Higher intake of ß-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of ß-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% ß-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of ß-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% ß-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% ß-glucan-fed individuals. Irrespective of ß-glucan intake, all the brooders recorded similar plasma 17ß-estradiol and maturation-inducing hormone (p>0.05). Higher intake of ß-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17ß-estradiol. However, plasma vitellogenin increased with increasing ß-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% ß-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% ß-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% ß-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% ß-glucan. This study demonstrated the stimulatory effect of ß-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Expression of genes encoding non-specific immunity, anti-oxidative status and aquaporins in ß-glucan-fed golden mahseer (Tor putitora) juveniles under ammonia stress.

The study investigated the effects of dietary administration of ß-glucan on aquaporins and antioxidative & immune gene expression in endangered golden mahseer, Tor putitora juveniles, exposed to ammonia stress. For that, fish were fed experimental diets having 0 (control/basal), 0.25, 0.5, and 0.75% ß-d-glucan for five weeks and then exposed to ammonia (10 mgL-1 total ammonia nitrogen) for 96 h. Administration of ß-glucan differentially influenced the mRNA expression of aquaporins, anti-oxidative, and immune genes in ammonia-exposed fish. For instance, the transcript abundance of catalase and glutathione-s-transferase in gill varied significantly among the treatment groups, with the lowest levels in 0.75% ß-glucan fed groups. At the same time, their hepatic mRNA expression was similar. Congruently, transcript abundance of inducible nitric oxide synthase considerably decreased in the ß-glucan fed ammonia-challenged fish. Conversely, the relative mRNA expression of various immune genes viz., major histocompatibility complex, immunoglobulin light chain, interleukin 1-beta, toll-like receptors (tlr4 and tlr5) and complement component 3 remained largely unchanged in ammonia-exposed mahseer juveniles that were fed with graded levels of ß-glucan. On the other hand, a significantly lower transcript level of aquaporins 1a and 3a was noticed in the gill of glucan-fed fish compared to ammonia-exposed fish that received the basal diet. However, branchial aquaporin 3b remained unaltered. Altogether, this study showed that dietary intake of 0.75% ß-glucan improved resistance to ammonia stress to a certain degree, probably through activating anti-oxidative system and reducing brachial ammonia uptake.

Molecular characterization of four innate immune genes in Tor putitora and their comparative transcriptional abundance during wild- and captive-bred ontogenetic developmental stages.

The current study was designed to characterize immune genes and compare their expression during ontogenetic developmental stages in progenies of wild-collected and captive-matured T. putitora. The full-length cDNA sequences of Tptlr2, Tpmyd88, Tpcd36, and Tpil8 were 2814, 1545, 1807, and 653 bp in length, with ORFs of 2379 bp, 855 bp, 1410 bp, and 297 bp, encoding for putative peptides of 793, 284, 469 and 98 amino acids, respectively. The predicted peptide sequences of the genes had high sequence homology and structural conservation with other teleost fishes, especially cyprinids. The expression of Tptlr2 was relatively low in both wild- and captive-bred offsprings during the early embryonic stages, but significantly increased later in development. The mRNA abundance of the Tpmyd88 gene was significantly low until the blastula stage, then increased notably from the gastrula stage to the advanced fry stage. The Tpcd36 expression elevated during later developmental stages, peaking at advanced fry stage in both. On the other hand, expression of Tpil8 was relatively low until the blastula stage and showed a moderate increase from the organogenesis stage onwards in wild-bred offsprings, whereas a significant upregulation was seen in the captive-bred offsprings from the organogenesis stage until the advanced fry stage, with its maximum expression at the pre-metamorphosis stage. Overall, the findings suggest the crucial role of Tpmyd88, Tptlr2, Tpcd36, and Tpil8 in inducing innate immunity in embryonic and larval stages of T. putitora. Further, the considerably higher expression of the immune genes in the embryonic and larval stages of captive-bred offsprings may indicate a stronger immune system.

Concurrent changes in thermal tolerance thresholds and cellular heat stress response reveals novel molecular signatures and markers of high temperature acclimation in rainbow trout.

The objective of this study was to better understand the molecular mechanisms which regulate acclimatory responses and thermal safety margins of rainbow trout (Oncorhynchus mykiss) at temperatures above physiological optimum. For this, we investigated the time course of changes in critical thermal tolerance thresholds and associated hepatic and renal transcript abundance of molecular markers related to cellular stress response, during high temperature acclimation. The experimental fish were initially acclimated to 17 °C and later exposed to a gradually raised elevated temperature regime (22 °C) for a period of 30 days. CTmax, CTmin and mRNA expression of candidate markers were examined before the thermal challenge (T0) and over the time-course (days) of high temperature exposure (T1, T3, T7, T15 and T30). With respect to organismal response, CTmax was significantly elevated at T3, but the degree of gain in heat tolerance was not persistent. Contrarily, we observed a gradual loss in cold tolerance with highest CTmin estimate at T30. Based on the time-course of mRNA expression, the studied markers could be categorized into those which were persistently elevated (hsp70a, hsp70b, hspa5, hsp90a, hsp90b, stip1 and serpinh1 in kidney and hsp90b in liver); those which concurred with changes in CTmin (hspbp1, hsp90b, stip1, gr1, hif1a, hyou1, tnfa and tlr5 in kidney); and those which concurred with changes in CTmax (hsp90a, serpinh1, tlr5 and lmo2 in liver). Apparently, transcriptional changes in kidney and liver reflected CTmin and CTmax trend, respectively. Expression profile of stip1 and tlr5 suggest that they are potential novel markers which could reflect thermal limits in rainbow trout. Hepatic metabolic markers were either initially elevated (alt, glud, g6pase1) or down-regulated at different time-points (ast2, gls1, fas, cpt1b, mtor), linked to gluconeogenesis and metabolic depression, respectively. Whereas, growth-axis markers showed no significant differences. Overall, this time-course analysis has revealed potential associations in organismal and tissue-specific cellular response to high temperature acclimation in a thermally sensitive coldwater ectotherm.

Dietary ß-glucan influences the expression of testicular aquaporins, antioxidative defence genes and sperm quality traits in endangered golden mahseer, Tor putitora (Hamilton, 1822).

The effect of dietary ß-glucan on seminal plasma composition, sperm characteristics, expression of aquaporins, and antioxidative defence genes of golden mahseer was evaluated. For that, four experimental diets containing 0 (control), 0.5, 1, and 1.5% ß-glucan were fed to male golden mahseer brooders for 130 days. Feeding of 0.5% ß-glucan was found to improve sperm characteristics, viz. sperm count, motility, viability, and morphology with no effect on gonadosomatic index and seminal plasma energy resources. The marked down-regulation in the transcript abundance of testicular aqp3a noticed in 1.5% ß-glucan fed brooders corresponds to their poor sperm quality. Further, the mRNA expression of genes encoding antioxidant enzymes, namely gst and sod1, was lowest in 0.5% ß-glucan fed brooders. In contrast, control and higher ß-glucan (1 and 1.5%) groups displayed relatively higher expression levels of testicular gst and sod1. On the other hand, the higher seminal plasma total antioxidant capacity observed in 0.5 and 1% ß-glucan fed brooders indicated increased scavenging ability of reactive oxygen species. Overall, supplementation of 0.5% ß-glucan improved sperm quality and antioxidative potential, but the higher inclusion (1.5%) negatively affected sperm characteristics. Collectively, dietary ß-glucan (0.5%) can be a practical approach to developing quality broodstock of golden mahseer.

ß-glucan modulates non-specific immune gene expression, thermal tolerance and elicits disease resistance in endangered Tor putitora fry challenged with Aeromonas salmonicida.

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of ß-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of ß-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of ß-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1ß were significantly upregulated, while ifn-γ and il-10 were unaffected by ß-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, ß-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of ß-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CTmax and lower CTmin than the control group. After feeding ß-glucan for eight weeks, the fish were bath challenged with a bacterial pathogen, Aeromonas salmonicida. The challenge study results revealed that ß-glucan intake improved most of the studied immune parameters, resulting in lower mortality. Overall, dietary inclusion of ß-glucan (0.5-1.0%) was efficient in improving the immune responses, thermal tolerance, and disease resistance of T. putitora fry.

Dietary soy lecithin augments antioxidative defense and thermal tolerance but fails to modulate non-specific immune genes in endangered golden mahseer (Tor putitora) fry.

A 70-day experiment was carried out to assess the effect of different levels (0, 1 and 2%) of soy lecithin in the diet on growth, survival, antioxidant defense markers, immune gene expression and thermal tolerance limits of golden mahseer, Tor putitora fry. Percentage weight gain, specific growth rate (SGR %) and survival of mahseer fed lecithin supplemented diets were not significantly different from those of the control group. Also, the mRNA expression levels of different immune related genes such as tnfα, il-1ß, il-10, complement-3, interferon-gamma (ifnγ) and tlr4 were unaffected by dietary lecithin supplementation. Nevertheless, superoxide dismutase (SOD) activity was significantly greater in the lecithin-fed groups than the control fish. The glutathione-S-transferase (GST) activity was exceptionally high in the 2% lecithin supplemented group compared to the rest two groups. This increase in antioxidant status with dietary lecithin supplementation, however, was not reflected in the whole body malonaldehyde (MDA) levels, as it did not vary significantly among the dietary groups. Importantly, dietary inclusion of soy lecithin significantly increased upper thermal tolerance limits as evidenced by higher CTmax and LTmax values. Likewise, golden mahseer fry fed with lecithin supplemented diets (both 1 and 2%) registered significantly lower critical and lethal thermal minimum (CTmin and LTmin) values than the control group, indicating higher cold tolerance capacity. Our results thus demonstrate that the dietary inclusion of soy lecithin could enhance the upper and lower thermal tolerance limits and antioxidant status of golden mahseer fry and failed to enhance immune related gene expression.

Temperature and oxygen related ecophysiological traits of snow trout (Schizothorax richardsonii) are sensitive to seasonal changes in a Himalayan stream environment.

In this study, we investigated the seasonal changes in key eco-physiological traits of a wild population of snow trout, Schizothorax richardsonii from river Gola in the Indian Himalayan region over one year. Live specimens (5.8-31.4 g) were electro-fished from their natural habitat during representative months of four seasons with notable differences in water temperature, oxygen concentration and saturation. After 24-72 h of captive-acclimation, the fishes were examined for upper and lower critical thermal limits (CTmax and CTmin), incipient lethal oxygen thresholds (ILOC and ILOS), apparent routine and maximum oxygen consumption rates (MO2rout and MO2max), and blood haemoglobin-haematocrit. Across the seasons, mean CTmin and CTmax values ranged from ∼0 to 34.6 °C, suggesting a relatively wide acute thermal tolerance range for this predominantly cold-water fish. Changes in the habitat's thermal condition during winter to summer was reflected in the CTmin (∼0-2.4 °C) and CTmax (31.7-34.4 °C) estimates, while the highest thermal scope (CTmax-CTmin; 33.2 °C) was recorded in autumn. Concurrently, the incipient lethal hypoxia threshold observed in autumn (ILOS-2.6% and ILOC-0.19 mgO2/L) was significantly lower than the other three seasons, possibly linked to warm-acclimation. The reduction in blood haemoglobin-haematocrit levels during winter could limit the oxygen carrying capacity, with possible reciprocations in thermal tolerance and aerobic metabolism. Concerning body mass corrected oxygen consumption, the apparent MO2rout was found to increase in a temperature-dependent manner from 150.3 mgO2/kg/h at 12 °C to 315.2 mgO2/kg/h at 26 °C, with Q10 ranging from 1.6 to 2.2. Whereas, changes in MO2max was not temperature sensitive (Q10 of 0.7-1.3), except during spring-summer (Q10-2), with lowest and highest measurements in spring and autumn (934 and 1514 mgO2/kg/h), respectively. Collectively, these data form the first information report on the seasonal plasticity in thermal and respiratory physiology of a Schizothoracine fish species, bearing significance for their conservation, aquaculture and habitat monitoring.

Molecular characterisation and transcriptional regulation of muscle growth regulatory factors myogenin and myogenic factor 6 in the Trans-Himalayan cyprinid fish Schizothorax richardsonii.

In vertebrates, myogenic regulatory factors (MRFs) play an important role in muscle growth through the processes of cell determination and differentiation. Herein, we report the molecular characterisation of two MRFs, myogenin (myog) and myogenic factor 6 (myf6) in Indian snow trout Schizothorax richardsonii. The full length mRNA sequence of Srmyog and Srmyf6 was 1437 and 1296 bp, with an ORF of 762 and 720 bp, encoding a putative protein of 253 and 239 amino acids, respectively. In silico analysis and predicted tertiary protein structure revealed that both these nuclear localized MRFs contained the highly conserved basic helix loop helix motif, to potentially aid in dimerization with functional partners and DNA binding. Phylogenetically, the deduced protein sequences of Srmyog and Srmyf6 closely clustered with high altitude dwelling cyprinids, suggesting common ancestry. Tissue-wise, transcripts of Srmyog and Srmyf6 were abundant in skeletal muscle, affirming their muscle-specific role. Moreover, Srmyog was highly expressed in females as compared to males, whereas Srmyf6 expression was higher in older-bigger individuals as compared to younger-smaller fish. Nutritional status (fed-starved-refed) and diet composition (protein and lipid levels) had no significant influence on Srmyog expression. But, Srmyf6 expression was elevated in fishes re-fed after 3 weeks of starvation and in those fed low protein diet. Only rearing temperature was found to coherently influence Srmyog and Srmyf6 transcripts, with highest expression at 18 °C, which favoured growth. Overall, molecular characterisation of the structure and regulation of these MRFs is the first step taken towards deciphering slow growth in this important Himalayan cyprinid.

Physiological changes in Labeo rohita during nitrite exposure: detoxification through dietary vitamin E.

This study investigated the effect of sub-lethal nitrite-nitrogen exposure on Labeo rohita. Fishes fed with different levels of vitamin E (VE) for 60days were exposed to nitrite for another 45days with same feeding regime. There were four treatment groups, viz., VE100-N, VE100+N, VE150+N and VE300+N. After 45days of exposure, lowest specific growth rate was observed in VE100+N and highest in VE100-N. Reno-somatic index and methaemoglobin reductase activity were significantly increased by nitrite exposure. Highest Hb and Hct were observed in VE100-N and significantly decreased upon nitrite exposure. Significant differences were observed in the activities of catalase and SOD as well as serum potassium and chloride levels among different treatments. However, serum calcium and osmolality was not significantly varied. Nitrite exposure caused marked increase in nitrite concentration in gill, liver and muscle. In liver and muscle dietary supplementation of higher amounts of VE found to reduce nitrite accumulation. It was noticed that nitrite exposure has adversely affected growth, haematological variables, ionic balance and dietary supplementation with additional amounts of VE found to overcome the adverse effects of nitrite-nitrogen. Detoxification of nitrite by methaemoglobin reductase system was enhanced by dietary supplementation of additional amounts of VE.

Alterations in serum electrolytes, antioxidative enzymes and haematological parameters of Labeo rohita on short-term exposure to sublethal dose of nitrite.

An experiment was conducted to study the effects of short-term exposure to sublethal levels of nitrite on electrolyte regulation, antioxidative enzymes and haematological parameters in Labeo rohita juveniles. The fishes were exposed to graded levels of nitrite (0-15 mg l(-1)) for different duration (0, 12, 24, 48 and 96 h). The 96-h LC(50) value for L. rohita (avg. wt, 66.5 ± 0.5 g) was found to be 11.28 mg l(-1). Activities of antioxidative enzymes (catalase and superoxide dismutase), acetylcholine esterase (AChE) and methaemoglobin reductase, serum electrolytes (sodium, potassium and chloride), haematological parameters and blood glucose level significantly varied (P < 0.05) in a dose-dependent manner. With increasing nitrite concentration and exposure period, a progressive reduction in the total erythrocyte count and haemoglobin were observed. With increase in nitrite concentration, a significant (P < 0.05) increase in activities was evidenced in catalase and superoxide dismutase in liver as well as gill, methaemoglobin reductase in blood, while progressive decline in AChE activity in brain was recorded. The serum sodium and chloride content showed a progressive decline, while potassium showed an increasing trend upon increase in nitrite concentration. The serum K(+) and Cl(-) after 96-h exposure demonstrated a linear relationship (Y = 0.221x + 2.542, R (2) = 0.938, P < 0.01 and Y = -5.760x + 129.5, R (2) = 0.952, P < 0.01, respectively) with nitrite concentrations. This study revealed that nitrite exposure causes alteration in all measured tissue enzymes, serum electrolytes and haematological parameters.