement of fingerlings' body composition and hematological indices. Present findings showed that probiotics supplementation at 2 g/kg level in linseed by-product-based diet was very useful for enhancing the overall performance of L. rohita fingerlings.

A indisponibilidade de probióticos em alevinos do sistema digestivo de peixes faz com que ele seja incapaz de digerir e absorver seus alimentos adequadamente. A presente pesquisa foi conduzida para investigar a influência de probióticos adicionados à dieta à base de farelo de linhaça (LMB) na hematologia e na composição da carcaça de juvenis de Labeo rohita. Os parâmetros hematológicos são diagnósticos essenciais usados para estimar o estado de saúde dos peixes. O uso de probióticos para a melhoria da saúde dos peixes está se tornando comum devido à maior demanda por sistemas de cultivo em água que não agridam o meio ambiente. Farinha de linhaça foi usada como ingrediente para preparar seis dietas de teste experimentais adicionando probióticos (0, 1, 2, 3, 4 e 5 g/kg) e 1% de óxido crômico indigestível por 70 dias. De acordo com seu peso úmido vivo, 5% de alimento eram dados aos alevinos duas vezes ao dia. Amostras de sangue e carcaça de peixes (corpo inteiro) foram coletadas para análise hematológica e de carcaça no final do experimento. A maior composição da carcaça (proteína bruta, 18,72%; gordura bruta, 8,80%; e energia bruta, 2,31 kcal/g) foi observada em peixes alimentados com a dieta teste II suplementada com probióticos (2 g/kg). Além disso, os números máximos de RBCs (2,62×106 mm-3), WBCs (7,84×103 mm-3), PCV (24,61), plaquetas (63,85) e hemoglobina (7,87) também foram relatados em alevinos alimentados com 2 g/kg de dieta suplementada com probióticos. Os resultados indicaram que a suplementação de probióticos tem um papel crítico na melhoria da composição corporal dos alevinos e índices hematológicos. As descobertas atuais mostraram que a suplementação de probióticos no nível de 2 g/kg em dieta à base de subproduto de linhaça foi muito útil para melhorar o desempenho geral de alevinos de L. rohita.

Partial replacement of fish meal with Moringa oleifera leaf meal in practical diets of Cirrhinus mrigala fingerlings / Substituição parcial da refeição de peixe com farinha de folha de Moringa oleifera em dietas práticas de dedos Cirrhinus mrigala

Fish protein is serving as a source of nutrition for protein starving world. However, sustainable aquaculture products require inexpensive plant by-products due to finite sources of fish meal. Therefore, this study was conducted to examine nutrient utilization, growth performance and hematological indices of Cirrhinus mrigala fingerlings fed on Moringa oleifera leaf meal (MOLM) based diets. Fish were fed with six isonitrogenous and isoenergetic diets having MOLM as a substitute of fish meal (FM) at the levels of 0%, 10%, 20%, 30%, 40% and 50% for the period of 90 days. Fingerlings having initial weight 6.35±0.04g were reared in triplicate tanks at the stocking density of 15 and hand fed at the rate of 5% of total biomass twice regularly. Chromic oxide inclusion level was 1% in diets. After analysis, maximum growth performance and improved digestibility of nutrients were found in fish fed with diet at 10% replacement level as compared to fish fed on control diet and other test diets. Additionally, it was found that the red blood cells, white blood cells, hemoglobin and mean corpuscular hemoglobin concentration of fish showed a significantly (p<0.05) inverse correlation with the increase in MOLM. In present research, it was concluded that MOLM has good potential to be used as a FM substitute in C. mrigala diet with maximum effect at 10% showing positive hematological indices.

A proteína do peixe está servindo como fonte de nutrição para o mundo faminto por proteína. No entanto, produtos de aquicultura sustentáveis requerem subprodutos vegetais baratos devido às fontes finitas de farinha de peixe. Portanto, este estudo foi conduzido para examinar a utilização de nutrientes, o desempenho do crescimento e os índices hematológicos de alevinos de Cirrhinus mrigala alimentados com dietas à base de farelo de folhas de Moringa oleifera (MOLM). Os peixes foram alimentados com seis dietas isonitrogênicas e isoenergéticas contendo MOLM como substituto da farinha de peixe (FM) nos níveis de 0%, 10%, 20%, 30%, 40% e 50% pelo período de 90 dias. Os alevinos com peso inicial de 6,35 ± 0,04g foram criados em tanques triplicados com densidade de 15 animais e alimentados à mão a uma taxa de 5% da biomassa total duas vezes regularmente. O nível de inclusão de óxido crômico foi de 1% nas dietas. Após análise, desempenho máximo de crescimento e melhor digestibilidade de nutrientes foram encontrados em peixes alimentados com dieta em nível de reposição de 10% em comparação com peixes alimentados com dieta controle e outras dietas teste. Além disso, verificou-se que as concentrações de hemácias, leucócitos, hemoglobina e hemoglobina corpuscular média dos peixes apresentaram correlação inversa significativa (p < 0,05) com o aumento do MOLM. Na presente pesquisa, concluiu-se que o MOLM tem bom potencial para ser utilizado como substituto do FM na dieta de C. mrigala com efeito máximo a 10% apresentando índices hematológicos positivos.

Chromium supplemented carbohydrate diets for Cirrhinus mrigala: effects on body composition, gut enzyme activity and hematological parameters / Dietas com carboidratos suplementados com cromo para Cirrhinus mrigala: efeitos sobre a composição corporal, atividade enzimática intestinal e parâmetros hematológicos

A ninety days nutritional trial was directed to explore the effects of dietary chromium on body composition, gut enzyme activity and physiological status of Cirrhinus mrigala by using G & NG corn. Six experimental diets were prepared by using different levels of chromium chloride hexahydrate (0, 0.2, 0.4 mg/kg, each with G & NG corn). For this experimental trial, 480 fingerlings, irrespective of sex were distributed in six aquariums each with replicate. Results revealed that gelatinized corn along with increasing level of Cr2Cl3.6H2O have a positive impact upon body composition of fish. Hematology was positively correlated with chromium chloride hexahydrate supplementation in gelatinized corn. Amylase gut enzyme also showed significant (P<0.05) increase in group fed with chromium chloride hexahydrate supplemented diet (G corn). However, corn with chromium chloride hexahydrate supplementation did not revealed any significant impact on gut protease enzyme activity. From these results it can be concluded that both chromium chloride hexahydrate and gelatinized corn in fish feed are very beneficial to improve body composition, enzymes activity and physiological health status of fish.

Um ensaio nutricional de 90 dias foi dirigido para explorar os efeitos do cromo dietético na composição corporal, atividade enzimática intestinal e estado fisiológico de Cirrhinus mrigala usando milho G & NG. Seis dietas experimentais foram preparadas usando diferentes níveis de cloreto de cromo hexa-hidratado (0, 0,2, 0,4 mg/kg, cada um com milho G e NG). Para este ensaio experimental, 480 alevinos, independentemente do sexo, foram distribuídos em seis aquários, cada um com réplicas. Os resultados revelaram que o milho gelatinizado, juntamente com o aumento do nível de Cr2Cl3.6H2O, tem um impacto positivo na composição corporal dos peixes. A hematologia foi positivamente correlacionada com a suplementação de cloreto de cromo hexa-hidratado em milho gelatinizado. A enzima amilase intestinal também apresentou aumento significativo (P <0,05) no grupo alimentado com dieta suplementada com cloreto de cromo hexa-hidratado (milho G). No entanto, o milho com suplementação de hexahidrato de cloreto de cromo não revelou nenhum impacto significativo na atividade da enzima protease intestinal. Desses resultados, pode-se concluir que tanto o cloreto de cromo hexa-hidratado quanto o milho gelatinizado na alimentação de peixes são muito benéficos para melhorar a composição corporal, a atividade de enzimas e o estado fisiológico de saúde dos peixes.

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.

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.

An Integrated biomarker approach for explaining the potency of exogenous glucose on transportation induced stress in Labeo rohita fingerlings.

Transportation of fish seed is a complex phenomenon associated with multiple kinds of stressors that simultaneously affect the fish in a confined environment, causing stress and mortality. The present study investigated the stress-relieving effect of exogenous glucose as a water additive in different concentrations (0.1, 0.2, 0.3, and 0.4%) during simulated transportation (12 h) of L. rohita fingerlings. The integrated biomarker response (IBR) index is a holistic tool to determine the optimum dose of exogenous glucose for mitigating transportation stress in fish. Based on selected biomarkers related to the stress hormone, serum biochemistry, oxidative stress, and HSP70 mRNA expression, the IBR index is calculated for each treatment and control group. The result showed a significant change in the level of stress hormone cortisol, enzymes (SGPT, LDH, MDH, SOD, CAT) and metabolites (serum glucose, triglyceride, creatinine) along with an upregulation in liver HSP70 mRNA expression. IBR index suggests that 0.2% glucose exhibited the lowest multi-biomarker stress response in comparison to other treatments and control. Therefore, the use of 0.2% glucose as a water additive will provide a solution to transportation induced stress in L. rohita fingerling and will underwrite the success of grow-out fish culture in days to come.

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.

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.

Investigation on Endocrine Disruption of the Larval Lampricide 3-Trifluoromethyl-4-Nitrophenol: Short-Term Reproduction Assay with Fathead Minnow (Pimephales promelas).

3-Trifluoromethyl-4-nitrophenol (TFM) has been used for more than 60 yr to control the invasive parasitic sea lamprey (Petromyzon marinus) in the Great Lakes Basin (USA/Canada). In the early 1990s, researchers reported that TFM induced vitellogenin in fish and that TFM was an agonist for the rainbow trout estrogen receptor. To support continued registration of TFM for sea lamprey control, regulatory agencies required further testing to evaluate potential endocrine disruption effects. Fathead minnow (Pimephales promelas) were exposed to TFM at measured concentrations of 0.0659, 0.181, 0.594, 1.79, and 5.11 mg active ingredient (a.i.)/L for 21 d. No-observable- and lowest-observable-effect concentrations (NOEC and LOEC, respectively) were determined to be 1.79 mg/L or greater for each endpoint. Male survival in the highest treatment group was reduced relative to the controls. Percentage of egg fertility was reduced in the highest treatment group, resulting in an estimated NOEC of 1.79 mg/L. Whereas no effect on the gonadosomatic index (GSI) was observed for males, female GSI was increased in the 5.11-mg/L treatment. Vitellogenin production was not altered relative to the controls for all TFM treatment groups. However, female testosterone was elevated in the 5.11-mg/L treatment. The results suggest that prolonged exposure to TFM at concentrations exceeding 1.79 mg/L has the potential to disrupt endocrine function. Biologically relevant effects were found at the highest exposure concentration following a 21-d exposure. However, the duration of exposure in our study is not consistent with typical treatment durations (12 h) for sea lamprey control. Environ Toxicol Chem 2020;39:1599-1607. © 2020 SETAC.

Acute toxicity and clotting times of anticoagulant rodenticides to red-toothed (Odonus niger) and black (Melichthys niger) triggerfish, fathead minnow (Pimephales promelas), and largemouth bass (Micropterus salmoides).

Anticoagulant rodenticides (ARs) are used in rat eradication efforts on island wildlife refuges. AR bait pellets can get into coral reef areas during broadcasting and lead to exposure of non-target organisms, such as marine fishes. The objective of this study was to determine the sensitivity of representative saltwater fishes, Red-toothed triggerfish (Odonus niger) and Black triggerfish (Melichthys niger), and common freshwater fishes, fathead minnow (Pimephales promelas), and largemouth bass (Micropterus salmoides) to first generation ARs, diphacinone (DPN) and chlorophacinone (CPN), as well as a second-generation AR, brodifacoum (BROD). Acute toxicity of ARs was evaluated by single dose, intraperitoneal injections. The median lethal dose (LD50) ranges were 137-175 µg DPN/g, 155-182 µg CPN/g, and 36-48 µg BROD/g for Red-toothed triggerfish and 90-122 µg DPN/g, 125-164 µg CPN/g, and 50-75 µg BROD/g for black triggerfish. Laboratory surrogate test fish species fathead minnow and largemouth bass were of similar sensitivity toward AR-induced toxicity compared to triggerfish based on LD50 values. Sublethal effects on elevated clotting time occurred in dose-dependent fashion in all fish tested. Fish appear to have low sensitivity to AR chemicals as compared to other taxa, in particular mammals and birds, based on across-taxa comparisons of species sensitivity distributions of whole body, single dose acute lethality (LD50 values). The sensitivity of fish to waterborne exposures of ARs has yet to be fully evaluated and indeed may prove more hazardous to fish.

Screening of Biomarkers Related to Ovarian Maturation and Spawning in Blunt Snout Bream (Megalobrama amblycephala) Based on Metabolomics and Transcriptomics.

In fish breeding practices, gamete maturity of females is vital to reproductive success. For some species, it is possible to estimate the female maturation status based on abdomen observation, but quite difficult for some species which mature at big size. To screen out the potential biomarker in fish blood relating to female maturation, we employed the approach integrating the UPLC-MS/MS and RNA-seq techniques to investigate the metabolites and genes reflecting the sexual maturation and spawning of female blunt snout bream Megalobrama amblycephala. The study included four groups, 1-year-old immature female individuals, 2-year-old immature female individuals, 2-year-old sexually mature female individuals, and 2-year-old sexually mature female individuals after 24 h of successful spawning. The upregulated metabolites in mature females were involved in "steroid hormone biosynthesis," "metabolic pathways," "glycerophospholipid metabolism," etc. compared with those of immature individuals. As the key intermediate of steroid hormone biosynthesis, 17α-hydroxypregnenolone exhibited the highest level in 2-year-old mature females than in the immature females. Meanwhile, the metabolites (i.e., dodecanoic acid and myristic acid) participating in fatty acid synthesis exhibited much lower levels in the females after spawning than those before spawning. In addition to the metabolites, the genes involved in ovarian steroidogenesis were significantly upregulated in the 2-year-old immature females compared to the 1-year-old immature females, indicating that the ovarian steroidogenesis plays important roles in ovarian development of M. amblycephala at the early stages. The significant upregulation of genes (i.e., itpr1, camk2, and mekk2) involved in the "GnRH signaling pathway" was observed in the mature females compared with the immature females, which indicated that the estrogen levels increased after female maturation in M. amblycephala. Moreover, many genes (e.g., gck, creb1, tf2-9, ryr2, asgr1, and creb1) regulating insulin secretion and thyroid hormone synthesis were significantly downregulated after female spawning. The dynamics of gene expression and metabolites observed in this study provide novel cues for guiding fish practical artificial reproduction.

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.

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.

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).

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.

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.

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.

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.