Terminalia arjuna Bark Powder as a Potential Immunomodulator in Labeo rohita: Enhanced Hematological, Adaptive, and Humoral Responses against Bacterial Pathogens and Concordant Liver Histomorphology.

This study investigated the dietary immunomodulatory effects of Terminalia arjuna bark powder (TABP) in Labeo rohita, a freshwater fish model. Four iso-nitrogenous and iso-caloric diets containing graded levels of TABP (0, 1, 10, and 15 g/kg were fed to fish for 90 days, followed by a 10 day challenge with pathogenic bacteria Aeromonas hydrophila and Edwardsiella tarda. An integrated biomarker response (IBR) approach assessed the impact of TABP on hematological, adaptive, and humoral immune parameters, along with liver histomorphology. Dietary TABP at 10 g/kg significantly enhanced (p < 0.05) hematological indices (hemoglobin, red blood cell count, hematocrit), specific immune parameters (lysosomal enzyme activity, phagocytosis, respiratory burst), and non-specific immune parameters (serum lysozyme, alternative complement activity), and exhibited improvements in liver architecture consistent with the enhanced immune response. Broken line regression analysis showed 11.5 g/kg to be an optimum dose. However, at 15 g/kg, a compromised trend was observed in some parameters. These findings suggest an optimal dosage range for TABP's immunomodulatory effects. The study highlights the potential of TABP as a natural immunomodulator in fish aquaculture. The improved immune response and concomitant liver health observed in Labeo rohita opens avenues for further research on TABP's applicability in animal health, using fish as a model organism. Additionally, the IBR approach proved effective in evaluating TABP's immunomodulatory properties, paving the way for similar studies on other natural products in aquaculture.

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.

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.

Immunogenic Effects of Dietary Terminalia arjuna Bark Powder in Labeo rohita, a Fish Model: Elucidated by an Integrated Biomarker Response Approach.

Utilizing agro-industrial waste and herbal products to create a circular bioeconomy is becoming increasingly popular. Terminalia arjuna is a significant ethnomedicinal plant that has not yet been exploited in animal feed. In the present study, nutritional Terminalia arjuna bark powder-based fish feed was created and supplied to a candidate fish species Labeo rohita at varied levels: 0% (0 g/kg), 0.5% (5 g/kg), 1% (10 g/kg), and 1.5% (15 g/kg). These treatment groups are denoted as CT, T1, T2, and T3, respectively. Utilizing a contemporary comprehensive biomarker response strategy, the study clarified the genomic influence of dietary herb inclusion. In response to bacterial infection, the immunogenic genes, STAT 1 (signal transducer and activator of transcription 1), ISG 15 (interferon stimulating gene), and Mx "myxovirus resistance gene", were shown to be elevated. The results of densitometry demonstrated a dose-dependent increase in STAT 1 and ISG 15, with Mx exhibiting maximal values at 1 g/kg TABP (Terminalia arjuna bark powder-based feed). This is the first study to identify TABP as an immunomodulator in fish and established the IBR (Integrated Bio-marker Response) as a reliable marker in evaluating the impact of multiple drivers in a holistic manner. Thus, the present study cleared the path for TABP to be utilized as an effective feed additive which enhances the specific adaptive immune system of the fish for the production of the Green fish product for a sustainable circular bioeconomy.

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.

Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium concentration.

This study assessed the responses of vitamin-D3 intraperitoneally injected to Rohu, Labeo rohita @ of 0 IU/kg bw (only solvent), 100 IU/kg bw and 500 IU/kg bw reared in 20 and 40 ppm of calcium (Ca) enriched water. The cellular changes in Corpuscles of Stannius (CS) gland, serum Ca, and inorganic phosphate (Pi) level were analysed up to the 60th day. Rohu administered with 100 IU/kg bw D3 and exposed to 40 ppm Ca-rich water exhibited notable hyperplasia of CS compared with their control groups. Notable changes with high serum Ca level (13.87 ± 0.3 mg/dl) was detected on the 5th day in fish exposed to 40 ppm Ca-rich water, while related values attained (13.74 ± 0.1 mg/dl) only after 7 days in 20 ppm Ca-rich water of 500 IU/kg bw vitamin D3 injection. Similarly, high serum Pi level (7.66 ± 0.2 mg/dl) in 40 ppm Ca injected with D3 at 500 IU/kg bw. The results demonstrated that the Ca homeostasis of Labeo rohita is influenced by intra-peritoneal vitamin D3. Progressive studies should be conducted by increasing the dose of vitamin D3 to investigate optimum dose/supplement in feed for commercially important aquaculture teleost Labeo rohita for maximum and sustainable absorption of Ca from the variable water Calcium levels to maintain Ca2+ homeostasis.

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.

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.

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.