Effects of Varying Dietary Protein Levels on Growth Performance, Survival, Body Composition, Haemato-Biochemical Profile, and Metabolic Responses of Hypselobarbus jerdoni (Day, 1870) Juveniles

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Introduction
Ornamental fshes are the most attractive and colourful pets, and keeping aquariums is the world's second preferred recreational activity after photography.Ornamental fsheries are generally considered as one of the most promising sectors within aquaculture that is rising day by day due to its great scope for enterprise development and income production.Moreover, the ornamental fsh industry is popularly known as a multibillion-dollar industry wherein 125 countries are engaged directly or indirectly in various trades [1].According to the recent report, more than 2 billion live ornamental fsh have been sold so far, with the global ornamental fsh trade being valued around US$ 15-30 billion [2,3].In addition, the global ornamental fsh trade involves roughly 2,500 species, of which more than 60% are freshwater species and the remaining species are marine.Tis industry is regarded as a sleeping giant in India.Being a tropical country, India is blessed with the vast biodiversity of freshwater ornamental fsh and ample natural freshwater resources.
Te Western Ghats are a line of hills that extend along India's west coast and are often regarded as one of the richest areas in the world in terms of biodiversity and endemism.Te Western Ghats occupy a total area of 136,800 km 2 and span a length of 1490 km from north to south, with a width ranging from 48 km to 210 km [4].According to a recent report, this biodiversity zone is home to 320 freshwater fshes from eleven orders, thirty-fve families, and 122 genera, including numerous migratory species [5].Of 320 species, 155 are recognized ornamental fshes, of which 117 are native to the Western Ghats [6].Te Western Ghats harbour several essential species, such as minor carps, barbs, minnows, loaches, rasboras, danios, and hill trouts, some of which are indigenous to this biodiversity hotspot region.
Te medium-sized barbs of genus Hypselobarbus are popularly known as a "catchall" genus, which encompasses near about twenty-two species, and they are native throughout this biodiversity hotspot region of India, occurring primarily in the diferent stretches of the rivers and their connected reservoirs [7,8].Tis particular genus contains economically important ornamental, as well as food fshes that are sold in local markets [9].Tese omnivorous, medium-sized fsh showing potamodromous migration, i.e., migrate upstream for spawning during the rainy season [10].Furthermore, the natural population of Hypselobarbus species is drastically declining as a result of the indiscriminate use of these species for aquarium and edible purposes in their native regions [11].Owing to such combined concerns (territory loss and over exploitation), most of the species of Hypselobarbus are now addressed as "threatened" on IUCN Red List categorisation [12].Among the twenty-two species, Jerdon's carp (H.jerdoni), locally known as Cha-meen, is a species of the Cyprinidae family endemic to India, mainly in the Western Ghat regions [7].Tis species inhabits freshwater, is benthopelagic and potamodromous [13], and can grow to a maximum length of 46 cm (18 inch) in total length [14].Moreover, as a consequence of the uncontrolled fshing and destruction of breeding ground due to anthropogenic activities, this species is now categorised as the "least concern" on the IUCN list [12].Tus, there is utmost necessity of developing captive breeding protocols followed by subsequent closing of a life cycle and widespread culture of this species for purposes of conservation and sustainable trade.
Conservation eforts may be manifold, of which formulation of a nutritionally balanced and cost-efective feed is of utmost importance to spearhead captive maturation and breeding programmes.Fish growth results from a higher rate of body protein synthesis than breakdown, which is dependent on an optimal dietary protein supply and ambient conditions [15].Among the several nutrients, dietary crude protein (DCP) is the most signifcant and costly ingredient in aquafeed, as well as a crucial component regulating fsh growth [16].Besides, dietary protein being of paramount importance should be supplied in adequate quantities through exogenous feed for fulflling energy demands of the fsh in various phases of its life.If the diet is low in protein compared to an optimum requirement level, it will reduce the growth rate of fshes [17][18][19].Alternatively, if the diet is rich in dietary protein relative to the optimum level, then excess DCP will be used for the production of energy and simultaneously, fshes will excrete more ammonia into the water, which leads to poor growth in fshes [20].Alternatively, if the fsh diet contains more protein than necessary, not only does it pollute the environment by producing nitrogenous wastes but also raises the price of feed without providing any additional benefts for growth [21].Terefore, dietary protein optimisation of a species at various life stages is critical towards making aquaculture lucrative and sustainable.Accordingly, various authors documented the optimum protein requirement of various barb species, such as 500 g protein/kg for tinfoil barb fry [22], 300-350 g protein/kg for lemon fn barb hybrid fngerlings [22], 350 g protein/kg for Puntius vittatus [23], 300 g protein/kg for Puntius gonionotus [24], 297 g protein/kg for Labeo bata fry [25], 281.48-282.53g protein/kg for Cirrhinus reba fry [26], 250 g protein/kg for Osteobrama belangeri fngerlings [27], 290 g protein/kg goldfsh fry [28], and 318.0-327.6 g protein/kg for Hypselobarbus pulchellus fngerlings [29].In this regard, the present research explores the dietary protein requirement of Jerdon's carp (H.jerdoni) since it is a relatively new commercial ornamental fsh, and many aspects of its culture including its food and feeding habit as well as nutritional requirements have not been examined yet.Likewise, no available information on the optimum DCP requirement at the early life stages (especially the juvenile nutrition with reference to protein) has yet been documented.In this context, the current research to determine the optimal dietary protein requirement of Jerdon's carp, H. jerdoni juveniles in relation to its growth performance, is justifed for its conservation and eforts for its captive maturation and breeding.

Experimental Fish Procurement and Acclimatization.
Six hundred and ffty (650) Jerdon's carp (H.jerdoni) fngerlings (weight 5.62-6.01g; length 6.5-7.1 cm) were obtained from a small hatchery in Mangalore, Karnataka, India.Ten, the fshes were carefully transported to the wet lab at the ICAR-Central Institute of Fisheries Education in Mumbai, India, in polythene bags comprising oxygenated water.On reaching the wet laboratory, the fshes were frst treated with KMnO 4 solution (4 mg/L) for 2 min to remove the possible pathogens, and fnally, mild salt (20 g/L) treatment was given for the duration of 2-3 min to overcome the transportation mediated stress.Following that, the treated fsh were transferred to rectangular tanks (1.05 m 2 × 0.89 m, 1000 L capacity) with vigorous aeration.Te fshes were then acclimatized for 2 Journal of Applied Ichthyology 15 days in the same tank, and during this period, they were fed with 30% crude protein feed at satiation level twice daily.
During the acclimatization period, 30% water was replaced with freshwater in every 2 days interval.

Formulation and Preparation of the Experimental Diets.
Six heteronitrogenous (200-450 g CP/kg), isocaloric (around 400 kcal digestible energy/100 g), and isolipidic (60.80 g/kg) experimental diets containing graded levels of DCP, viz., such as 200, 250, 300, 350, 400, and 450 g CP/kg, were formulated and prepared (Table 1).Previously, dried ingredients were then crushed using a pulverizer, sieved, weighed, and securely stored in separate containers, according to the formulation.After adding the necessary amount of water, all the ingredients (excluding additives and oil) were mixed properly to make the dough.Te dough was fnally sealed in a heat-resistant polythene bag and steamcooked inside a pressure cooker at least for 20 minutes, after which it was kept at room temperature for cooling.Ten, the remaining ingredients, viz., vitamins-minerals mixture, betaine hydrochloride, oil, BHT, vitamin E, and Stay-C, were mixed properly with the cooled dough (cooked), and another dough was remade.Te resulting dough was then put through a pelletizer to produce pellets (diameter of a pellet: 1.5 mm), which were then maintained at ambient temperature for 24 hours before drying in an oven (40 °C) until the amount of moisture in the pellet went down below 100 g/kg (<10%) feed.At last, the dried pellets were packed into airtight polyethylene zipper bags and stored at 4 °C till further use.

Setting Up of the Experimental Unit.
Eighteen circularshaped FRP (fbre glass-reinforced plastic) tanks with 300 L (1 m diameter × 0.8 m height, 200 L water volume) capacity were taken for the present study.Te experimental tanks were disinfected with 4 ppm KMnO 4 solution followed by thorough washing with freshwater and fnally dried well under sunlight.Following that, freshwater was introduced to the tanks, and continuous aeration was maintained.Ten, using a completely randomised design (CRD), two hundred and seventy healthy and well-acclimatized fngerlings of Jerdon's carp (6.12-6.18g) were dispersed randomly in six diferent treatments in triplicates using a stocking density (SD) of 15 fsh/tank.During the entire experimental period of 60 days, the animals were fed daily two times at 9: 00 am and 6: 00 pm with their respective diet to the apparent satiation level.Every morning, faeces from each tank were siphoned out, and the siphoned water was reflled with freshwater to maintain a uniform amount of water in all tanks.At every 14 days interval and after 60 days, the experimental animals from each tank were carefully weighed after subjecting to one day starvation for adjusting the satiation feeding level.Mortality of fsh from every tank was counted daily during the entire experimental period to calculate the survival percentage.Troughout the 60-day experiment, a waterproof digital thermometer (Labart, India) and a pH probe (HANNA Instruments, Singapore) were used every day to measure the variety of water quality parameters, including temperature and pH, whereas dissolved oxygen, total hardness, total alkalinity, total ammoniacal nitrogen, nitrate-nitrogen, nitrite-nitrogen, and free carbon dioxide were measured at a 7-day interval following the standard protocols of APHA [30].

Sample Collection and
Processing.Before starting of the feeding trial, initial body weight was measured.Te survival percentage (%) was calculated by counting the living animals, and the fnal body weight of fsh from each tank was assessed after a 24-hour fasting period following the conclusion of the feeding trial.Afterward, six fshes were randomly collected from each tank and anaesthetized with the help of clove oil solution (50 μl/L).After that, three fsh were kept for carcass composition analysis, and the remaining three fsh were thoroughly dissected to obtain liver, gill, muscle, and intestine samples.Following that, diferent organs were weighed separately and representative quantity were selected for homogenization with chilled sucrose solution (0.25 M) in a glass tube using a tissue homogenizer (REMI instrument) to prepare 5% tissue homogenates.Ten, the 5% tissue homogenates were kept at 10,000 rpm for 15 min at 4 °C for centrifugation (Termo Fisher Scientifc), and fnally, the supernatants were collected in 2 ml vials and kept in a freezer (−20 °C) for further analysis.

Proximate Analysis.
Standard procedures were utilised to perform the proximate composition of six diferent experimental diets and whole carcass [31].Te amount of moisture in the samples was analysed with the help of oven drying at 102 °C until they reached a constant weight.Te remaining proximal components were calculated on a dry matter basis.Te sample's nitrogen content was evaluated by using an automated micro-Kjeldahl analyser (PELICAN, India), and the CP level was obtained by multiplying the total nitrogen content by 6.25.Lipid content of the samples was calculated by following the method of solvent extraction.Te Fibretec instruments (Tulin, India) were used to analyse the crude fbre of the diet samples.Furthermore, a mufe furnace was used to obtain the ash content of a sample (550 °C for 6 hours, WIT; Australia).(1) Treatment groups having graded levels of protein, i.e., TCP20 (200 g/kg dietary protein), TCP25 (250 g/kg dietary protein), TCP30 (300 g/kg dietary protein), TCP35 (350 g/kg dietary protein), TCP40 (400 g/kg dietary protein), and TCP45 (450 g/kg dietary protein).b GNOC: groundnut oil cake, procured from the local market, India.c Composition of vitamin-mineral mix (PRE-MIX PLUS) (quantity/kg): vitamin A, 5,500,000 IU; vitamin D3, 1,100,000 IU; vitamin B2, 2,000 mg; vitamin E, 750 mg; vitamin K, 1,000 mg; vitamin B6, 1,000 mg; vitamin B12, 6 mg; calcium pantothenate, 2,500 mg; nicotinamide, 10 g; choline chloride, 150 g; Mn, 27,000 mg; I, 1,000 mg; Fe, 7,500 mg; Zn, 5,000 mg; Cu, 2,000 mg; Co, 450 l-lysine, 10 g; DL-methionine, 10 g; selenium, 125 mg; vitamin C, 2,500 mg.d CMC: carboxymethyl cellulose, procured from HiMedia Ltd., India.e BHT: butylated hydroxytoluene, procured from HiMedia Ltd., India.[33] and Drapeau [34] were followed to measure the intestinal amylase and protease activity.Cherry and Crandall [35] techniques were followed to conduct the intestinal lipase enzyme activity.

Oxidative Stress-Related or Antioxidant Enzymes.
Te superoxide dismutase (SOD) assay was carried out using the Misra and Fridovich [36] method.Te Takahara et al. [37] method was used to conduct the catalase (CAT) enzyme activity.

Protein Metabolic Enzymes.
Te aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in muscle and liver tissue homogenates were determined by using commercial kits from ERBA, India.

Carbohydrate Metabolic Enzymes. Te Wroblewski
and Ladue [38] method was used to evaluate the lactate dehydrogenase (LDH) activity.Te malate dehydrogenase (MDH) activity was measured using the Ochoa [39] technique.

Haemato-Biochemical Parameters
2.8.1.Haematological Parameters.Te Hendricks [40] method was applied to count red blood cells (RBCs) using a Neubauer hemocytometer.Te Shaw [41] method was used to estimate white blood cells (WBCs) in fsh blood samples.Te van Kampen and Zijlstra [42] method was applied to estimate the haemoglobin (Hb) content in the blood samples.A haematocrit centrifuge was used to analyse the haematocrit value at 1000 g for 10 min [43].(2) 2.9.Statistical Analysis.Te experimental results were put through one-way ANOVA (analysis of variance) using the SPSS 22.0 statistical analysis tool to establish the means and average standard error of means (SEM).Te overall treatment efects were evaluated, and then, the linear and quadratic efects of graded protein levels were measured using polynomial contrast analysis.Duncan's multiple range test with post hoc analysis was used to assess and determine the signifcant diference between means at the 5% probability level (p < 0.05).Finally, the data were presented as the means and average standard error of means (SEM).Furthermore, broken-line linear regression [44] and secondorder polynomial regression analysis were conducted based on the WG% to optimise the DCP level [45].7).Serum total protein and albumin in fsh fed with 200 g/kg protein (TCP20) and 250 g/kg protein (TCP25) were signifcantly lower (p < 0.05) than in other dietary groups.However, the total globulin level increased signifcantly (p < 0.05) as dietary protein levels increased to 300 g/kg and remained unchanged in afterward high protein-fed groups.Te fsh received 200 g protein/kg showed a similar P/E value (p > 0.05) in TCP25 and TCP30 groups and signifcantly higher (p < 0.05) than the other groups.

3.7.
Oxidative Stress or Antioxidant Enzymes.In all, feeding of graded levels of DCP had a signifcant (p < 0.05) efect on the linear, quadratic, and overall trends of SOD and CAT activity (Table 8).Te fsh of TCP20 and TCP25 groups showed signifcantly (p < 0.05) higher SOD and CAT activity than those of other treatments.

Carbohydrate Metabolic Enzymes. Liver and muscle
LDH and muscle MDH activities were signifcantly (p < 0.05) afected by graded levels of DCP overall, linearly, and quadratically (Table 9).Te fsh of TCP20 and TCP25 groups showed signifcantly (p < 0.05) higher hepatic and muscle LDH and muscle MDH activities than those of other dietary groups.However, the graded level of DCP did not afect hepatic MDH activity signifcantly (p > 0.05).
3.9.Protein Metabolic Enzymes.DCP levels had no efect on muscular AST and ALT activities (p > 0.05), whereas DCP levels had an efect (p < 0.05) on the overall and quadratic trends of hepatic ASTand ALTactivities (Table 9).Te fsh of TCP30 and TCP35 groups showed signifcantly (p < 0.05) higher hepatic AST and ALT activities than those of other dietary groups.

Optimum Dietary Protein Level.
Te optimal DCP requirement of H. jerdoni juveniles was evaluated using broken-line linear and second-order polynomial regression analysis, and the optimum DCP level of H. jerdoni juveniles was found to be 309.72 and 316.40 g/kg, respectively (Figure 1).[46] values of the various diets demonstrated that the diets were heteronitrogenous, isolipidic, and isoenergetic, supporting the premise of the protein requirement study [20,47,48].

Water Quality Parameters.
Water quality parameters have a signifcant impact in maintaining physiological equilibrium in animals, including teleosts [48].Among the several physicochemical properties of water, temperature (optimal 26-30 °C) is the most critical factor in sustaining metabolic activity in cold-blooded animals including teleosts [49,50].Troughout the experiment, the temperature was kept between 27.16 and 29.92 °C.Widiyati et al. [50] reported that the optimal pH range for a fsh culture is 6-9, while the pH value fuctuated from 6.99 to 7.44 during the whole study.In the present experiment, the dissolved oxygen (DO) level was kept within a range of 5.62-6.41mg/L, which falls closer to the optimal DO (>5 mg/l) level for fsh production Journal of Applied Ichthyology    Mean values (n � 3) in the same column with diferent superscripts difer signifcantly (p < 0.05).a Treatment groups having graded levels of protein, i.e., TCP20, 200 g/kg protein; TCP25, 250 g/kg protein; TCP30, 300 g/kg protein; TCP35, 350 g/kg protein; TCP40, 400 g/kg protein; TCP45, 450 g/kg protein.
b GOT, glutamate oxaloacetate transaminase activity is expressed as nanomoles of oxaloacetate released/min/mg.c GPT, glutamate pyruvate transaminase activity is expressed as nanomoles of sodium pyruvate released/min/mg protein.d LDH, lactate dehydrogenase activity is expressed in unit/mg protein/min.e MDH, malate dehydrogenase activity is expressed in unit/mg protein/min.

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Journal of Applied Ichthyology [51,52].Te recommended total alkalinity value for the fsh culture, according to Santhosh and Singh [53], is 50-300 mg/ L, and the total alkalinity (mg/l) value in the current study was kept within that range.Te concentration of calcium (Ca +2 ) and magnesium salts (Mg +2 ), which are generally coupled with bicarbonates and carbonates (temporary hardness) and sulphates, chlorides, and other anions (permanent hardness) governs the total hardness of water [54].
In the present experiment, total hardness of diferent experimental units was observed within a range of 183.78-199.93 mg/L, which falls within that optimum range for the fsh culture [55].Total ammonium-nitrogen, nitritenitrogen, and nitrate-nitrogen concentrations were found to be within the acceptable range for aquaculture in the current experiment [56][57][58][59].

Growth and Nutrient Utilisation.
In general, animal growth, including teleosts, is a phenotypic display of muscle hyperplasia that is infuenced by various factors where environmental and nutritional factors are more crucial among them [48].In a nutritional study, CP is the most vital dietary component in animals including fsh as because its presence in the diet in adequate quantities not just supports growth but also delivers energy via amino acid breakdown (catabolism).Generally, many species growth is directly proportional to the DCP levels, but quantities of protein higher or lower than the optimum level cause growth retardation [60,61] probably due to catabolism of more amino acids instead of body protein synthesis with reduced protein conversion efciency in this situation at the cost of growth [62].Furthermore, a lack of nonprotein energy in the diet lowered the fsh growth performance despite having high protein in the diet probably due to preferential catabolism of dietary protein-derived amino acids as fsh feed on energy satiation [63].
In the current experiment, diferent growth parameters such as WG, FBW, WG%, and SGR of H. jerdoni were observed to be raised with the raising levels of DCP up to 300 g CP/kg diet, but an increase in the DCP level in the diet reduces growth performance.Tis implies that excess dietary protein above the optimal amount with higher P : E values in the diet does not aid in growth rather assists in energy generation.Many other cyprinid species have shown growth retardation as a result of consuming protein at higher than optimal levels.Barlaya et al. [29] reported that of 20, 25, 20, 35, and 40% crude protein-fed groups, the peninsular barb H. pulchellus showed best growth in the 35% CP group at the end of 60 days.Similarly, other minor carp such as C. reba fngerlings showed best growth at 30% CP of 20, 25, 30, 35, and 40% CP-fed groups [26].While rearing in fow through aquaria for 6 weeks, Lochmann and Phillips [28] reported that of 21.2, 25.3, 28.9, 31.1, and 34.5% crude protein-fed groups, the goldfsh of the 28.9% CP-fed group exhibited the highest growth rate in terms of various growth indices.
Te FCR and PER of the feed generally show how well the fsh utilise the food item and the amount of protein it contains for growth.Te FCR value reduced considerably (p < 0.05) with rising DCP levels up to 300 g CP/kg diet, but further addition of DCP in the diet increases the FCR of fsh.Fish belongs to TCP30 (300 g CP/kg) and TCP35 (350 g CP/ kg) groups with protein (P): energy (E) values of 74.52 and 86.87 mg protein/kcal DE, respectively, showing higher PER than in TCP40 and TCP45 groups.Tese data suggested that optimal dietary protein and P : E values may be a signifcant dietary element for efective protein utilisation, resulting in higher growth in fsh.Similar kinds of results were also conveyed by Singha et al. [48], Santiago et al. [64], and Siddiqui et al. [65].Moreover, Mohanta et al. [66] reported that silver barb showed lower FCR at the 30% CP level when fed with 20, 25, 30, 35, and 40% CP for a period of 90 days.In this study, PER is showing a declining trend with the escalation of protein in the feed which might be due to the reason that in lower protein-fed groups, probably almost all the dietary amino acids may be used in the formation of body proteins for achieving somatic growth.Alternatively, a signifcant proportion of dietary amino acids in the higher protein-fed groups may be used for the production of energy rather than synthesis of body protein, resulting in lower PER.Tis fnding was similar to the outcomes of other studies [29,66].
4.4.Carcass Composition of Fish.Te higher carcass CP content of H. jerdoni juveniles was observed in the TCP30 (300 g CP/kg) group than in the other protein-fed groups.Furthermore, when DCP levels exceeded 300 g/kg, there was a decreasing trend in body crude protein deposition.Many authors found similar patterns in carcass protein as a result of feeding varied levels of DCP [26,29,66].Te increasing trend of carcass protein of H. jerdoni juveniles up to an optimum level with an optimum P : E value demonstrated efective dietary protein utilisation for maximizing growth.
However, the increasing level of DCP beyond an optimum level reduced growth probably due to excess amino acids produced from dietary protein used for the production of energy rather than synthesis of body protein that ultimately results in a decrease or no change in the carcass protein level.
Our fndings have been supported by Yadav et al. [26], who reported that the escalation of DCP up to 30% enhanced the CP deposition in carcass in case of C. reba.In this study, other parameters such as moisture, lipid, and ash remain unchanged (p > 0.05) among the diferent protein-fed groups.Similar inferences were also documented by many authors [26,29,67].In this study, the graded level of DCP has no efect on the carcass lipid level which might be due to the efect of species diferences or feeding isolipidic experimental diets.However, in contrast to the present study, Singh et al. [68] demonstrated the highest level of lipid deposition in fsh of the optimum protein-fed group.Te HSI value is directly proportional to the dietary energy sources and level as reported by many researchers [69,70].
In this study, the HSI value of fsh was signifcantly higher in lower protein-fed groups (TCP20 and TCP25) than in higher protein-fed groups.Tis disparity is most likely due to the reduction in digestible carbohydrate levels when protein levels in diets increase.It has been observed that lipogenesis from carbohydrate is a recognized biological phenomenon, where the excess dietary carbohydrate can be reserved as lipid in the liver, ultimately leading to an increase in the liver size or HIS [71,72].A similar kind of result was also demonstrated by several authors [73,74].

Digestive Enzyme Activities.
Digestive enzymes provide information about the whole digestion process and how efciently fsh digest the food item for their growth [75,76].
Te intestinal protease activity increased with rising DCP levels, which might be attributed to the availability of greater amounts of protein as a substrate in the intestine.Te current fnding supported the fndings of Bazaz and Keshavanath [77] in Tor khudree, Singha et al. [48] in GIFT, and Jayant et al. [78] in Pangasianodon hypophthalmus, respectively.In case of amylase activity, a completely opposite trend was found; i.e., a negative correlation was evident between amylase enzyme activity with the DCP levels which may be attributed to less availability of starch or vice versa.Many authors have been found the similar kind of result [29,78].Finally, the DCP levels had no efect on intestinal lipase activity (p > 0.05) which might be attributed to feeding of all groups with isolipidic diets.Tis fnding was as per the observation of Singha et al. [48] in GIFT and Barlaya et al. [29] in H. pulchellus.
4.6.Haematological Parameters.Haematological parameters are considered as vital indicators of fsh health [79], and it also guides the biologist in identifying the physiological stress in fsh caused by an abrupt changes in diet as well as in the environment [76,80].In this study, the RBC count showed an increasing trend with DCP levels up to 350 g/kg, which may have occurred due to its early release from the storage pool in the spleen [81,82].Ten, again, a low RBC count probably caused less oxygen supply to the cells of a lower protein-fed group, which leads to poor growth in fshes [83].Similarly, the haematocrit (Hct) value increased with increasing the dietary CP level up to 350 g/kg, which indicates the well transport of oxygen in the body, leading to improved fsh health and growth [73].On the other hand, the unchanged WBC count among diferent protein-fed groups in this study verifed the results of Baruah et al.

Serum Biochemical Profle. Serum biochemical indices
(albumin, globulin, and serum total protein) constitute a dietary element that provides an excellent insight into a fsh's immunological capacity under various environmental conditions [86].Te most important protein, albumin, is typically synthesised in the hepatocytes and plays a vital role in the transport of several essential biological components, including hormones, vitamins, and enzymes, as well as maintain the osmotic homeostasis in case of fshes [79].On the other hand, globulins (gamma-globulins) help in maintaining the healthy immune system in case of teleosts [87].Te total protein, globulin, and albumin were signifcantly higher in moderate (TCP30 and TCP35) and high protein-fed groups (TCP40 and TCP45) than in low protein-fed groups (TCP20 and TCP25), which can be attributed to increased absorption of amino acids from protein digestion [88].Many factors infuence serum indices, including digestion efciency, fsh biomass, dietary composition, and temperature [89,90].A high protein level in the diet increases the activity of serum indices as well as protein metabolic enzyme function which generally refects higher protein catabolism.Excess amino acids from protein-rich diets cannot be stored efectively in fsh, but they can potentially be deaminated and transformed into energy molecules.[91].In this current study, the increase in serum indices with dietary protein might be attributed to an increase in digested protein [92].A similar kind of result was reported by Lu et al. [93] in red swamp crayfsh.Moreover, a lower A/G ratio was observed in moderate and high protein-fed groups, which may be due to higher DCP levels in the diet which helps in terms of increasing the lymphocyte proliferation and subsequent immunoglobulin production in the body of fsh and gives better immunological barrier to the fshes [94].
4.8.Antioxidant Enzymes.Continuous cellular metabolic activity produces free radicals or ROS, which cause oxidative stress, the breakdown of numerous macromolecules in cells, and tissue damage in animals [95].Te SOD-CAT enzymatic antioxidant mechanism is considered as the frst line of defence against ROS in animals where SOD frst encourages the dismutation of the superoxide anions (O 2 − ) into molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) [96].Following that, another enzyme catalase is responsible for the conversion of toxic hydrogen peroxide (H 2 O 2 ) into H 2 O and O 2. Terefore, higher activity of these enzymes denotes that animals are in a stressful condition.Accordingly, increased SOD and CAT activity in lower protein-fed groups (TCP20 and TCP25) in this study might indicate high dietary carbohydrate-driven stress in fsh.In support with the current conclusion, higher SOD and CAT activities were recorded in GIFT of lower protein-fed groups [48,79].4.9.Activities of Carbohydrate Metabolic Enzymes.Te MDH and LDH activities are closely related to anaerobic metabolism of carbohydrate and generally occur during the conditions of energy crisis, when malate is transformed into oxaloacetate and pyruvate into lactate (or vice versa), respectively, for giving energy via gluconeogenesis [97,98].In this study, higher LDH activity in low protein-fed groups (TCP20 and TCP25) might be attributed to high dietary carbohydrate-mediated oxidative stress in animals, which eventually triggers the anaerobic metabolism of pyruvate for energy production via lactate.Similarly, higher MDH activity in TCP20 and TCP25 groups might indicate the high carbohydrate-mediated oxidative stress with high-energy demand, which could be fulflled via gluconeogenesis [98].A similar kind of result was reported by many researchers, such as Singha et al. [48] in GIFT, Tok et al. [99] in Pangasianodon hypophthalmus, and Shimeno et al. [100] in C. carpio.4.10.Activities of Protein Metabolic Enzymes.AST and ALT are the two transaminase enzymes that play an important role in amino acid metabolism [101].In this experiment, the high hepatic ALTand ASTactivities in low (TCP20 and TCP25) and high (TCP40 and TCP45) protein-fed groups might indicate the synthesis of new amino acids, which, in former groups, probably took part in synthesis and accretion of body protein, but maybe due to insufcient dietary supply of amino groups, the sufcient quantity of new amino acids could not be synthesised to support optimum growth of fsh; on the other hand, in latter groups, newly synthesised amino acids probably could be oxidized followed by gluconeogenesis for energy supply at the cost of growth of fsh.However, the moderate activities of these enzymes in the fsh of TCP30 and TCP35 groups indicate the synthesis of new amino acids, which probably could take part in synthesis and accretion of body protein to accelerate growth of fsh.Tis fnding might be due to dietary supply of optimum protein and protein-derived amino acids with an optimum dietary P : E ratio in the presence of sufcient energy from nonprotein sources (lipid and carbohydrate).Te present fndings corroborated the higher activities of hepatic ALT and AST in GIFT fed with varying dietary CP [48,79].

Conclusion
In aquaculture, understanding the nutritional requirements of any species is critical for the formulation and preparation of cost-efective and environmentally friendly feed to optimise output in a sustainable manner.Based on broken-line and second-order polynomial regression analysis in relation to WG%, the optimum dietary crude protein levels for H. jerdoni juveniles reared for 60 days were found to be 309.72 and 316.40 g/kg.Furthermore, DCP within the 300-350 g/kg range gives a better condition for growth, nutrient utilisation, and physiological wellbeing of the fsh.However, a further increment in DCP beyond the optimum level causes growth retardation in case of Jerdon's carp fngerlings.Tese observations will provide baseline data for developing nutritionally balanced diets for the intensive and semi-intensive culture of this fsh species.

2. 6 .
Growth, Nutrient Utilisation, Survival Rate, and Body Indices.Te growth and body indices were calculated using the following formulas: Journal of Applied Ichthyology weight gain(WG) � final body weight(FBW) -initial body weight(IBW) weight gain percentage(WG%) � (FBW-IBW) IBW × 100 specific growth rate(SGR) � [(Ln FBW) -(Ln IBW)] × 100 feed conversion ratio(FCR) � intake of feed in dry form(g) wet weight gain (g) feed efficiency ratio (FER) � wet weight gain (g) intake of feed in dry form protein efficiency ratio (PER) � wet weight gain(g) intake of protein in dry form(g) survival(%) � animals harvested in live form animals stocked in live form   × 100 hepato − somatic index (HSI %) � liver weight of the animal total weight of the animal   × 100.

Figure 1 :
Figure1: Te broken-line linear and second-order polynomial regression analysis to optimise dietary crude protein requirement in relation to weight gain percentage (WG%) of H. jerdoni juveniles reared for 60 days and fed with graded levels of dietary crude protein.

Table 1 :
Formulation and proximate composition of the experimental diets (g/kg, dry matter basis).
[32]mount of tissue protein in all the treatment samples was calculated by following the method of Bradford[32].

Table 2 :
Physicochemical parameters of water in diferent experimental units during 60 days of the feeding trial.DO, dissolved oxygen.c TA-N, total ammonia nitrogen.d NO −2 -N, nitrite nitrogen.e N0 −3 -N, nitrate nitrogen.f Free CO 2, free carbon dioxide.ND, not detected. b

Table 3 :
Growth, nutrient utilisation, and percentage survival of H. jerdoni juveniles fed diets containing varying levels of dietary protein for 60 days.

Table 4 :
Digestive enzyme activities of H. jerdoni juveniles fed diets containing varying levels of dietary protein for the experimental period of 60 days.Amylase activity is expressed as the micromole of maltose released/min/mg protein.cProteaseactivity is expressed as millimole of tyrosine released/min/mg protein.dLipaseactivity is expressed as units/min/mg protein.Te haematological traits of fsh fed with graded levels of DCP are displayed in Table6.Te concentration of haemoglobin and the number of white blood cells were not afected signifcantly across dietary groups, but the overall and quadratic trends of the RBC count and overall, linear, and quadratic trends of the haematocrit value were signifcantly afected (p < 0.05) by graded levels of DCP.Te RBC count and the haematocrit value signifcantly increased (p < 0.05) with increasing the DCP level up to 300 g/kg, and a further increase in DCP signifcantly reduced (p < 0.05) the RBC count and the haematocrit value in fsh.

Table 5 :
Whole-body proximate composition (on g/kg, wet weight basis) and body indices of H. jerdoni juveniles fed diets with graded levels of dietary protein for the experimental period of 60 days.

Table 6 :
Haematological indices of H. jerdoni juveniles fed diets containing varying levels of dietary protein for the experimental period of 60 days.
b Hb, haemoglobin.c RBC, red blood cell.d WBC, white blood cell.e Hct, haematocrit.

Table 7 :
Haemato-biochemical parameters of H. jerdoni juveniles fed diets containing varying levels of dietary protein for the experimental period of 60 days.

Table 8 :
Oxidative stress enzyme activity of H. jerdoni juveniles fed diets containing varying levels of dietary protein for the experimental period of 60 days.

Table 9 :
Carbohydrate and protein metabolic enzyme activity of H. jerdoni juveniles fed diets containing varying levels of dietary protein for the experimental period of 60 days.