Multifarious Biomarker Approach Elucidate Stocking Density Mediated Stress, Modulates Growth Attributes in Cage Reared Rohu, Labeo Rohita (Hamilton)

doi:10.21203/rs.3.rs-951694/v1

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Multifarious Biomarker Approach Elucidate Stocking Density Mediated Stress, Modulates Growth Attributes in Cage Reared Rohu, Labeo Rohita (Hamilton)

https://doi.org/10.21203/rs.3.rs-951694/v1

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The present study was conducted for 240 days to evaluate the effects of stocking density based on growth attributes, digestive enzymes, muscular composition, biochemical and physiological responses of Labeo rohita fingerlings in tropical inland open water cages. L. rohita (30.35±1.08 g) were randomly distributed into three treatments, namely low stocking density, LSD (10 m^− 3), medium stocking density, MSD (20 m^− 3) and high stocking density, HSD (30 m^− 3) in triplicates. Fish were fed twice daily with CIFRI CAGEGROW® floating feed (crude protein-28%, crude fat-4%). Fish growth and feed efficiency was higher (P<0.05) in LSD, however MSD registered higher yield. Amylase and protease activity reduced whereas lipase activity increased with increasing stocking density. Muscle crude protein and crude fat formed inverse correlation. The fillet quality deteriorated at higher stocking densities based on Muscle pH, drip loss and frozen leakage rate. The stress biomarkers level (glucose, cortisol, superoxide dismutase and catalase) increased in serum under crowding condition. Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in serum was significantly increased in HSD. Serum protein level decreased with increase in stocking densities. Body ionic imbalance (Na⁺, Cl⁻ and K⁺) observed under crowding stress. Based on growth attributes and multiple biomarker responses, L. rohita @ 10 m^− 3 found to be optimum density for inland open water cage culture.

General Biochemistry

Agroecology

Environmental Engineering

Environmental Policy

Cage

stocking density and Labeo rohita

Stocking density optimization is a prerequisite for the development of protocol for practice of any candidate fish species and it differs based on species and its life stages, types of production system and management practice followed ¹. Over stocking produces significant stress which leads to growth retardation, poor health, lower survivability and yield loss ^2,3. The stocking density and growth are inversely correlated owing to the concurrence for food and space which in turn elucidate stress in fish ⁴. However, understocking leads to poor production due to underutilization of available space and resources ^2,5. As the cage culture is an intensive farming system, the apparent efficiency of culture systems can be maximized by increasing the densities to optimal level ^6,7.

Stressor in aquaculture covers a broad and diverse range of biotic and abiotic factors. Stocking density mediated stress has known physiological and behavioural consequences for the cultured fish ⁸. The biochemical responses of fish to menacing stimuli are regularly controlled by physiological modification of the nervous and humoral system in order to maintain body homeostasis ⁹. Fish subjected to stressors can drop the muscular flesh quality ¹⁰, decreased in digestive enzymes ¹¹ and altered the serum biochemical parameters ¹². Stress can also increases adrenaline which rise blood pressure resulting increase the blood flow through gill lamellae and alter the exchange of ions ¹³.

Cage culture in inland open water is considered as relatively recent aquaculture innovation, has rapidly expanded during the past decades in fresh, estuarine, and marine open water bodies. Due to pressures for aquatic products across the globe, cage culture is presently undergoing swift changes ^14,15. In India, reservoirs and wetlands are the untapped open water resources suitable for freshwater cage culture ¹⁶. India is bestowed with 19,370 reservoirs covering 15 states with an estimated area of 3.51 million ha surface area at its full capacity ¹⁷. Utilizing a modest fraction of their surface area, cage culture can accord notable quantity of fish production to the total fish production basket of the country. At present, Pangasianodon hypophthalmus occupies the centre stage in inland cage culture in India. However, problems and prospects of this species in cage culture cannot be neglected. Farmed P. hypophthalmus from India fetches low prices in the domestic market and export as well ¹⁸. Hence diversification of species in inland cages in India, which is having good market demand, is need of the hour ¹⁷.

Indian major carp is the mainstay of the Indian aquaculture contributing 80% of the total country aquaculture production ¹⁹, of which rohu (Labeo rohita) is in greater preference by the consumers. Despite a huge prospective for species diversification, the culture of L. rohita has been only restricted in pond and tanks and no scientific information available on its performances in cage culture. With this background, the present study was undertaken to bring out the optimum stocking density of L. rohita based on its growth attributes and by examining variety of endpoints to measure its physio-biochemical responses in inland open water cage culture. This is the first report on grow out cage culture of L. rohita in reservoirs. The study will be extremely beneficial for aquaculturist and researcher for standardizing the cage culture protocol of L. rohita in tropical reservoirs of India and Southeast Asian countries.

Growth performance and feed efficiency

The growth characteristics among the groups were significantly differed (P<0.05) (Table 1). Higher growth performances in terms of FBW, WG and AGR were recorded in lower stocking density (P<0.05). The value of SGR was (1.29±0.01 g) in LSD followed by MSD (1.21±0.01 g) and HSD (1.07±0.03 g). Increase in stocking density led to decline in fish survival percentage (P<0.05) from LSD (88±0.11%) to HSD (66±0.18%). Fish reared at LSD reported higher (P<0.05) feed and protein utilization (PER and FCE) than MSD and HSD. On contrary, the lowest FCR (1.96±0.01) displayed in LSD while medium and high density showed significantly deleterious FCR value (P<0.05).

Serum biochemical indices

Post hoc analysis signifies that the level of serum glucose markedly increased (P<0.05) with increase in stocking density (Fig. 2A). The glucose concentration was ranged from 25.14±1.02 mgdL^-1 to 42.19±0.68 mgdL^-1 from LSD to HSD. The SOD level was increased significantly (P<0.05) in HSD (39.31±0.43 U mg protein^-1) and the extent of increase in SOD was 15% from LSD (Fig. 2A). There was no significant (P>0.05) changes in SOD level among lower and medium density. The CAT value was significantly (P<0.05) increased in MSD (29.93±0.81 U mg protein^-1) and HSD (35.69±0.06 U mg protein^-1), than LSD (26.80±0.58 U mg protein^-1) (Fig. 2A). Cortisol level demonstrated apparently higher in HSD with a factor of 29.89%, in comparison to LSD (P<0.05). However cortisol level did not vary among MSD and HSD (Fig. 2A).

The concentration of SGOT and SGPT in LSD and MSD varied significantly from HSD (P<0.05) as presented in table 2, however the level did not vary among low and medium density. The sum of total serum protein decreased significantly (P<0.05) with increasing stocking density from 3.29±0.01 gdL^-1 in LSD to 2.33±0.08 gdL^-1 in HSD (Table 2). Albumin, which is an important carrier for various hormones like thyroid, steroids and fatty acids varied from 0.96±0.01 gdL^-1 to 0.72±0.01 gdL^-1 from lower to higher stocking density. Serum globulins responsible for blood clotting and immunological functions proclaimed a decreasing trend from lower to higher stocking density. The ratio of albumin and globulin was found to be non-significant (P>0.05) amongst LSD and MSD but significant with HSD (Table 2).

The serum electrolytes like Na⁺, K⁺ and Cl^- were monitored and their values were represented in table 2. The level of Na⁺, altered among lower and higher density treatments. Chloride ion decreased with the increased stocking density. Significant difference of K⁺ and Cl^- values (p<0.05) have been observed in the lower and higher stocking density.

The serum thyroid level was estimated using the parameters T3 and T4 (Fig. 2B). The T3 value was not differed (P>0.05) significantly except in HSD (2.16±0.03 ngmL^-1), however T4 was decreased (P<0.05) with increase in stocking density and the lowest value recorded in HSD (3.68±0.06 ngmL^-1). The IGF1 value was highest at LSD (155.52±3.92 ngmL^-1) and it was significantly (P<0.05) declined in both MSD and HSD (Fig. 2C).

Digestive enzymes

The gut amylase, protease and lipase activities were analyzed and shown in figure 2D. Amylase was found to be inversely proportional to the stocking density and the value ranged from 19.30±0.35 U mg protein^-1 to 22.48±0.41 U mg protein^-1 from HSD to LSD. Protease, which is responsible for breakdown of proteins into smaller polypeptides or single amino acids did not differ (P>0.05) among LSD (10.25±0.12 U mg protein^-1) and MSD (9.46±0.18 U mg protein^-1) however it was markedly differed in HSD (7.88±0.05 U mg protein^-1). The lipase activity was found to be indifferent among MSD and HSD but varied in LSD (P<0.05).

Chemical composition of muscle and flesh quality

The muscular chemical compositions at different stocking densities were measured and presented in table 3. The crude protein content showed significant variation between HSD and LSD. The decreasing trend of crude lipid content among the treatments was observed though the values were insignificant between LSD and MSD. Stocking density had no impact (P>0.05) on moisture and ash content. The muscular pH declined with increase in stocking densities where the LSD showed the highest pH value (6.50±0.23) and lowest at HSD (5.8±0.17) as shown in table 3. Muscular DL and FLR decreased with increasing stocking density and the values are significantly differ between LSD and HSD (Table 3).

Nucleotide ratio

The ratio of RNA:DNA is an important sign of stress in the fishes as shown in the figure 2E. The quantity of DNA did not vary significantly (P>0.05), however the quantity of RNA varied significantly (P<0.05) among LSD (1.33±0.08 µgmg^-1) and HSD (1.56 ±0.12 µgmg^-1). The nucleotide ratio (RNA:DNA) was found to be decreased with increasing stocking density (P<0.05).

Univariate and multivariate statistical analysis

Pearson correlations among the parameters are depicted in figure 3A. The correlation coefficient suggested that correlated parameters are closely associated and their behaviours are inter-dependent to each other. This correlation was further confirmed by principal component analysis (PCA). Two PC were obtained having eigenvalues >1. PC1 and PC2 constitutes eigenvalues 32.23 and 4.76 and the variance 87.12% and 12.88% respectively. PC1 showed strong positive correlation among FBW, WG, SGR, AGR, survival, PER, FCE, amylase, protease, K⁺, Cl^-, RNA, RNA:DNA, moisture, CF, pH, serum protein, albumin, globulin, T3, T4, and IGF1 (Fig. 3B). However, a negative correlation exist among FCR, lipase, CP, DL, FLR, serum glucose, cortisol, SGOT, SGPT, SOD and CAT (Fig. 3B). PC2 didn’t show any significant correlation among the vital parameters. Hierarchical clustering dendrogram formed two groups among the three stocking densities. Cluster one noticed between LSD and MSD while HSD formed a separate cluster (Fig. 3C).

Stocking density, nutrition and appropriate culture environment are considered as vital factor which affects the fish growth and production ²⁰ however overcrowding negatively affect the growth of fish ²¹. In the present study, stress led by crowding has obvious effect on growth attributes like FBW, WG, SGR, AGR and survival which are significantly different among LSD, MSD and HSD. The present findings proclaimed that the feed utilization and protein efficiency ratio was found significantly greater in lower stocking density, however, higher density had a deleterious impact on FCR, FCE and PER. Growth retardation, poor production and feed utilization in HSD could be the result of crowding which triggered a raising demand for energy to activate the physiological functions to combat the stress by decreasing appetite and food intake, and led to reduction in the available energy for growth ^22–24. The major studies on evaluation of stocking density were observed on various tropical as well as temperate fishes and also in different culture systems. The similar effect of crowding stress on growth and feed utilization was also observed in common carp, Cyprinus carpio ^25–27; grass carp, Ctenopharyngodon idella ^28,29; olive barb, Puntius sarana ⁷; Amazon fish, Colossoma macropomum ³⁰ and Nile tilapia, Oreochromis niloticus ^31,32. On contrary, many authors could not establish any relation between stocking density and growth attributes in silver perch, Bidyanus bidyanus ³³, Atlantic sturgeon, Acipenser oxyrinchus ³⁴. Thus stocking density (10 m⁻³) of L. rohita ensured the best growth performances in term of weight gain, survival and feed and protein utilization.

Glucose is considered as an indicator of secondary stress response in fishes ^35,36. Stressors such as transportation, stocking density, confinement and bad handling have found to be responsible for increase in blood-glucose and whole body-glucose level. The increased level of catecholamine results in activation of glycogenolysis and gluconeogenesis, which ultimately leads to rise of glucose level in blood ³⁷. It is a secondary stress response which shifts the glucose to body tissue in order to cope with restoration of energy demanding activity. In the present study, increased blood glucose level (P<0.05) from LSD to HSD was observed with an increment in stocking density followed by crowding stress. Similar findings on stocking density mediated stress elevates blood glucose level observed in olive barb, P. sarana ⁷, Gilthead seabream, Sparus auratus ³⁸; Senegalese sole, Solea senegalensis ³⁹; Chinese sturgeon, Acipenser sinensis ⁴⁰; common carp, C. carpio ^25,26 and Asian seabass, Lates calcarifer ⁴¹.

Free radicals and reactive oxygen species (ROS) are produced continuously under stressful conditions to combat the damage of antioxidant abilities and also act as a scavenger of excessive superoxide generated in the body. SOD and CAT played a key antioxidant enzyme in the animal defence system, to function against oxidative stress, and with the increase in stress; its value tends to ascend ^42,43. SOD level in serum of L. rohita at higher density increased significantly. Similar to the observations with an increment of SOD and CAT with increased stocking density observed in cage reared olive barb, P. sarana ⁷, common carp, C. carpio ^25,26, Nile tilapia, O. niloticus ¹¹, GIFT strain of Tilapia ⁴⁴, and dourado, Salminus brasiliensis ⁹.

Cortisol released as an indicator of chronic and acute stress which elevates the expenditure of energy and hence the stored somatic energy expenses also subsequently increase, which ultimately led to growth retardation in fish ^25,41. The cortisol level of L. rohita elevated at high density (P<0.05). Moreover increase the level of cortisol and glucose were associated with the reduction in muscular fat. In concordance with the present study, growth retardation and cortisol level positively correlated in olive barb, P. sarana ⁷, common carp, C. carpio ^{25–27, 45}. GIFT strain of Tilapia ⁴⁴,. In contrast to the present finding, cortisol level remained unaffected in African catfish, Clarias gariepinus ⁴⁶ at higher density, but decreased in Nile tilapia, O. niloticus in biofloc based systems ¹¹.

The major thyroid hormones, triiodothyronine (T3) and thyroxine (T4) play an important role in growth and development of fishes ⁴⁷ which are majorly affected by crowding stress, hypoxic condition and starvation ⁴⁸. The crowding stress plays a major role in elevation of cortisol level and the increase in cortisol level has a negative feedback mechanism on hypothalamus-pituitary-interrenal axis thus decreasing the peripheral circulation of T3 and T4. Unless the thyroid hormones are not in free form, they cannot be utilized by the fish, leading to poor growth. The elevated crowding stress led the fishes towards growth retardation, also towards less availability of thyroid hormone in fish serum, which was observed in the present study. In our study, the decrease in level of T3 and T4 with increase in stocking density was supported by the earlier findings in Amur sturgeon (Acipenser schrenckii), mosquito fish (Gambusia holbrooki), channel catfish (Ictalurus punctatus) and olive barb (P. sarana) ^7,49−51 respectively.

The IGF1 axis has a prominent role in regulations of various physiological responses in fishes ^52,53. Due to the secretion of IGF 1, growth hormone has a prominent impact on growth regulation in fishes ⁵⁴. Due to the stress factors like overcrowding in the present study, the somatic growth was inhibited significantly in MSD and HSD; also these groups of fishes were encountered with significantly lower IGF 1 concentration in their serum. The findings were consistent with earlier studies where stress affects the secretion of IGF in tilapia, Oreochromis mossambicus; Chinese sturgeon, A. sinensis; Amur sturgeon, A. schrenckii; Senegalese sole, S. senegalensis ^40,55−57 respectively. The result divulged that high cortisol level under stressful conditions ,inhibition the secretion of growth hormone and IGF 1 ⁵⁸.

SGOT and SGPT, both are ubiquitous aminotransferases in the mitochondrion of fish and used as indicator of hepatic ruination ⁵⁹. The enzymes are coerced to be released into bloodstream when the liver gets damaged. In the present finding, SGOT and SGPT activity upraised in L. rohita due to crowding stress in HSD; however the change in such values among LSD and MSD was not varied significantly. This result might also reflect that increase of these enzymes in the serum was occurred due to over utilization of hydrocarbons in order to meet the stress mediated energy demands by fish. Similar types of outcome also observed in olive barb, P. sarana ⁷, GIFT strain of Tilapia ^21,60, Channel catfish, Ictalurus punctatus ⁵¹, Marbled eel, Anguilla marmorata ⁶¹, Amur sturgeon, A. schrenckii ⁶², Nile tilapia, O. niloticus ⁶³ and rohu, L. rohita ^64,65.. Those means any kind of unpleasant living condition or stress situations could lead to the injury of the hepatic function.

Total protein, albumin and globulin content in the serum plays a pivotal role in fish innate immune response; chiefly during the stressful conditions such as dietary irregularity, high stocking density, infections due to disease and other environmental stress factors ⁶⁶.These parameters are also used as indicator of humeral defence system ⁶⁷. The drop in serum protein, albumin and globulin and their ratio in the higher stocking density (HSD) was a clear image of compromised innate immunity which may be due to inhibition of protein synthesis, liver cell lesions, kidney dysfunction or malnutrition. The crowding stress has also badly affected the serum protein and albumin concentration in Nile tilapia, O. niloticus ³¹ and common carp, C. carpio ²⁵.

Neuroendocrine system controls the acute and chronic stress responses which release catecholamines and cortisol ⁶⁸, both of the steroid are responsible for controlling the ionic regulation in fish body by the ion concentrations and their exchange between the body and surrounding environment called osmoregulation ⁶⁹. The responses of fish to the stress are related to the neuroendocrine system, which is a critical part of osmoregulatory adaptations ⁷⁰. This present study showed that the Na⁺ level did not vary (P>0.05) among the treatments except LSD and HSD however, the levels of Cl⁻ and K⁺ decreased with increasing stocking density and the lowest value were noticed in high density. The result may be attributed by excessive blood flow in gills and the permeability of the epithelium, resulting in ionic losses in freshwater fish ⁷¹. A similar finding was recorded in Atlantic salmon, Salmo salar ⁷², however contrasting results have seen in case of salmon smolts, Salmo salar ⁷³ and rainbow trout, Oncorhynchus mykiss ⁷⁴.

The digestive enzymes were affected by increase in stocking density, since the crowding stress could force the fish body metabolism to channelize their energy towards coping of stress conditions. Thus, it can also imply that the digestion and utilization of feed in fishes could be affected by crowding stress caused by higher stocking densities ²⁵. In present study, the amylase activity was decreased significantly with increasing stocking density. Crowding stress also caused decline in amylase activity in olive barb, P. sarana ⁷; common carp, C. carpio, ²⁵; rainbow trout, Oncorhynchus mykiss ⁷⁵ and turbot, Scophthalmus maximus ⁷⁶. The decrease in lipase activity with increase in stocking density among the treatments with L. rohita indicates that, the utilization of body fat during crowing stress led to increase in lipase activity. In present study, due to crowding stress, protease and lipase activity in fishes were found declining with increase in stocking density, although the change was not significant among LSD and MSD. The crowding stress has also been found affecting the digestive enzymes of fishes like common carp, C. carpio ²⁵; marbled eel, A. marmorata ⁶¹ and rainbow trout, Oncorhynchus mykiss ⁷⁵.

Energy metabolism in fish body can also be indicated by flesh composition ⁷⁷. In present study, deterioration in growth parameters suggests the decline in metabolism. A number of factors can be a reflection to change in body flesh composition of the stocked fishes, including water quality, crowding stress, nutrient availability, feed intake and the follow up utilization ³². In current study, significant decline of lipid extract was noticed in HSD which can be attributed due to chronic crowd stressing in cages followed by lesser uptake of feed resulting lower accumulation of lipid in fish fleshes. In other hand, crowding stress enhanced the process of lipid metabolism in order to meet growing demand of energy followed by decreased lipid content in some other fish species as Amur sturgeon, A. Schrenckii⁷⁸; juvenile blunt snout bream. M. amblycephala²³ and Nile tilapia, O. niloticus⁷⁹. In contrast, lipid content increased with increasing stocking density in African catfish, Clarias gariepinus and rainbow trout, O. mykiss^80,81. The dissimilar results of these above studies could be due to the differences in fish species, age, size and the exposure time of crowding stress. Crude protein content of fish muscle was increased significantly in HSD in comparison to LSD. The present result corroborate with ⁶¹ in A. marmorata where crude protein content increases (P>0.05) with increment in stocking density.

The muscle pH is an important flesh quality parameter and under stressful condition, fishes produce low pH muscle ^10,82,83. In present study, lower pH was exhibited in higher densities. It is possibly induced by chronic crowding stress which stimulates lactate acid formation in muscle ^51,83. Similar findings of lower pH and water holding capacity were obtained in case of rainbow trout, O. mykiss ⁸¹. The DL and FLR in HSD displayed a significant increasing trend, which indicate the deterioration of flesh quality attributed by lower muscular pH. According to ⁵¹, DL and FLR is inversely correlated to muscular pH in channel catfish, I. punctatus.

RNA, as a reflection of protein synthesis and expected to increase with an increase in the growth of somatic tissues ⁸⁴, whereas DNA, which is a genetic carrier molecule, and the quantity remains constant ⁸⁵. The RNA : DNA is sensitive to nutrition and as an index of the cell’s metabolic intensity and closely related to protein synthesis ⁸⁶. In the present experiment, the concentration of DNA did not vary (P>0.05) among the three stocking densities of L. rohita however the RNA and RNA to DNA ratio was significantly decreased (P<0.05) with an increase in stocking densities. The present study was endorsed by previous studies in Japanese flounder, Paralichthys olivaceus ⁸⁷; rohu, L. rohita ^88,89 and catla, C. catla ⁹⁰.

In the present study, Pearson correlation coefficient and PCA analysis depicted that growth attributes like FBW, WG, SGR, AGR, survival, PER and FCE and biochemical parameters namely serum protein, albumin, globulin, T3, T4, and IGF1 are strongly correlated. However a negatively correlation established among serum glucose, cortisol, SGOT, SGPT, SOD and CAT. These correlated parameters are interdependent to each other and their responses are similar under crowding stress. Similar observation reported in P. sarana reared in cages ⁷. The outcome of PCA analysis revealed that stress responses negatively influenced while, decreased in the level of digestive enzymes (amylase and protease), serum protein and thyroid hormones (T3, T4 and IGF1) are positively influence on the growth attributes. Cluster analysis depicted a separate cluster by HSD indicates the bio-markers are significantly differ in HSD comparison to LSD and MSD.

Ethical statement

The study protocol and the experiment conducted was approved by the ethical committee of ICAR-Central Inland Fisheries Research Institute, Barrackpore (IAEC/2020/04). The present study is in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (http://arriveguidelines.org/).

Experimental facilities and animals

The present study was carried out in Salia reservoir (N19°48.3887ʹ and E85°03.6983ʹ), Odisha, India (Fig. 1a and 1b) in floating rectangular cages made of high density polyethylene (HDPE) of dimension 6m x 4m x 4m with an effective volume of 84 m^-3 for a period of 240 days. Advance fingerlings of L. rohita (14.25±0.19 cm and 30.35±1.08 g) was stocked at three different stocking density, namely LSD (10 m^-3), MSD (20 m^-3), and HSD (30 m^-3) in triplicates as per complete randomized design (CRD). Fishes were fed with commercial extruded floating feed (28% protein and 4% fat) at a rate of 3 % of body weight twice (09.00 hour and 16.00 hour). The feed rations were adjusted based on the estimated fish biomass in every week. During the study, antibiotics, chemicals and medicines were strictly avoided. The health status of fish was monitored at regular basis (Fig.1c)

Growth performance and feed efficiency

The growth performances were assessed in every week by randomly lifting 30 fishes from cages in order to adjust the feed ration however other parameters were studied at the end of the experiment. Prior to sampling, the feeding was withheld for 24 h. The parameters such as weight gain (WG), absolute growth rate (AGR), specific growth rate (SGR), total yield and survival were calculated as follows: weight gain (g) = FBW (g) – IBW (g) where IBW = Initial body weight (g), FBW = Final body weight (g), absolute growth rate = [FBW (g) – IBW (g)]/culture period (days), specific growth rate (%) = (In FBW – In IBW) ˟ 100/culture period (days), yield (kgm^-3) = total biomass of fish harvested per m³ volume at the end of experiment, survival (%) = (number of fish harvested / number of fish stocked) ˟ 100. The feed efficiency was assessed by feed conversion ratio (FCR), feed conversion efficiency (FCE) and protein efficiency ratio (PER) and were calculated as follows: FCR = dry weight of diet fed (g)/wet weight gain of fish (g), FCE = wet weight gain of fish (g)/dry weight of diet fed (g), PER = wet weight gain of fish (g)/crude protein intake (g).

Serum sample collection and preservation

Five fish from each cage were randomly collected and anesthetized using clove oil @ 50 µL per liter water. The blood sample was collected using 2 mL sterile disposable syringe, by puncturing the caudal vein and kept in 1.5 mL eppendorf tube for 30 minute for coagulation. The blood samples were centrifuged @ 4000 rpm for 10 minute at 4 °C, the straw colored serum was pipetted out. The serum samples were carried to laboratory in dry ice and stored at -80 °C for further analysis.

Serum biochemical indices

The automated blood biochemistry analyzer (Transaia-Erba, EM-2000, USA) was used to measure serum parameters such as protein, albumin, glucose, serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) using necessary reagents provided by manufacturer. With additional use of ISE module in the same instrument, the electrolytic balance of fish such as Na⁺, K⁺ and Cl^- were estimated. Serum globulin was calculated by subtracting serum albumin from serum protein, and A/G ratio was calculated as albumin/globulin.

Superoxide dismutase (SOD) was assayed using carbonate-bicarbonate buffer (0.1 M) with epinephrine (3 mM) ⁹¹. The change in optical density was measured at 480 nm (BioTek’s Epoch^TM 2). Catalase (CAT) activity was analyzed using phosphate buffer (50 mM, pH 7.0) with H₂O₂solution ⁹². The change in OD value was measured at 240 nm (BioTek’s Epoch^TM 2).

Cortisol, tri-iodothyronine (T3), thyroxine (T4) and insulin-like growth factor 1 (IGF1) in serum were quantified using enzyme-linked immune survey assay (ELISA) kit (BT Bio Assay, Shanghai, China) as per the manufacturer’s instruction and the final OD value was taken at 450 nm (BioTek’s Epoch^TM 2).

Digestive enzyme assay

The fish were dissected from each treatment and the gut tissues were kept in 0.25M sucrose solution. The samples were homogenized using tissuelyser (Qiagen, Hilden, Germany), and centrifuged at 10000 rpm for 10 minutes in 4 °C and the supernatant was collected to store in -80 °C for further analysis. Amylase activity was assayed using 3,5-dinitrosalicylic acid method ⁹³ by observing reducing sugar production by the gluco-amylase and α-amylase. Lipase activity was measured using phenolphthalein indicator based titration method ⁹⁴. The casein digestion method (triphosphate buffer- pH 7.8, trichloroacetic acid) was performed to measure protease activity ⁹⁵.

Chemical composition of muscle and flesh quality

Fish flesh samples (n = 3) from all the treatments were collected. The proximate composition assessed as per ⁹⁶. Moisture content was estimated via desiccation in an oven at 105 ^°C for 30 minutes and then cooling and weighing to a constant weight. Crude protein (nitrogen × 6.25) was determined by the Kjeldhal method using kjeltec System (Tecater 1002 Distilling Unit), crude fat content was evaluated by using extraction with ether by soxtech system (Tecater 1043 Extraction Unit). The ash content was assessed by incineration in a muffle furnace at 550 ± 10 ^˚C for 12 h. Muscular pH (n = 6) was measured in three different parts of the body (two dorsal sides and one caudal side), then average was calculated. The fish was dissected and insert a pH probe (Thermo orion A211) into the muscular part. Before every use, the probe was calibrated with pH 4 & pH 7 buffer. Drip loss (DL) was calculated as per ⁹⁷, the sample was weighed and kept in a vacuum polythene bag and stored at 4℃ for 72 hour. After thawing and the fish fillets were taken out from the bag and wiped with a paper towel and weighed. The calculation of the drip loss was based on difference of initial sample weight and weight after 72 hr. DL = {100 ˟ (Initial weight-weight after 72 hr)/Initial weight}. For FLR, the sample was weighed and kept in a vacuum polythene bag and stored at -20˚C for 72 hours ⁹⁸. After thawing, the fish fillets were taken out from the bag and wiped with a paper towel and weighed. The calculation of FLR was as follows; FLR = [100*(Initial weight-weight after 72 hr)/Initial weight].

Nucleotide ratio

Fish muscle tissue were collected in RNAlater and 70% ethanol for RNA and DNA isolation. DNA and RNA from muscle tissue were extracted using DNeasy and RNeasy kit (Qiagen Hilden, Germany) respectively as per manufacturer instruction. The concentration of DNA and RNA samples were measured by Nanodrop plate at 260/280 (BioTek’s Epoch^TM 2, USA). After quantification, RNA/DNA ratio was calculated.

Statistical analyses

The data generated from all the parameters were subjected to one way analysis of variance (ANOVA) and Duncan’s multiple range test to determine significant differences among the means and undergone principal component analysis, using statistical software SPSS 22.0, in which P<0.05 considered statistically significant. Pearson correlation and hierarchical clustering analysis was carried out using PAST, 4.03 software.

In conclusion, the present study accentuates that stocking density has a distinctive effect on fish physio-biochemical responses and importance of evaluating density stress, towards determining optimal density to warrant the fish production.The stocking density mediated crowding stress negatively affects the growth attributes like WG, SGR and percentage survival. Poor digestive enzymes (amylase and protease) activity and flesh quality, fall of thyroid activity (T3, T4 and IGF1) and serum electrolytes imbalance has been perceived with increment in stocking density. However, elevated levels of the stress response like SOD, CAT, SGOT, SGPT, serum cortisol and glucose were encountered at higher stocking density. Based on growth attributes and multiple biomarker responses it is suggested that the optimum stocking density of Indian major carp (L. rohita) for tropical inland open water cage culture is 10 m^-3. This is the first ever attempted in inland cage reared fish to optimize stocking density based on multiple biomarker responses.

Acknowledgement

Authors are thankful to the Director, ICAR-CIFRI, Kolkata, India and ICAR-CIFE, Mumbai, India for providing all kind of facilities and financial support, The DoF, Odisha, India are highly acknowledged for providing the cage for the experiment and the local hospitality.

Conflict of interest

Authors have no conflict of interest

Author’s contribution

HSS: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft; BKD: conceptualization, validation, review and editing, supervision, funding acquisition, AU: formal analysis, data curation, writing – review and editing; MHR: formal analysis, data curation, writing – review and editing, VK: software, data curation, writing – review and editing, DKM: writing – review and editing, formal analysis, data curation, UKS: writing – review and editing, supervision, NKC: writing – review and editing, supervision, KDR: writing – review and editing, supervision.

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Table 1. Growth performance and feed utilization efficiency of L. rohita stocked at three different stocking densities

Treatment	IBW (g)	FBW (g)	WG (g)	SGR (%)	AGR (g)	Survival (%)	FCR	PER	FCE	Yield (kgm^-3)
LSD	30.35±1.08	680.69±21.30^a	650.34±21.30^a	1.29±0.01^a	2.70±0.08^a	88±0.11^a	1.96±0.01^c	1.69±0.01^a	0.50±0.02^a	5.98±0.53^b
MSD	30.35±1.08	560.52±25.98^b	530.17±25.98^b	1.21±0.01^a	2.20±0.10^b	72±0.14^b	2.56±0.02^b	1.29±0.01^b	0.38±0.02^b	8.059±0.71^a
HSD	30.35±1.08	405.45±32.79^c	375.1±32.79^c	1.07±0.03^b	1.56±0.13^c	66±0.18^c	2.87±0.01^a	1.16±0.02^c	0.34±0.03^c	8.050±1.13^a

Values are mean±SE. Means in the same column within each classification bearing different superscripts are significantly different at (P <0.05).

Table 2. Serum biochemical indices of L. rohita stocked at three different stocking densities

Treatments	SGOT (UL^-1)	SGPT (UL^-1)	Protein (gdL^-1)	Albumin (gdL^-1)	Globulin (gdL^-1)	A:G ratio	Na⁺ (mmolL^-1)	K⁺ (mmolL^-1)	Cl^- (mmolL^-1)
LSD	58.16±1.72^b	3.88±0.04^b	3.29±0.01^a	0.96±0.01^a	2.33±0.01^a	0.41±0.01^a	131.13±0.54^a	2.52±0.02^a	112.50±4.04^a
MSD	60.01±2.89^b	4.00±0.04^b	2.96±0.05^b	0.85±0.01^b	2.11±0.04^b	0.40±0.01^a	129.50±0.28^b	2.58±0.07^a	102.40±3.41^ab
HSD	66.09±1.90^a	4.51±0.08^a	2.33±0.08^c	0.72±0.01^c	1.61±0.06^c	0.44±0.01^b	130.50±0.28^ab	2.12±0.05^b	98.16±1.30^b

Values are mean±SE. Means in the same column within each classification bearing different superscripts are significantly different at (P <0.05).

SGOT- Serum glutamate oxaloacetate transaminase

SGPT- Serum glutamate pyruvate transaminase

Table 3. Muscle chemical composition and flesh quality of L. rohita stocked at three different stocking densities

Treatments	Moisture (%)	Crude protein (%)	Crude fat (%)	Ash (%)	Muscle pH	Drip loss (%)	FLR (%)
LSD	77.67±0.74^a	13.82±0.11^b	2.03±0.05^a	3.72±0.01^a	6.5±0.11^a	2.1±0.17^b	0.7±0.05^b
MSD	77.03±0.26^a	14.45±0.52^ab	1.90±0.06^a	3.74±0.01^a	6.0±0.05^b	3.2±0.11^a	1.1±0.11^ab
HSD	75.61±1.62^a	15.28±0.22^a	1.63±0.06^b	3.72±0.02^a	5.8±0.17^b	3.8±0.40^a	1.4±0.17^a

Values are mean±SE. Means in the same column within each classification bearing different superscripts are significantly different at (P <0.05).

FLR – Frozen leakage rate

No competing interests reported.

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Multifarious Biomarker Approach Elucidate Stocking Density Mediated Stress, Modulates Growth Attributes in Cage Reared Rohu, Labeo Rohita (Hamilton)

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