Induction of trained immunity using ß-glucan and its protective responses in Nile tilapia, Oreochromis niloticus.

Emerging and re-emerging diseases in fish cause drastic economic losses in the aquaculture sector. To combat the impact of disease outbreaks and prevent the emergence of infections in culture systems, understanding the advanced strategies for protecting fish against infections is inevitable in fish health research. Therefore, the present study aimed to evaluate the induction of trained immunity and its protective efficacy against Streptococcus agalactiae in tilapia. For this, Nile tilapia and the Tilapia head kidney macrophage primary culture were primed using ß-glucan @200 µg/10 g body weight and 10 µg/mL respectively. Expression profiles of the markers of trained immunity and production of metabolites were monitored at different time points, post-priming and training, which depicted enhanced responsiveness. Higher lactate and lactate dehydrogenase (LDH) production in vitro suggests heightened glycolysis induced by priming of the cells using ß-glucan. A survival rate of 60% was observed in ß-glucan trained fish post challenge with virulent S. agalactiae at an LD50 of 2.6 × 107 cfu/ml, providing valuable insights into promising strategies of trained immunity for combating infections in fish.

Effect of sulfate on the osmoregulatory and physio-biochemical responses of GIFT (Oreochromis niloticus) juveniles reared in potassium-deficient medium saline waters.

The inland saline waters were continuously observed to have low potassium concentrations compared to their seawater counterpart of the same salinity. We hypothesize that the toxic effect of sulfate may manifest in low potassium saline (LPSW) waters compared to brackish water of the same salinity. Thus, LC50 trials were performed in GIFT (genetically improved farmed tilapia) fry (0.5 ± 0.02 g) to determine the acute sulfate toxicity in freshwater (FW, 0.5 g L-1), artificial seawater (ASW, 10 g L-1), and LPSW (10 g L-1). The median lethal concentrations (96h LC50) of sulfate ion in FW, LPSW, and ASW for the GIFT were 5.30 g L-1, 2.56 g L-1, and 2.98 g L-1, respectively. A second experiment was conducted for 21 days, exposing fish to a sub-lethal level of sulfate ion (SO42-) concentration (1000 mg L-1, one-fifth of FW LC50) with different types of waters (FW, freshwater, 0.5 g L-1; ASW, artificial seawater, 10 g L-1; LPSW, low potassium saline water, 10 g L-1) with and without sulfate inclusion to constitute the treatments as follows, (FW, FW + SO4, ASW, ASW + SO4, LPSW, LPSW + SO4). The effect of sulfate on GIFT reared in sulfate-rich potassium-deficient medium saline water was evaluated by focusing on the hematological adjustments, stress-induced oxidative damage, and osmoregulatory imbalances. The survival was not altered due to the sulfate concentration and K+ deficiency; however, there were significant changes in branchial NKA (Na+/K+-ATPase) activity and osmolality. The increase in NKA was highest in LPSW treatment, suggesting that internal ionic imbalance was triggered due to an interactive effect of sulfate and K+ deficiency. The cortisol levels showed a pronounced increase due to sulfate inclusion irrespective of K+ deficiency. The antioxidant enzymes, i.e., SOD (superoxide dismutase), catalase, GST (glutathione-S-transferase), and GPX (glutathione peroxidase), reflected a similar pattern of increment in the gills and liver of the LPSW + SO4 groups, suggesting a poor antioxidant status of the exposed group. The hepatic peroxidation status, i.e. TBARS (thiobarbituric acid reactive substances), and the peroxide values were enhanced due to both K+ deficiency and sulfate inclusion, suggesting a possible lipid peroxidation in the liver due to handling the excess sulfate anion concentration. The hematological parameters, including haemoglobin, total erythrocyte count, and hematocrit level, reduced significantly in the LPSW + SO4 group, indicating a reduced blood oxygen capacity due to the sulfate exposure and water potassium deficiency. The hepatic acetylcholine esterase activity was suppressed in all the treatments with sulfate inclusion, while the highest suppression was observed in the LPSW + SO4 group. Thus, it is concluded that sulfate-induced physiological imbalances manifest more in potassium-deficient water, indicating that environmental sulfate is more detrimental to inland saline water than freshwater or brackish water of the same salinity.

Βeta-glucan stimulation induces trained immunity markers in common carp, Cyprinus carpio.

Trained immunity refers to the memory acquired by innate immune cells, leading to cross-protection and non-specific responses to subsequent infection, thereby improving host survival. Trained immunity induction is a combined effect of immune signaling, metabolic changes, and epigenetic modifications. The present study evaluated the induction of markers of the phenomenon of trained immunity in common carp, which is trained using ß-glucan. The mammalian target of rapamycin (mtor) and hypoxia-inducible factor (hif1α), the metabolic basis of trained immunity; the histone deacetylase (hdac7), one of the markers of epigenetic modifications, metabolic activity of activated cells and expression profiles of proinflammatory cytokines viz. il6a, tnfαa2, and ifnγ were targeted in the study and analyzed in vivo. Besides in vivo analysis, in vitro analysis of mtorc2, hif1α, hdac7, and ifnγ were analyzed. In vitro analyses were performed on head kidney macrophages isolated and maintained in L-15 media and double trained with ß-glucan at 100µg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with ß-glucan at 20 mg/kg body weight for training followed by a resting phase for 6 days and were restimulated with the same dose. Head kidney was collected from the fish post-induction as well as post-restimulation. The expression profile of mtorc2, hdac7, and hif1α were found elevated post-stimulation of ß-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-ß-glucan stimulation was confirmed by the high lactate and LDH production detected in the cell culture supernatant. Overall, the study revealed the expression profile of the trained immunity markers and the increased metabolic activity in cells induced with ß-glucan, which further validates that the action of trained immunity is indispensable in fish on encounter with a potential ligand. The study supports the existing reports on trained immunity in teleost fish with evidence at the genomic level. However, further studies are required to understand the responses and actions of trained immune cells during infection in detail.

Effect of potassium supplementation on osmoregulatory and stress response of Pangasianodon hypophthalmus (Sauvage, 1878) with Spinacia oleracea L. in aquaponics.

This study evaluated the effect of different potassium supplementation dosages on the physiological responses of Pangasianodon hypophthalmus reared in an aquaponic system with Spinacia oleracea L. for 60 days. The system comprised of a rectangular fish tank of 168 l capacity (water volume = 100 l) with nutrient film technique (NFT)-based hydroponic component with fish to plant ratio of 2.8 kg m-3 : 28 plants m-2 in all the treatments. The osmoregulatory and stress parameters of P. hypophthalmus at four different potassium dosages of T1 (90 mg l-1 ), T2 (120 mg l-1 ), T3 (150 mg l-1 ) and T4 (180 mg l-1 ) were compared with C (control, 0 mg l-1 ) to examine the potassium level to be applied to aquaponics. The water quality parameters and fish production were found to have no adverse impact due to potassium supplementation. The spinach yield during two harvests, i.e., before and after potassium supplementation, revealed that the yield was significantly higher (P < 0.05) after supplementation with the highest yield in T3 and T4. The osmoregulatory parameters such as plasma osmolality, Na+ , K+ ATPase activity in gill and plasma ionic profile (Cl- , Ca2+ and Na+ ) showed an insignificant variation (P > 0.05) between control and treatments except for higher plasma potassium concentration (1.98 ± 0.19 mmol l-1 ) in T4. The stress and antioxidant enzyme analysis exhibited significantly higher plasma glucose and superoxide dismutase (SOD) activity in gill and liver in T4, whereas cortisol and catalase showed an insignificant difference (P > 0.05). The experimental findings demonstrated that the potassium dosage up to 150 mg l-1 could be suggested as optimum for P. hypophthalmus and spinach aquaponics without impairing the health and oxidative status of P. hypophthalmus.

Taurine and/or inorganic potassium as dietary osmolyte counter the stress and enhance the growth of GIFT reared in ion imbalanced low saline water.

The effects of dietary osmolytes for alleviating osmotic stress and enhancing growth are not well elucidated in fish reared in inland saline water. The present study evaluated the effects of dietary taurine or potassium (K+) individually or in combination on growth, ionic homeostasis, and stress response of GIFT tilapia reared in potassium deficient low saline water (PDLSW, 10 ppt salinity) mimicking inland saline water. Isonitrogenous and isoenergetic diets supplemented with five potassium concentrations (0, 0.3, 0.45, 0.6 and 0.75 %), two taurine (T) concentrations (0.5 and 1.0 %) and two combinations of both (K+ 0.1 % + T 0.5 % and K+ 0.2 % + T 0.5 %) were fed to GIFT juveniles (4.4 ± 0.02 g body weight) and reared in PDLSW for 45 days. The fish fed on the diet fortifying with K+ 0.2 % + T 0.5 % showed the highest growth performance among the controls and other treatment groups. Dietary supplementation had no effects on PDLSW induced increase in osmoregulatory endpoints. The optimum dietary potassium requirement of GIFT reared in PDLSW was 0.57 and 0.599 g/100 g diet. Dietary K+ down-regulated the PDLSW induced expression of NKAa1, AQP1, and ClC2, whereas inhibited taurine-induced up-regulation of AQP1 and CLC2, which is the first report in tilapia. In addition, dietary K+ and taurine modulated antioxidant and metabolic enzyme activities for easing stress and balancing energy requirements. Thus, blending of potassium (0.2 %) and taurine (0.5 %) in the diet appears best to mitigate stress and enhance GIFT growth reared in inland saline water.

Preparation and characterization of glucose-conjugated super-paramagnetic iron oxide nanoparticles (G-SPIONs) for removal of Edwardsiella tarda and Aeromonas hydrophila from water.

The present research was conducted to prepare efficient G-SPIONs by co-precipitation to remove Edwardsiella tarda and Aeromonas hydrophila from the aqueous solution. The synthesized G-SPIONs were characterized by UV-Vis spectrophotometer, DLS, FEG-TEM, FT-IR, XRD, and VSM analysis. The results showed that the synthesized G-SPIONs had super-paramagnetic properties (58.31 emu/g) and spherical shape (16 ± 3 nm). The antibacterial activity was assessed in sterilized distilled water at different G-SPIONs concentrations viz. 0, 1.5, 3, 6, 12, 24, 48, 120, and 240 mg/L against E. tarda and A. hydrophila with various bacterial loads viz. 1 × 103 , 1 × 104 , 1 × 105 , 1 × 106 , and 1 × 107 CFU/ml at different time intervals 15, 30, 45, and 60 min. At a lower bacterial load of E. tarda and A. hydrophila 1 × 103 -1 × 104 CFU/ml, 100% bacterial load was removed by 15 min exposure with NPs concentration 6-48 mg/L and 1.5-6 mg/L, respectively. Cent percent bacterial removal was observed in both the bacterial species even at higher bacterial load (1 × 105 -1 × 107 CFU/ml) by increasing exposure time (15-60 min) and nanoparticle concentration as well (24-240 mg/L). At an initial bacterial load of E. tarda and A. hydrophila (1 × 103 -1 × 107 CFU/ml), the EC50 ranged between 0.01-6.51 mg/L and 0.02-3.84 mg/L, respectively, after 15-60 min exposure. Thus, it is concluded that the antibacterial effect of G-SPIONs depends on concentration and exposure time. Hence, G-SPIONs can be used as an antibacterial/biocidal agent to treat Edwardsiellosis and Aeromonosis disease in aquaculture. HIGHLIGHTS: The glucose-conjugated super-paramagnetic iron oxide nanoparticles (G-SPIONs) synthesized by rapid and high yield co-precipitation method shows bacterial removal efficiency against Edwardsiella tarda and Aeromonas hydrophila The bacterial removal efficiency of G-SPIONs depends on concentration and exposure time to both the bacterial species The 100% bacterial removal efficiency can be achieved even at the highest bacterial load (1 × 107 CFU/ml) against E. tarda and A. hydrophila after 45 and 30 min exposure Due to the high bacterial removal efficiency of G-SPIONs against E. tarda and A. hydrophila, it can be used for the treatment of Edwardsiellosis and Aeromonosis in fish.

Dietary arginine attenuates hypoxia- induced HIF expression, metabolic responses and oxidative stress in Indian Major Carp, Cirrhinus mrigala.

Hypoxia is a common stressor in aquaculture systems, which causes severe physiological disturbances, ultimately leading to mortality or reduced productivity. Arginine, as a precursor of NO, has a role in enhancing oxygen delivery. Thus, an experiment was conducted to evaluate the effect of dietary arginine (Arg) in Cirrhinus mrigala exposed to hypoxia. The fish were fed with different levels of arginine for 60 days and exposed for 72 h to a sublethal level of hypoxia (0.50 ± 0.16 mg/L dissolved oxygen [DO]). The six treatment groups with three replicates were N0 (0% Arg + Normoxia), H0 (0% Arg + Hypoxia), N0.7 (0.70% Arg + Normoxia), H0.7 (0.70% Arg + Hypoxia), N1.4 (1.40% Arg + Normoxia), H1.4 (1.40% Arg + Hypoxia). Eighteen experimental units with twelve animals (5.8 ± 0.18 g) each were used for the trial.The results indicated that supplementation of arginine at 0.7 and 1.4% enhanced the hypoxia tolerance time, although the high dose (1.4%) did not yield any further increments. The exposure to hypoxia up-regulated Hypoxia Inducible Factor (HIF)-1α mRNA expression and supplementation of arginine significantly decreased hypoxia induced up-regulation of HIF at 1.4%. Arginine supplementation partially or completely normalised the hypoxia induced changes in the metabolic enzymes of C. mrigala. The fish exposed to hypoxic conditions exhibited significantly higher (P < 0.05) lipid peroxidation levels than those maintained under normoxic conditions, while arginine feeding significant in reducing lipid peroxidation. Antioxidant enzyme activities were significantly higher (P < 0.05) in hypoxia-exposed carp, indicating increased oxidative stress during the hypoxic exposure, that was improved in Arg-supplemented groups. However, arginine did not modulate erythrocyte countsalthough itreduced the erythrocyte fragility. We conclude arginine supplementation is effective in ameliorating hypoxia induced metabolic alterations and improving antioxidant defences in fish.

Extra-Fortification of Zinc Upsets Vitellogenin Gene Expression and Antioxidant Status in Female of Clarias magur brooders.

The present experiment was designed to evaluate the effect of graded level of zinc on Vitellogenin gene (Vtg) expression and antioxidant enzymes in threatened catfish, Clarias magur (C. magur). One hundred and eighty female C. magur with an average weight of 145 ± 5 g were allocated in twelve cemented tanks with dimension 4.5 × 2 × 1 m for a period of 60 days. Fish were distributed in four groups with three replicates following the completely randomised design. The first group treated as control (C) fed with basal diet contained normal zinc level, and remaining groups were fed with basal diets having 50, 200 and 300 mg/kg zinc acetate and treated as T1, T2 and T3 respectively. To evaluate the effect of dietary zinc supplementation on Vtg gene expression, three sampling were carried out, I sampling (April, before starting the experimental trail), II sampling (May, after 1 month of feeding trail) and III sampling (June before breeding season). In the present study, a dose-dependent relationship between Vtg gene expression and zinc inclusion in the diet of threatened catfish, C. magur, was reported. Vtg gene expression increased in all groups from I sampling to II sampling but the highest Vtg gene expression was found in T1 group and the lowest in T3 group at II sampling. Vtg gene expression among the treatments differs significantly (P < 0.05) in each sampling. Accumulation of zinc was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) in C. magur and it was reported that the significantly higher (P < 0.05) zinc was accumulated in the liver and ovary of T3 group as compared to other groups. The antioxidant enzyme activities (superoxide dismutase, SOD, catalase and GST) were also measured in different tissues (liver, gill and ovary) to evaluate the effect of extra-supplementation of zinc on the antioxidant status. In T3 group, SOD, catalase and GST activities were significantly higher than those in other groups. In the current study, serum glucose level was also measured and it was found in increasing trend with inclusion of zinc in the diet of C. magur. In the present study, it can be concluded that the zinc exhibits beneficial effect only up to 50 mg/kg. Thus, it is concluded that supplementation of zinc at 200 mg/kg or more disrupts Vtg gene expression and antioxidant status.

Effect of Dietary Lipid Level on Growth Performance, Body Composition, and Physiometabolic Responses of Genetically Improved Farmed Tilapia (GIFT) Juveniles Reared in Inland Ground Saline Water.

A 60-day feeding trial was carried out to determine the effect of dietary lipid levels on growth and physiometabolic responses to optimize the dietary lipid requirement for maximizing the growth of Genetically Improved FarmedTilapia (GIFT) juveniles reared in inland ground saline water (IGSW) of medium salinity (15 ppt). Formulation and preparation of seven heterocaloric (389.56-449.02 Kcal digestible energy/100 g), heterolipidic (40-160 g/kg), and isonitrogenous (410 g/kg crude protein) purified diets were done for conducting the feeding trial. Random distribution of 315 acclimatized fish (mean weight 1.90 ± 0.01 g) was made in seven experimental groups such as CL4 (40 g/kg lipid), CL6 (60 g/kg lipid), CL8 (80 g/kg lipid), CL10 (100 g/kg lipid), CL12 (120 g/kg lipid), CP14 (140 g/kg lipid), and CL16 (160 g/kg lipid) with 15 fish per triplicate tank (fish density, 0.21 kg/m3). Respective diets were used for feeding the fish at satiation level three times daily. Results indicated that weight gain percentage (WG%), specific growth rate (SGR), protein efficiency ratio, and protease activity significantly increased up to 100 g lipid/kg fed group, and then the values significantly decreased. Muscle ribonucleic acid (RNA) content and lipase activity were highest in 120 g/kg lipid-fed group. RNA/DNA (deoxyribonucleic acid) and serum high-density lipoproteins levels of 100 g/kg lipid-fed group were significantly higher than 140, and 160 g/kg lipid-fed groups. The lowest feed conversion ratio was found in the 100 g/kg lipid-fed group. The amylase activity was significantly higher in 40 and 60 g lipid/kg fed groups. The whole-body lipid level was increased with increasing the dietary lipid levels, whereas, there was no significant difference in whole-body moisture, crude protein, and crude ash contents of all groups. Highest serum glucose, total protein and albumin, and albumin to globulin ratio and lowest low-density lipoproteins level were found in 140 and 160 g/kg lipid-fed groups. Serum osmolality and osmoregulatory capacity did not vary significantly, whereas carnitine palmitoyltransferase-I and glucose-6-phosphate dehydrogenase showed an increased and decreased trend, respectively, with the increasing dietary lipid levels. According to second-order polynomial regression analysis based on WG% and SGR, the optimum dietary lipid for GIFT juveniles in IGSW of 15 ppt salinity was found to be 99.1 and 100.1 g/kg, respectively.

Insulin-like growth factor signalling and its significance as a biomarker in fish and shellfish research.

The insulin-like growth factor signalling system comprises insulin-like growth factors, insulin-like growth factor receptors and insulin-like growth factor-binding proteins. Along with the growth hormones, insulin-like growth factor signalling is very pivotal in the growth and development of all vertebrates. In fishes, insulin-like growth factors play an important role in osmoregulation, besides the neuroendocrine regulation of growth. Insulin-like growth factor concentration in plasma can assess the growth in fishes and shellfishes and therefore widely applied in nutritional research as an indicator to evaluate the performance of selected nutrients. The present review summarizes the role of insulin-like growth factor signalling in fishes and shellfishes, its significance in aquaculture and in evaluating growth, reproduction and development, and discusses the utility of this system as biomarkers for early indication of growth in aquaculture.

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.

Dietary GABA enhances hypoxia tolerance of a bottom-dwelling carp, Cirrhinus mrigala by modulating HIF-1α, thyroid hormones and metabolic responses.

The Indian major carp, mrigal (Cirrhinus mrigala), is a bottom-dwelling fish that can survive hypoxic episodes in its natural environment. We hypothesise that it can better survive hypoxic conditions by altering metabolic responses through GABA (Gamma-aminobutyric acid) supplementation. In the first experiment, the hypoxia tolerance time of the fishes was evaluated under extreme anoxic conditions after feeding with GABA, which showed that GABA had improved survival time under hypoxia. To study the response of dietary GABA in hypoxia-exposed fish, the branchial HIF-1α expression levels, serum thyroid hormone levels and hepatic metabolic responses were assessed in the subsequent experiment. The treatment groups were fed for 60 days with experimental diets containing 4 levels of GABA (0.00% G, 0.50% G, 0.75% G and 1.0%G) and were subjected to 72-h hypoxia exposure (0.5 ± 0.02 mg L-1 dissolved oxygen (DO)) whereas a control group was maintained under normoxic conditions (6.0 ± 0.21 mg L-1 DO). The five treatment groups with three replicates were C0 (0% G + normoxia), H0 (0% G + hypoxia), H0.5 (0.50% G + hypoxia), H0.75 (0.75% G + hypoxia) and H1.0 (1.00% G + hypoxia). The results indicated that GABA supplementation triggered downregulation of HIF 1 alpha expression. When compared with the control group, decreased thyroxine (T4) and triiodothyronine (T3) levels were observed in the GABA-fed hypoxic groups. However, TSH (thyroid stimulating hormone) level remained unchanged in all the treatments. The LDH (lactate dehydrogenase) level in hypoxia-exposed groups was decreased by GABA supplementation. Our study demonstrated that GABA supplementation restores acute hypoxia-induced HIF-1α expression, thyroid hormone levels and LDH activities. On the other hand, it enhanced the citrate synthase (CS) activities at 0.5-1.00%, which showed a sharp decline in hypoxia. Hypoxia caused increase in the serum metabolites such as glucose, lactate, cholesterol and triglycerides. However, GABA supplementation was partially effective in reducing glucose and lactate level while triglycerides and cholesterol values remained unchanged. Overall, our results suggested a potential role of GABA in suppressing metabolism during hypoxia exposure, which can increase the chances of survival of the species Cirrhinus mrigala during hypoxia.

Molecular and computational analysis of Ghrelin, growth hormone Secretagogues receptor and mRNA expression of Growth-related genes after exogenous administered ghrelin peptide in Labeo rohita.

Ghrelin is a peptide hormone secreted primarily by the stomach and is involved in controlling growth by governing different functions in vertebrates including feed intake and metabolism in vertebrates. This work was aimed to identify sequences of ghrelin gene and growth hormone secretagogue receptor (GHSR) in Labeo rohita. The full-length cDNA sequence of ghrelin is 453 bp including 5'-untranslated region (UTR) of 65 bp, 3'-UTR of 76 bp with a poly-A frame. An open reading frame (ORF) is 312 bp, which encodes a peptide of 103 amino acid residues. A secondary structure of GHSR protein consists of alpha helix 66.0%, 16% disordered and 43% transmembrane helix. Molecular docking and interaction between synthetic ghrelin peptides and GHSR in the contact map revealed 19 amino acid residues closer than 4.5 Šdistance. The mRNA expression level of ghrelin, leptin, GHSR, growth hormone releasing hormone (GHRH) and insulin growth factor-I (IGF-1) revealed significant changes (p < 0.05), in the different treatments. The outcome of the present work contributes to understanding the role of ghrelin and its mechanism of action in regulating the expression of growth-related genes and feed intake in fish.

Pseudobranch mimics gill in expressing Na+K+-ATPase 1 α-subunit and carbonic anhydrase in concert with H+-ATPase in adult hilsa (Tenualosa ilisha) during river migration.

In hilsa (Tenualosa ilisha), pseudobranch comprises a row of parallel filaments bear numerous leaf-like lamellae arranged on both sides throughout its length. The purpose of this study was to elucidate involvement of pseudobranchial Na+, K+-ATPase (NKA) 1 α-subunit, and carbonic anhydrase (CA) in concert with H+-ATPase (HAT) compared to their branchial counterparts in freshwater acclimation of hilsa during spawning migration from off-shore of the Bay of Bengal to the Bhagirathi-Hooghly zones of the Ganga river system in India. Adult hilsa fish were collected from seawater (SW), freshwater 1 (FW1), and freshwater 2 (FW2) locations, where the salinity level was 26-28‰, 1-5‰, and 0-0.04‰, respectively. Hilsa migrating through freshwater showed a consistent decrease in the plasma osmolality, sodium (Na+) and chloride (Cl-) ion levels indicates unstable ionic homeostasis. The mRNA expression and activity of NKA 1 α-subunit in pseudobranch as well as in true gills declined with the migration to upstream locations. The pseudobranchial CA activity almost mirrors its branchial counterpart most notably while hilsa entered the freshwater zone, in the upstream river suggesting its diverse role in hypo-osmotic regulatory acclimation. Nevertheless, the H+-ATPase activity of both the tissues increased with the freshwater entry and remained similar during up-river movement into the freshwater environment. The results confirm that the pseudobranchial NKA 1 α-subunit mRNA expression and activity mimic its branchial counterpart in the process of ionoregulatory acclimation during migration through salt barriers. Also, the increase in the activities of pseudobranchial and branchial CA in concert with H+-ATPase (HAT) during freshwater acclimation of hilsa suggests their critical involvement in ion uptake.

Expression of Growth and Hunger Related Genes and Physio-Biochemical Responses in Labeo rohita (Hamilton, 1822) Fed with Lysine and Betaine.

BACKGROUND/AIMS: The growth promoting effect of lysine and betaine as well as the expression of candidate genes reflecting their efficacy, such as ghrelin, leptin, Growth Hormone Secretagogue Receptor (GHS-R), Insulin like Growth Factor (IGF- 1) and Growth Hormone Releasing Hormone (GHRH) was examined in Labeo rohita fingerlings. METHODS: One hundred eighty healthy juveniles from a homologous population were randomly distributed to 15 rectangular tanks of 150 litres capacity. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. At the end of trial, the growth parameters such as weight gain, SGR, PER were estimated from the weight of the triplicate groups. The digestive, metabolic and antioxidant enzymes were analysed using spectrophotometric methods. The intestine, brain and liver were sampled from the treatments and expression of different genes ghrelin, leptin, GHSR, IGF-1 and GHRH was also performed by realtime PCR. RESULTS: A significant (P<0.05) increase in weight gain, SGR, PER and lowest FCR was found in T4 group which was significantly (p < 0.05) different from other experimental groups. The highest mRNA expression levels of expression were found in T4 group which was similar to that of ghrelin gene mRNA of T2 group. The significantly (p<0.05) highest GHSR, GHRH and IGF-1 gene expression levels were found in T4 treatment group compared to other groups. CONCLUSION: The present study reveals that the lysine and betaine stimulate growth and expression of ghrelin GHRH, GHS-R and IGF-1 genes. The increase of IGF-I mRNA expression with lysine and betaine supplementation revealed that these compounds act as growth modulators. However, lysine was found to be a more potent modulator of growth compared to betaine.

Expression and activity of key lipases during the larval development of walking catfish (Clarias magur).

The study was conducted to investigate the expression and activity of key lipolytic enzymes during the ontogenetic development of Clarias magur. After partial characterization, the messenger RNA (mRNA) expression analysis of lipoprotein lipase (LPL), pancreatic triacylglycerol lipase (PL), and bile salt-activated lipase (BAL) genes along with the specific lipase activity were performed in larvae from Day 1 after hatching till 34-day posthatch (dph). Heterogeneous patterns of mRNA expression were shown by the important lipolytic enzymes and were detected before first exogenous feeding during the yolk-sac stage. LPL started increasing from 13 dph and peaked at 16 dph followed by a declining trend till 34 dph. However, the PL observed to be peaking at 9, 22, and 30 dph. Similarly, BAL showed an increasing trend from 11 to 22 dph with a significantly high level of mRNA expression at 16 dph. Later, the specific lipase activity was evaluated which appears at Day 1 after hatching with a progressive increase from 7 to 16 dph and a further declining trend afterwards with a peak at 22 dph. The results indicated the development of exocrine pancreas at 16 dph. Furthermore, the transcript levels and the activity of lipases were regulated with the age. Hence, the present study can be helpful in devising different strategies containing optimum lipid levels at a suitable stage of development for improving the survival during larval rearing. Furthermore, the study could be a baseline for elucidating the optimized dietary lipid levels of this catfish during its larval rearing.

Temporal changes in superoxide dismutase, catalase, and heat shock protein 70 gene expression, cortisol and antioxidant enzymes activity of Labeo rohita fingerlings subjected to starvation and refeeding.

A short term starvation and refeeding experiment was conducted to study the temporal changes in SOD, CAT and HSP70 gene expression of Labeo rohita fingerlings. The study was carried out for 15 days with initial 7 days of starvation and then refeeding up to 15th day of the experimental trial. The expressions of SOD and CAT genes of liver and gills were significantly up-regulated after 7 days of starvation, down-regulated after 3 days of refeeding, and returned to the basal values after 8 days of refeeding. The HSP70 gene expression was significantly (p < 0.05) increased after starvation, with highest mRNA expression found on 7th day and reduced to the levels of control on refeeding. The activities of antioxidant enzymes, SOD and CAT were also studied to correlate with the results of gene expression. The changes in activities of SOD and CAT were found significantly (p < 0.05) higher in the starved group compared to the fed group. The dynamics of AST and ALT in serum revealed a progressive increase till the 7th day and decreased upon refeeding, cortisol level also has shown significant increase up to 7th day of starvation and sharp decline on refeeding. The concentration of blood glucose level start declining on 3rd day onwards with lowest level found on 7th day of starvation and was quickly restored to the levels of control on refeeding. The present study reveals that starvation elicits oxidative stress response as revealed by enhanced expression and activities of antioxidant enzymes, HSP 70 and serum biochemical alterations. However, these alterations were restored upon refeeding of L. rohita within 7 days.

Regulation of compensatory growth by molecular mechanism in Labeo rohita juveniles under different feeding regimes.

A study was carried out to assess the regulation of compensatory growth under different restriction feeding regimes in Labeo rohita juveniles by the interaction of various feed intake and growth regulating genes. A 60 day feeding trial was conducted with five treatment groups, Control (3% body weight, bw), T1 (alternate days), T2 (0.5% bw), T3 (1% bw) and T4 (2% bw) and feeding was done for first 30 days of the trial. For next 30 days, all the treatment groups were fed at a rate of 3% bw as in the control group. There was significant (p < 0.05) difference in the weight gain among the treatment groups with lowest FCR and highest PER was found in T2 group. Ghrelin gene mRNA levels were upregulated during first 30th days of the trial with highest expression levels in the T2 group. The expression levels of leptin gene mRNA were found significantly different (p < 0.05) among the treatments, which was down-regulated during initial 30 days and upregulated as the experiment progress towards 60th day. The IGF-1 mRNA expression levels were upregulated more in liver compared to the muscle tissue. The results of the study suggest that increased ghrelin levels and decreased leptin levels lead to hyperphagia during the onset of refeeding, which further triggers the compensatory growth in L. rohita. The present study describes the molecular mechanism behind the compensatory growth following a different feed restriction regime in L. rohita which is regulated due to the interaction of different energy homeostasis and growth regulating genes.

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species.