Effect of temperature on growth performance and immuno-biochemical changes in endangered golden mahseer, Tor putitora (Hamilton, 1923).

The present study to evaluated the effect of different thermal regimes on growth performance, oxidative stress, antioxidant enzymes, and immuno-biochemical responses of endangered golden mahseer, Tor putitora. A total of 144 healthy fingerlings were randomly distributed into four experimental groups (13 °C, 16 °C, 19 °C, 22 ± 0.5 °C) in triplicate rectangular fiberglass reinforced plastics tanks. The highest and lowest Growth rate, Specific growth rate, Daily growth index, Thermal growth coefficient, Viscera-somatic index, and Growth hormone were observed at 19 °C and 13 °C, respectively. The lowest SGR and VSI were observed at 22 °C. The feed conversion ratio was significantly higher in the control group (p < 0.05), whereas there was a significant decrease in all the treatment groups. The highest and lowest hepato-somatic index was observed at a temperature of 13 °C and 16 °C, respectively, while no significant (p > 0.05) impact of temperature regimes were observed on the length-weight relationship. Regarding hematological indices results, the hematocrit, hemoglobin, total erythrocyte count, and total leucocyte count were significantly higher at 13 °C, whereas the lowest was observed in all the treatment groups (p < 0.05). Higher myeloperoxidase activity and lysozyme activity were observed at 13 °C, whereas lowest at 22 °C. Anti-oxidant potential, SOD, and catalase (muscle) were significantly higher in the control group, whereas they gradually decreased with an increasing temperature. Catalase (liver) level was significantly higher at 22 °C. GST content was significantly higher at 22 °C compared to 13 °C, and GST content increased gradually with an increased temperature. The lipid peroxidation level in the liver and muscle was significantly higher at 22 °C and 13 °C, respectively. The present study concluded that the different phenotypic, antioxidative, and immuno-biochemical modulations of golden mahseer fingerlings in response to different temperature regimes could be used for better management and propagation.

Transgenerational effects of ß-glucan on thermal tolerance, growth performance, and immune gene expression of endangered cyprinid Tor putitora progeny.

Nutritional programming signifies a process in which broodstock feeding approaches have long-term effects on the subsequent progeny. The present study aimed to elucidate whether supplementing golden mahseer, Tor putitora broodstock diets with ß-glucan affects progeny growth performance, survival, thermal tolerance, and non-specific immunity. Initially, the growth performance of progeny produced from brooders fed with different levels of ß-glucan was non-significant. However, on the 15th and 35th DPH, the maximum weight was observed in fry obtained from the brooders fed with 0.5% followed by 1.0% ß-glucan. Furthermore, on 50th DPH, significantly higher weight was registered in the fry from the 0.5% ß-glucan fed group while 1.0% ß-glucan group had no transgenerational effect on growth. The condition factor of fry obtained from golden mahseer brooders fed with a 0.5% ß-glucan diet was greater than the control and 1.0% ß-glucan fed group. On the other hand, we did not find any significant transgenerational influence of ß-glucan on the survival of the progeny. The thermal tolerance of fry produced from brooders fed with ß-glucan was significantly modulated at both end-points (CTmax and CTmin). Expression of interleukin-1ß was significantly up-regulated in fry obtained from ß-glucan fed brooders. In contrast, the expression level of tumor necrosis factor-α was significantly higher only in fry produced from 1.0% ß-glucan fed brooders. The expression of immunoglobulin light chain and serum amyloid A gene was significantly higher in fry produced from 0.5% ß-glucan fed brooders. Overall results suggest that the dietary provisioning of ß-glucan in golden mahseer brooders can be a strategy to produce healthy and robust fry in captivity for stock enhancement and conservation programs.

Molecular characterization of non-specific immune genes of endangered golden mahseer (Tor putitora) and their expression during embryonic and larval development.

The present study was undertaken to characterize and analyze the expression of non-specific immune genes to get an insight into the early immune status of endangered golden mahseer. In this study, the full-length mRNA sequence of IFNγ, TNFα, C3, and IL10 was 927, 1409, 5125 and 1177 bp with an ORF of 558, 765, 4938, and 540 bp, encoding a putative protein of 185, 254, 1645, and 179 amino acid residues, respectively. The deduced amino acid sequences of these genes shared highly conserved structures with those from other cyprinids. Ontogenic real-time qPCR results indicated that expression of IFNγ and TNFα was lower until the morula stage and increased from blastula stage and found maximum at the organogenesis stage. Expression of the C3 gene was lower until the gastrula stage, followed by a linear increase from organogenesis to the pre-metamorphosis stage. The expression of IL10 was significantly lower during early developmental stages (till gastrula stage) and reached maximum at organogenesis. The level of IL1ß was found maximum in unfertilized eggs and remained elevated till the morula stage. TLR4 expression remained lower during the initial developmental stages and reached the maximum at the organogenesis stage. The expression level of defensin1 was substantially low until the organogenesis stage. In comparison, hepcidin1 was found considerably high until the blastula stage and remained significantly lower during later stages of development. Overall, the data generated improves knowledge on the immune status of endangered golden mahseer during embryonic and larval development, which may help develop effective immunomodulatory interventions during nursery rearing of golden mahseer to produce fry with better fitness.

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.

Reproductive dysfunction in females of endangered golden mahseer (Tor putitora) in captivity.

The present study was undertaken to gain insight on the physiological basis underlying the constraints in attaining maturity of endangered golden mahseer (Tor putitora) in captivity. Selected hormone levels and stress biomarkers were analysed in wild and captive reared brooders to address the above objectives. As compared to their captive counterparts, plasma 17ß-estradiol was significantly (p<0.05) higher in wild caught females. A concurrent trend was observed for plasma vitellogenin, aromatase, 17α, 20ß-dihydroxy progesterone (17α, 20ß DHP), luteinizing hormone (LH) and11-ketotestosterone (11-KT) indicating a weak hormone response in captive females that potentially hindered maturity. To the contrary, the plasma11-KT levels were not significantly different between wild and captive males. Plasma 17α, 20ß DHP level was found significantly (p<0.05) higher in wild caught females compared to females reared in captivity. However, both males of wild and captivity registered significantly higher 17α, 20ß DHP than captive females. Plasma 11-KT level was significantly higher in males compared to females. However, the captive females had higher level of 11-KT than captive males. Stress biomarkers viz., cortisol, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were also estimated both in wild and captive brooders. There was no significant difference in plasma cortisol levels of wild and captive reared brooders. However, plasma GPx and SOD activity were significantly higher in captive reared T. putitora as compared to wild brooders counterpart manifesting prevailing oxidative stress in captivity. Overall results showed endocrine and stress differences between wild and captive reared brood fishes during early spawning period which highlighted the endocrine failure of female reproductive maturity in captivity.

Morpho-histological and ultra architectural changes during early development of endangered golden mahseer Tor putitora.

Ultrastructural and histological changes in the embryonic and larval surface during ontogenesis of the endangered golden mahseer Tor putitora is studied here for the first time. Embryonic development was completed 91-92 h after fertilization at an ambient temperature of 23° ± 1° C (mean ± s.d.). The gastrula stage was characterized by presence of the Kupffer's vesicle, notochord, ectoderm and endoderm cells. Primordial germ cells were clearly identifiable from c. 55 h post-fertilization at the organogenesis stage. Mean total length of newly hatched larvae was 7·0 ± 0·5 mm. Scanning electron microscopy of newly hatched larvae demonstrated vitelline arteries, microridged epithelial cells and mucous gland openings over much of the body surface. Eye, oral cavity, pharyngeal arches, heart, intestinal loop, prosencephalon, cephalic vesicle and nasal epithelium were clearly distinguished in 3 day old hatched individuals. In 6 day old individuals, caudal-fin rays and internal organs were evident. The dorsal fin became prominent at this stage and larvae began swimming at the surface. The reserved yolk material was totally absorbed 8-11 days after hatching and larvae began feeding exogenously. Tor putitora exhibited a longer early developmental period than other cyprinids reared at similar temperatures.

Thermal tolerance, oxygen consumption and haemato-biochemical variables of Tor putitora juveniles acclimated to five temperatures.

A 30-day acclimation trial was conducted using Tor putitora to elucidate its thermal tolerance, oxygen consumption, haemato-biochemical variables and selected enzymatic activities at five acclimation temperatures (AT). Juveniles of T. putitora were randomly distributed among five treatment groups (20, 23, 26, 29 and 32 ± 0.5 °C). There was a significant curvilinear increase in critical thermal maxima (CT(max)) (y = -0.0693x² + 1.7927x + 34.628, R² = 0.996) and lethal thermal maxima (LT(max)) (y = -0.1493x² + 2.3407x + 35.092, R² = 0.991) with increasing AT. The oxygen consumption rate increased significantly with increasing AT. The Q10 values were 1.16 between 20 and 23 °C, 3.09 between 23 and 26 °C, 1.31 between 26 and 29 °(C) and 1.76 between 29 and 32 °C of AT. The acclimation response ratios were ranged between 0.37 and 0.59. Catalase, superoxide dismutase and ATPase activities were increased linearly in liver, gill and kidney, while brain acetylcholine esterase activity decreased linearly with increasing AT. Blood glucose remained unchanged up to AT of 26 °C and increased significantly at AT of 29 and 32 °C. Haemoglobin content was increased linearly with increasing AT. The highest WBC count was observed at 20 °C, and no significant changes found till AT of 26 °C and significantly decreased at 32 °C. Total serum protein and globulin were significantly decreased with increasing AT. Highest values were observed at 20 °C and remained consistent till 26 °C, then decreased significantly. There was no significant change in A/G ratio through the AT 20-29 °C and increased significantly at 32 °C. The increase in CT(max), LT(max) and oxygen consumption rate with increasing AT may suggest that the thermal tolerance of T. putitora is dependent on its prior thermal exposure history, and it could adapt to higher AT by altering its haemato-biochemical variables.

Nitrite-induced alterations in sex steroids and thyroid hormones of Labeo rohita juveniles: effects of dietary vitamin E and L-tryptophan.

An experiment was conducted to study the effect of sub-lethal nitrite exposure on sex steroids (testosterone and estradiol), cortisol and thyroid hormones (T3 and T4) of Labeo rohita juveniles. Fishes previously fed with normal or elevated levels of vitamin E (VE) and tryptophan for 60 days were exposed to sub-lethal nitrite for another 45 days with same feeding regime. There were nine treatment groups, viz. VE0TRP0-N, VE0TRP0+N, VE100TRP0-N, VE100TRP0+N, VE100TRP0.75+N, VE100TRP1.5+N, VE150TRP0+N, VE300TRP0+N and VE200TRP1+N. Except the groups VE0TRP0-N and VE100TRP0-N, all other groups were exposed to nitrite. At the end of the 45 days of nitrite exposure, serum samples were assayed for sex steroids, cortisol and thyroid hormones. The serum T3 and T4 levels decreased to the extent of 84.5 and 94.06%, respectively, upon nitrite exposure. Dietary supplementation with additional amounts of VE and tryptophan appears to reduce the decline of the production of T4. The serum testosterone and estradiol decreased 97.31 and 92.86%, respectively, upon nitrite exposure. Supplementation with additional amounts of VE was found to reverse nitrite-induced inhibition of testosterone and estradiol production. Serum cortisol increased upon nitrite exposure and unexposed (VE100-N) group showed lower levels, which were comparable to groups fed with elevated levels of VE. The overall results of the present study revealed that environmental nitrites have a negative impact on steroidogenesis, which can be overcome by dietary supplementation of elevated amounts of VE (minimum of 150 mg VE Kg diet(-1)) and to a lesser extent by tryptophan (only at the level of 1.5% of the diet).

Physiological responses of dietary tryptophan fed Labeo rohita to temperature and salinity stress.

Two experiments were conducted to elucidate the possible effects of dietary L-tryptophan (TRP) in Labeo rohita based on growth performance and physio-biochemical responses. In the experiment I, a 60-day feeding trial was carried out to elucidate the effects of dietary TRP enrichment on growth performance and physio-biochemical responses. In the experiment II, the TRP pre-fed L. rohita, from experiment I, was exposed to temperature and salinity stress to evaluate stress-mitigating efficacy of TRP. In L. rohita, dietary supplementation of TRP showed significant effect on weight gain percentage and feed conversion ratio but not on blood glucose. A significant increase in RNA content and RNA/DNA ratio upon TRP supplementation was observed and was positively correlated with growth performance. The results of experiment II indicated that weight gain percentage, serum T3 and T4 levels were significantly reduced in groups that were exposed to temperature and salinity stress and fed diets without TRP supplementation. However, dietary supplementation of TRP significantly augmented weight gain percentage in stress-exposed groups. Tryptophan supplementation helped in bringing back T3 and T4 levels comparable with control. A significant increase in superoxide dismutase, catalase, Adenosine triphosphatase, blood glucose and serum cortisol was observed in temperature- and salinity-exposed groups fed without TRP-supplemented diets. However, TRP supplementation was found to be effective in restoring the above parameters. The results of these experiments suggest that dietary TRP supplementation augments growth, lowers energy demand and helps in mitigating thermal and salinity stress in L. rohita.

Effects of dietary pyridoxine on haemato-immunological responses of Labeo rohita fingerlings reared at higher water temperature.

A sixty-day feeding trial was carried out to elucidate the effect of dietary pyridoxine (PN) on hemato-immunological parameters in Labeo rohita fingerlings exposed to an elevated temperature (ET) of 33 °C. Two hundred and seventy fingerlings (6.71 ± 0.32 g) were randomly distributed into six treatments in triplicates (15 fish/tank). Five iso-nitrogenous (356.3 ± 2.7 g/kg crude protein) purified diets were prepared with graded levels (0, 10, 50, 100 and 200 mg/kg diet) of PN. Six treatment groups were P10T26 (10 mg PN + ambient temperature (26 °C), P0T33 (0 mg PN + ET), P10T33 (10 mg PN + ET), P50T33 (50 mg PN + ET), P100T33 (100 mg PN + ET) and P200T33 (200 mg PN + ET). The responses of L. rohita fingerlings to dietary PN were assessed in terms of growth and by analysing some hemato-immunological parameters. When PN supplementation increased from 10 to 200 mg/kg diet, a significant improvement in weight gain percentage was observed. Erythrocyte (RBC) count, leucocyte (WBC) count, haemoglobin content and respiratory burst activity were significantly (p < 0.05) higher in 100 mg PN/kg diet-fed group than their non-PN-fed counterpart. Also, serum albumin, globulin and lysozyme activity were found significantly (p < 0.05) higher in PN-fed groups. The overall results indicated that dietary PN supplementation at 100 mg/kg diet may reverse the negative effects caused by ET and may protect the haemato-immunological status of L. rohita fingerlings reared at higher water temperature.