Effect of Dietary Lipid Level on Growth Performance, Body Composition, and Physiometabolic Responses of Genetically Improved Farmed Tilapia (GIFT) Juveniles Reared in Inland Ground Saline Water

A 60-day feeding trial was carried out to determine the effect of dietary lipid levels on growth and physiometabolic responses to optimize the dietary lipid requirement for maximizing the growth of Genetically Improved FarmedTilapia (GIFT) juveniles reared in inland ground saline water (IGSW) of medium salinity (15 ppt). Formulation and preparation of seven heterocaloric (389.56-449.02 Kcal digestible energy/100 g), heterolipidic (40-160 g/kg), and isonitrogenous (410 g/kg crude protein) purified diets were done for conducting the feeding trial. Random distribution of 315 acclimatized fish (mean weight 1.90 ± 0.01 g) was made in seven experimental groups such as CL4 (40 g/kg lipid), CL6 (60 g/kg lipid), CL8 (80 g/kg lipid), CL10 (100 g/kg lipid), CL12 (120 g/kg lipid), CP14 (140 g/kg lipid), and CL16 (160 g/kg lipid) with 15 fish per triplicate tank (fish density, 0.21 kg/m3). Respective diets were used for feeding the fish at satiation level three times daily. Results indicated that weight gain percentage (WG%), specific growth rate (SGR), protein efficiency ratio, and protease activity significantly increased up to 100 g lipid/kg fed group, and then the values significantly decreased. Muscle ribonucleic acid (RNA) content and lipase activity were highest in 120 g/kg lipid-fed group. RNA/DNA (deoxyribonucleic acid) and serum high-density lipoproteins levels of 100 g/kg lipid-fed group were significantly higher than 140, and 160 g/kg lipid-fed groups. The lowest feed conversion ratio was found in the 100 g/kg lipid-fed group. The amylase activity was significantly higher in 40 and 60 g lipid/kg fed groups. The whole-body lipid level was increased with increasing the dietary lipid levels, whereas, there was no significant difference in whole-body moisture, crude protein, and crude ash contents of all groups. Highest serum glucose, total protein and albumin, and albumin to globulin ratio and lowest low-density lipoproteins level were found in 140 and 160 g/kg lipid-fed groups. Serum osmolality and osmoregulatory capacity did not vary significantly, whereas carnitine palmitoyltransferase-I and glucose-6-phosphate dehydrogenase showed an increased and decreased trend, respectively, with the increasing dietary lipid levels. According to second-order polynomial regression analysis based on WG% and SGR, the optimum dietary lipid for GIFT juveniles in IGSW of 15 ppt salinity was found to be 99.1 and 100.1 g/kg, respectively.

A 60-day feeding trial was carried out to determine the effect of dietary lipid levels on growth and physiometabolic responses to optimize the dietary lipid requirement for maximizing the growth of Genetically Improved FarmedTilapia (GIFT) juveniles reared in inland ground saline water (IGSW) of medium salinity (15 ppt). Formulation and preparation of seven heterocaloric (389. .02 Kcal digestible energy/100 g), heterolipidic (40-160 g/kg), and isonitrogenous (410 g/kg crude protein) purified diets were done for conducting the feeding trial. Random distribution of 315 acclimatized fish (mean weight 1:90 ± 0:01 g) was made in seven experimental groups such as CL 4 (40 g/kg lipid), CL 6 (60 g/kg lipid), CL 8 (80 g/kg lipid), CL 10 (100 g/kg lipid), CL 12 (120 g/kg lipid), CP 14 (140 g/kg lipid), and CL 16 (160 g/kg lipid) with 15 fish per triplicate tank (fish density, 0.21 kg/m 3 ). Respective diets were used for feeding the fish at satiation level three times daily. Results indicated that weight gain percentage (WG%), specific growth rate (SGR), protein efficiency ratio, and protease activity significantly increased up to 100 g lipid/kg fed group, and then the values significantly decreased. Muscle ribonucleic acid (RNA) content and lipase activity were highest in 120 g/kg lipid-fed group. RNA/DNA (deoxyribonucleic acid) and serum high-density lipoproteins levels of 100 g/kg lipid-fed group were significantly higher than 140, and 160 g/kg lipid-fed groups. The lowest feed conversion ratio was found in the 100 g/kg lipid-fed group. The amylase activity was significantly higher in 40 and 60 g lipid/kg fed groups. The whole-body lipid level was increased with increasing the dietary lipid levels, whereas, there was no significant difference in whole-body moisture, crude protein, and crude ash contents of all groups. Highest serum glucose, total protein and albumin, and albumin to globulin ratio and lowest low-density lipoproteins level were found in 140 and 160 g/kg lipid-fed groups. Serum osmolality and osmoregulatory capacity did not vary significantly, whereas carnitine palmitoyltransferase-I and glucose-6-phosphate dehydrogenase showed an increased and decreased trend, respectively, with the increasing dietary lipid levels. According to second-order polynomial regression analysis based on WG% and SGR, the optimum dietary lipid for GIFT juveniles in IGSW of 15 ppt salinity was found to be 99.1 and 100.1 g/kg, respectively.

Introduction
Intensification of aquaculture leads to more dependence on quality feed as it accounts for around 50-60% of the total operational cost, and judicial use of nutrients in feed can easily sustain the growth of the aquaculture sector [1,2]. Therefore, the most crucial consideration is an adequate nutritional information, especially the knowledge of the dietary nutrient requirements of species in relation to age and environmental conditions [3]. Dietary lipid is one of the crucial nutrients after protein to spare protein for energy supply, and thus contributes an important role in optimizing the dietary protein for the growth of living organisms [4]. Dietary lipid also plays an important role in providing essential fatty acids, phospholipid, and fat-soluble vitamins to fish [5,6]. Fish prefer to catabolize dietary protein to satiate the physiometabolic energy needs than other nutrients [7]. Nevertheless, a diet with a higher amount of protein and lower levels of nonprotein energy sources accelerates ammonia production through preferential amino acid catabolism, resulting in the deterioration of the water quality and poor growth of fish [2,8]. Therefore, during feed formulation, optimization of dietary protein to energy ratio (P : E) by increasing lipid content can spare protein for promoting the growth of fish with minimized nitrogenous output in the environment [1]. High dietary lipid levels can hamper the feed manufacturing process and reduce feed intake, thus resulting in the animal's poor growth performance [9]. Likewise, excess dietary lipid levels beyond the optimum level can cause abdominal fat deposition and fatty liver syndrome due to increased leptin levels [10], which affects the general wellbeing of the fish and the characteristic muscle quality [11]. So, lipid must be included in the fish diet at an optimum level to maximize protein utilization for better growth [4].
Lipid content of the diet can affect the digestibility of nutrients, activities of substrate-specific digestive enzymes, and the ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio in the muscle of the fish [12]. The gastrointestinal digestive enzyme activity is an important biochemical indicator of feeding activity [13]. The digestive enzyme activities of fish are positively correlated to the dietary nutrient digestion and absorption for growth [1] and are particularly regulated by specific substrates present in the fish's gut [14]. In fish, new tissue protein synthesis and deposition for growth are interlinked with muscle RNA levels corresponding to RNA to DNA ratio, and thus considered a suitable biomarker for evaluating the growth metrics of fish [15,16].
Higher growth rate, tolerance of a wide range of salinity, efficient feed conversion efficiency, ease of spawning, resistance to diseases, and increased consumers preference have made tilapia as the world's second-largest farmed species after carps [17]. Unlike brackish, estuarine and marine water, inland ground saline water (IGSW) is deficient in potassium (K + ) ion but high in calcium (Ca 2+ ) and magnesium (Mg 2+ ) ions [14,18], which affects the physiological homeostasis and growth performance of fish [19]. Although growth rate is high in freshwater, Nile tilapia is least tolerant to salinity compared to other species of tilapia. However, it can tolerate a salinity up to 25 ppt [20], tilapia, especially the GIFT strain of Nile tilapia (Oreochromis niloticus), can be a suitable species for culturing in IGSW of 15 ppt as they have higher growth rate and similar salinity tolerance when compared to Nile tilapia [21]. However, tilapia can be cultured in IGSW without fortifying the K + ion in IGSW [18,22].
Different studies have reported that optimum dietary crude lipid for maximum growth of GIFT can be different under freshwater culture conditions in different age groups such as 85.6 g/kg for larvae [23], 93.4 g/kg for juveniles [24], and 75 g/kg for adult male [25]. Additionally, Hybrid tilapia [26] and GIFT [27] requires 120 and 92.2 g/kg dietary lipid, respectively, at the culture system of 10 and 8 ppt salinity. Reports on dietary lipid requirements of GIFT are available in freshwater and brackish water but limited for IGSW. Salinity difference is reported to influence the requirement of different energy nutrients [18]. Keeping the views mentioned in mind, the current study is aimed at optimizing the dietary lipid for juveniles of GIFT reared in IGSW of 15 ppt ambient salinity conditions with respect to growth, nutrient utilization, digestive enzyme activities, and muscle RNA/DNA ratio.

Fish Husbandry, Experimental Facilities, and Feeding
Trial. The GIFT juveniles (mean weight 1:50 ± 0:01 g) were purchased and transported from Rajiv Gandhi Centre for Aquaculture (RGCA), India, in polythene bag containing oxygenated water to ICAR-Central Institute of Fisheries Education (ICAR-CIFE), Rohtak station, Haryana, India. Stocking of fish was made in a cemented tank (4 m × 2:5 m × 1 m; 12,000 L capacity), containing freshwater with the facility of continuous aeration. An underground pump collected IGSW of identical salinity, passed through a filtration unit (100 μm), and kept into tanks (3 m × 2 m × 1:5 m, 9000 L capacity) for settlement. After two weeks, the IGSW was filled into storage tanks (1:06 m 2 × 0:88 m, 950 L capacity) and used in the experiment. Fish were fed with a commercial diet (350 g/kg protein and 60 g/kg lipid, Avanti Feeds, India) on a satiation basis three times per day for two weeks acclimation. Then, the addition of IGSW was done with rising of 1 ppt per day until achieving the final salinity of 15 ppt, in which the fish were further acclimatized for three weeks with the same feeding schedule and continuous aeration facility.
After acclimatization, 315 fish (mean weight 1:90 ± 0:01 g) were arbitrarily dispersed in seven experimental groups such as CL 4 (40 g lipid/kg), CL 6 (60 g lipid/kg), CL 8 (80 g lipid/kg), CL 10 (100 g lipid/kg), CL 12 (120 g lipid/kg), CP 14 (140 g lipid/kg), and CL 16 (160 g lipid/kg) in triplicates following completely randomized design (CRD). Fifteen fish were stocked (stocking density 0.21 kg/m 3 ) in each circular tank (93 cm dia and 44 cm height, 325 L capacity with 225 L water volume) fitted with continuous aeration, and 12 h photoperiod was maintained. Respective diets were used for feeding the fish on a satiation basis three times per day (10.00, 14.00, and 18.00 h). At every fifteen days 2 Aquaculture Nutrition interval, the body weight was checked to judge the growth rate. Faeces from each tank were siphoned out prior to morning first feeding, and IGSW was replenished with an equal volume of siphoned water from the storage tank. About 20-25% experimental water from every tank was exchanged with IGSW of 15 ppt salinity from the storage facility at the interval of three days during the trial period. The animal experiment conducted were strictly adhered to the recommended guidelines for animal care and use after approval of Research Ethics Committee, ICAR-CIFE, Mumbai, India (University under Sec. 3 of University Grants Commission Act, and ISO 9001 : 2008 certified), while conducting the current study. The approval of the synopsis was obtained with no: FNT-PA7-04 from the university. Everyday water quality parameters like pH, temperature, and salinity were estimated by pH meter (Aquafins Instruments, India), thermometer (Merck Millipore, Germany), and refractometer (Aquafins Instruments, India). Dissolved oxygen (DO), total alkalinity, total hardness, free carbon dioxide (CO 2 ) , nitrate-N, ammonia-N, nitrite-N, Ca 2+ , and Mg 2+ ion concentrations were estimated following "APHA" procedures [28]. K + concentration was estimated in a flame photometer (Essico Instruments, India). The analysis of the water quality parameters was done at two day's intervals.

Experimental Diet Formulation and Preparation.
Ingredients were used for formulation ( Table 1) and preparation of seven isonitrogenous (410 g/kg crude protein, CP) and heterolipidic (40-160 g/kg crude lipid, CL) purified experimental diets such as CL 4 (40 g/kg lipid), CL 6 (60 g/kg lipid), CL 8 (80 g/kg lipid), CL 10 (100 g/kg lipid), CL 12 (120 g/kg lipid), CP 14 (140 g/kg lipid), and CL 16 (160 g/kg lipid). All the milled ingredients except oils, choline chloride, betaine, butylated hydroxytoluene (BHT), stay C (protected vitamin C), and vitamin-mineral mixture were mixed uniformly followed by addition of required quantity of water to form dough. Dough was then steam cooked for 25 min in a pressure cooker (121°C). After cooling, rest of the raw materials was added, thoroughly mixed and dough was prepared. Pellets (1 mm diameter) were then prepared by a pelletizer (S.B. Panchal & Co., India) and dried under air for overnight. On the very next day, the pellets were mechanically dried in the drier (42°C) till reaching around 90-100 g/kg moisture level. The pellet strands were properly crushed to adjust with mouth size of experimental fish followed by packaging in polythene bags, labelling, and storing at 4°C until used for feeding [29].

Growth Performance and Survival
Rate. Following oneday feed restriction, the weight measurement of the whole fish was performed by electronic weighing balance at the beginning and after the completion of trial to calculate growth parameters as follows [30]: Lipid efficiency ratio LER ð Þ= Wet body weight gain g ð Þ Lipid intake on dry matter basis g ð Þ : ð5Þ After completion of the trial, live fish of every experimental tank was counted for calculation of survival as follows: Survival % ð Þ= Total number of live fish harvested at the end of feeding trial Total number of fish stocked at the commencement of feeding trial × 100: 2.4. Proximate Composition. Initially, whole 20 fish were pooled to one sample and after the end of 60 days trial period with overnight feed restriction, 15 fish from each treatment (5 from each tank) were pooled to one sample as for proximate analysis. Methods of AOAC [31] were employed for proximate analysis of whole-body and experimental diets. Samples were kept in a drier at 80°C till constant weight was achieved to determine the moisture content. The micro-Kjeldahl method (Kjeltech, Pelican Instrument, India) was followed for the estimation of the crude protein (CP), but lipid was estimated by solvent extraction (petroleum ether) method using soxhlet apparatus (SOCS plus, Pelican Instrument, India). Samples were incinerated by a muffle furnace (Wilser and Tetlow, Australia) (550°C for 6 h) for determining the crude ash (CA) content of samples. The crude fibre (CF) content analysis of fatfree samples was performed through acid and alkali digestion method in crude fibre assembly (Tulin Equipment, India) followed by burning digested sample in a muffle furnace at 550°C for 6 h. The subtraction method was employed to calculate NFE (nitrogen-free extract) of experimental 3 Aquaculture Nutrition diets and TC (total carbohydrate) of fish samples.

NFE
g kg = 1000 -CP g kg + EE g kg TC % ð Þ = 100 -CP% + EE% + CA% ð Þ f g : Determination of gross energy (GE) of diet was performed in a Bomb Calorimeter (Changsu Instrument, China) assembly. Digestible energy (DE) [32] and protein to energy ratio (P : E) of diets were determined based on the following equations: P : E mg protein 2.5. Assays of Digestive Enzymes. After trial completion, three fish were randomly collected from every experimental tank and anaesthetized by clove oil emulsion (Himedia Laboratories, India, 50 μL/L) [18]. A small piece from the extreme anterior portion of intestine was then dissected out [33] and pooled to prepare the intestinal tissue homogenate under the iced condition to analyse the digestive enzyme activities.
2.5.1. Tissue Homogenate Preparation. The collected intestinal tissue was immediately mixed with a chilled solution of sucrose (0.25 M), and a 5% tissue homogenate was prepared under iced condition using a homogenizer (Remi Equipment, India) coated with Teflon. Then, centrifugation (4°C) of the samples was performed for 10 min at 5000 rpm using a centrifuge (ThermoFisher Scientific, USA), and after  [34], and the obtained value was used to calculate digestive enzymes activities.
2.5.3. Digestive Enzyme Assay. The activity of protease (millimole of tyrosine released/min/mg protein) was estimated as per Drapeau [35]. The activity of amylase (micromole of maltose released/min/mg protein) was analysed according to Rick and Stegbauer [36]. The titrimetric method of Cherry and Crandall [37] was followed to analyse lipase activity (unit/h/mg protein).
2.6. RNA and DNA Quantification and RNA-DNA Ratio.
Muscle sample was carefully dissected out following the same procedure of intestinal digestive enzyme activities and immediately a 20% tissue homogenate was prepared. The quantification of muscle nucleic acids were done according to Schneider [38]. DNA was estimated by the diphenylamine method and RNA estimated by the orcinol. Briefly, 2.5 mL of cold 10% trichloroacetic acid (TCA) was added with 1 mL of 20% muscle homogenate and centrifuged at 5000 rpm for 10 min. The precipitate was washed by adding same volume of TCA. Then, precipitate was extracted twice with 5 mL of 95% ethanol by centrifugation at 5000 rpm for 10 min. Next, 2 mL 1 N potassium hydroxide (KOH) was added with lipoid compound free sample and incubated at 37°C for 20 h. After incubation, DNA and protein were precipitated by the addition of 0.4 mL 6 N hydrochloric acid (HCl) and 2 mL 5% TCA and centrifuged. After centrifugation, supernatant containing RNA is separated, and 0.2 mL supernatant was diluted by adding 1.5 mL distilled water. Then, diluted sample was heated by 1.5 mL orcinol reagent for 10 min and the absorbance was measured at 660 nm using a spectrophotometer (Thermo-Fisher Scientific, USA) for RNA content. On the other hand, sediment containing DNA was brought in to solution by heating with 2 mL 5% TCA. After cooling, 2 mL diphenylamine added with 1 mL solution and heated for 10 min in boiling water and the absorbance was measured at 600 nm for DNA. The following formulae were used for the calculation.
DNA μ g mL = OD at 600 nm 0:019 , ð11Þ RNA μ g mL = OD at 660 nm + 0:008 . Globulin content (g/dL) was calculated by subtracting the total albumin value from the total protein value (g/dL), and the total albumin value (g/dL) was divided by total globulin value to compute the albumin to globulin ratio (A/G).

Hepatic Lipid Metabolic Enzymes.
Liver sample was collected in the same manner like intestinal digestive enzymes and a 5% tissue homogenate was prepared and stored in −20°C until used for hepatic lipid metabolic enzymes assay.

Discussion
Growth, the muscle hyperplasia of animals, including fish, is mainly regulated via dietary and environmental conditions [43]. Dietary lipid provides energy to the animals, thus sparing amino acids for energy production and making it available for fish growth. Therefore, optimum dietary lipid level reduces the dietary protein requirement with optimizing the protein to energy ratio (P : E), in which almost all dietary protein derived amino acids undergo synthesis and deposition of muscle protein to causes the maximum growth of fish with reduced environmental pollution in terms of nitrogenous waste [3]. Moreover, less dietary protein reduces the feed cost to make the aquaculture production economic. In the present study, the WG%, SGR, and PER of GIFT juveniles enhanced significantly with elevating dietary lipid levels up to 100 g/kg with lowest FCR and beyond that it caused significant reduction of growth and protein utilization with enhanced FCR. This finding clearly indicates that feeding excess lipid beyond the optimum level for GIFT reared in IGSW of 15 ppt salinity was not beneficial in terms of growth, which might be due to the reason that excess lipid with imbalanced P : E probably reduces the protein digestibility and availability of amino acids for production and deposition of somatic tissue protein leading reduced growth of fish [44]. Moreover, excess dietary lipid probably causes the metabolic burden to fish with the outcome of reduced growth [45]. In accordance with the present finding, Mohanta true [46] and Sivaramakrishnan true [11] reported growth retardation in pangas and silver barb, respectively, due to feeding of excess dietary lipid with imbalanced P : E. On the other hand, feeding of low dietary lipid, as found in the present study, also causes reduction of growth of fish may be owing to less availability of lipid as nonprotein energy source, which probably directs the dietary protein derived amino acids towards catabolism for energy production at the cost of body protein synthesis [47]. Therefore, optimum protein, lipid, and P : E of aquafeed maximize growth of fish with minimizing feed cost, less degradation of water quality through nitrogenous discharges, and profitable aquaculture production. In our study, based upon the second degree polynomial analysis of WG% and SGR,the optimum dietary lipid requirement of GIFT juveniles reared in IGSW of 15 ppt salinity was found to be 99.1 and 100.1 g/ kg, respectively, at the dietary protein level of 410 g/kg. In the line of the present finding, Mohammadi true [27] reported that 90 g/kg dietary lipid could be optimum at 360 g/kg dietary protein level for all male tilapia in brackish water of 8 ppt salinity. In the present study, the salinity (15 ppt) and ionic composition of inland saline water was different from that of brackish water used in the study by Mohammadi true [27], which resulted in the higher lipid requirement. However, optimum dietary lipid level for Nile tilapia [48], juvenile hybrid tilapia [26], adult GIFT [25], and larval GIFT [23] in freshwater condition was reported to be 91.0, 120.3, 87.9, and 85.6 g/kg, respectively. Therefore, the present finding is the first report of dietary lipid requirement for juveniles of GIFT reared under the IGSW of 15 ppt ambient salinity condition. FCR is the indicator of feed utilization in relation to the growth of animals, including fish that depends upon the feed quality, condition of fish, and environmental factors [14,43]. In the present study, FCR showed the opposite trend of fish growth in relation to dietary lipid levels. A similar observation was also made by Mohammadi true [27] in Nile tilapia (all male) in the brackish water of 8 ppt salinity. PER value indicates the utilization capacity of protein by animals, including fish. Thus, in our study, higher PER and growth in 100 g/kg lipid fed GIFT juveniles indicates maximum utilization of dietary protein-derived amino acids for production and deposition of somatic tissue protein. A similar observation was demonstrated by Qiang true [23] for GIFT and Mohammadi true [27] for all male tilapia in freshwater and brackish water conditions, respectively. In this study, complete survival of the animal in the entire experiment group suggests that neither water quality parameters nor the experimental diets were fatal to the GIFT juveniles in IGSW because water quality parameters in the present study were found within the recommended limits for inland saline water fish culture [14,18].

Aquaculture Nutrition
Whole body composition indicates the nutritional quality of feed and nutrient utilization efficiency, flesh quality, and wellbeing of fish [49]. In this study, increased body lipid was found in higher lipid-fed groups. In agreement with the present finding, Peres and Oliva-Teles [50], Yildirim-Aksoy true [51], Mohanta true [46], and Sivaramakrishnan true [11] demonstrated the positive correlation between dietary lipid levels and whole-body lipid content of fish indicating excess dietary lipid could cause the deposition of body fat irrespective of culture condition. A similar finding was also demonstrated in Nile tilapia [52], adult GIFT [25], and larval GIFT [23] in freshwater culture conditions. On the other hand, no significant changes were found in other compo-nents of the proximate composition of fish. In contrast to the present finding, other studies in several fishes [11,23,26,46] demonstrated the higher ash content of body was due to feeding of higher dietary lipid. However, Tian true [25] found higher whole-body ash content in the lower dietary lipid-fed group. But, the exact reason for these variable observations remains unclear. Decreased whole-body moisture content in rohu [53], silver barb [46], adult GIFT [25], and larval GIFT [23] of higher dietary lipid-fed group also opposed the present observation.
Digestive enzyme activities are positively linked with fish growth, probably through enhancing nutrient digestibility and utilization [54]. In our study, protease activity of 80 and 100 g/kg lipid-fed groups and lipase activity of 80, 100, and 120 g/kg lipid-fed groups were significantly higher, whereas amylase activity was significantly lower in fish fed with dietary lipid levels beyond 60 g/kg. The protease and lipase activity in relation to dietary lipid level indicates that 80-100 g/kg dietary lipid probably could be optimally utilized to spare dietary protein for energy production, and 410 g/kg dietary protein at this lipid level could be properly digested, and derived amino acids were almost available for somatic tissue synthesis leading to maximum growth [55] as proved in this study. Gangadhara true [56], Mohanta true [46], and Sivaramakrishnan true [11] demonstrated similar findings in rohu (Labeo rohita), silver barb (Puntius gonionotus), and sutchi catfish (Pangasiodon hypophthalmus), respectively. Lower lipase activity in the lower dietary lipid-fed groups might be due to the availability of limited substrate in the gastrointestinal tract, and probably higher dietary carbohydrates could suppress the protease activity of these groups. Lower protease and lipase activity in very high lipid-fed groups indicates that excess dietary lipid probably could suppress the activity of proteolytic and lipolytic enzymes [6,57]. On the other hand, the increased and    10 Aquaculture Nutrition decreased amylase activity in lower and higher lipid-fed groups of this study might be due to higher and lower levels of digestible carbohydrates intake [6].
Muscle RNA content and RNA-DNA ratio has a positive correlation with the tissue protein synthesis and accretion for the growth of fish, thus used as a growth marker [1]. In this study, higher muscle RNA concentration and RNA/ DNA in 80, 100, and 120 g/kg lipid-fed groups were positively correlated with higher growth of fish, probably due to the protein-sparing effect of dietary lipid at these levels to influence more body protein synthesis and accretion. In corroboration with this finding, Mohanta true [46] and Sivaramakrishnan true [11] observed that RNA/DNA ratio increased in the P. gonionotus and P. hypophthalmus with increasing the dietary lipid up to the optimum level to correlate the enhanced growth. In our study, muscle DNA content showed a nonsignificant variation among the treatment groups and was well supported by the observations of Mohanta et al. [46] and Kumar true [1] in P.gonionotus and L.rohita, respectively.
Serum glucose level is an important stress biomarker in fish [58]. In our study, serum GLU level increased with an increase in dietary lipid levels. This finding indicates that the fishes were stressed due to feeding a high lipid diet associated with higher metabolic burden and energy needs [59]. Wang true [24] also concluded a similar report in GIFT juveniles fed with varying dietary lipid levels reared in freshwater conditions. In fish, serum triglyceride level reflects the magnitude of lipid catabolism in the body for energy production [6]. Thus, in our study, the higher serum TAG values in very high-dietary lipid-fed groups probably could indicate the lower lipid utilization due to metabolic burden [25]. Similar observations were also reported by Wang true [24] in GIFT (O. niloticus), Jin true [60] in grass carp (Ctenopharyngodon idella), and Nayak true [61] in silver barb (P. gonionotous) to corroborate the present finding.
High-density lipoproteins (HDL) carry excess cholesterol to the liver, where it can be either used or excreted through bile [62]. Low-density lipoproteins (LDL) carries cholesterol to the living cells, but very high-serum LDL is indicator of poor health status of animals, including fish [63]. Serum T-CHO, HDL, and LDL levels can also be regulated via fish diet [64,65]. In the current study, serum T-CHO and HDL concentrations enhanced with dietary lipid levels up to 100 g/kg and then decreased with increased dietary lipid content. This decreasing trend of serum T-CHO and HDL levels in a high-fat diet beyond the optimum level may indicate poor liver function and chances of parenchymal liver disease [66]. In agreement with the present finding, Tian true [25] reported increased serum T-CHO and HDL levels in tilapia, Kikuchi true [67] and Wang true [24] in tiger puffer and GIFT, and Guo true [9] in largemouth bass (Micropterus salmoides) due to feeding of optimum level of dietary lipid. However, in our study, serum LDL exhibited a significantly decreasing trend with increasing dietary lipid and excessive decrease of serum LDL in very high lipid-fed groups might indicate the liver dysfunction [68]. However, a contrasting report by Deng true [6] described that dietary lipid levels did not influence the serum T-CHO, HDL and LDL concentration in Asian red-tailed catfish, Hemibagrus wyckioides.
Serum protein profile can be used as the indicator of nutritional, metabolic, and health status of fish [13,69]. In this study, the higher lipid-fed group showed significantly higher serum total protein concentrations than the lower lipid-fed groups. This increment of serum total protein might be owing to the transportation of excess dietary lipid as lipoprotein in the blood of higher lipid-fed groups. Lim true [52] and Yildirim-Aksoy true [70] reported similar observations in Nile tilapia and channel catfish, respectively. Among the serum proteins, globulin and albumin levels indicate the general well-being of fish [71,72]. Albumin content of serum exhibited significantly increased values with enhancing dietary lipid up to a maximum level, which was similar to the finding of Wang true [73] in Crucian carp. In the present experiment, the highest albumin and the lowest globulin content were found in the CL 14 and CL 16 groups, suggesting probable damage and malfunctioning of liver cells of GIFT in response to higher dietary lipid levels.
There were no significant differences in serum and water osmolality among the treatment, and the fact was that salinity did not affect serum osmolality of GIFT. The results indicated that GIFT adapted to the high water salinity of IGSW at 15 ppt during the culture periods. Moreover, the serum osmolality of fishes remains the same among the different treatment groups as osmolality is more a function of water salinity, which was constant in the experiment [2]. According to Verdegem true [74] and Jana true [3], the diet composition does not affect serum osmolality.
Carnitine palmitoyltransferase-I(CPT-I) is the main regulatory enzyme in mitochondrial fatty acid oxidation [75]. The hepatic CPT-I activity was significantly elevated with increasing dietary lipid levels in many fishes, and it has been reported that feeding high-fat diets will increase CPT-I expression compared with low-fat diets in large yellow croakers [76] and grass carp [77]. In the present study, the higher lipid levels beyond 120 g/kg suppressed the hepatic

Aquaculture Nutrition
CPT-I activity might be due to excess intake of fat. This result is in corroboration with the results reported by Guo true [9], who observed a reduced CPT-I activity with very high-dietary fat in largemouth bass. Similar observations were also reported in blunt snout bream, where the activity and expression of CPT-I were significantly downregulated in fish fed the high-fat diet [78].
Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in catalyzing nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) production, essential for hepatic fatty acid biosynthesis [79]. In the present study, the G6PDH activities of the liver in relation to increasing dietary lipid exhibited a reducing trend from lower lipid to high-lipid diets. Higher activity of liver G6PDH in fish fed the low-lipid content diet than other lipid-containing diets suggest that the lipid biosynthesis is active in this group. Similar results were observed in hybrid tilapia [26] and juvenile cobia [80]. Additionally, dietary carbohydrate supply is also known to affect enzyme activities [80]. It has been confirmed in many fish species that high-carbohydrate diets stimulate lipogenesis and are conversely suppressed by high-dietary lipid [80][81][82]. However, the decrease in this enzyme activity with higher-lipid levels reveals that the high-dietary lipid depressed lipogenic enzyme activities in the liver of tilapia, as suggested by Shimeno true [83]. In the present experiment, the higher dietary carbohydrate content might be higher G6PDH activity of dietary lipid in fish fed with a 40 g lipid/kg diet.

Conclusion
Knowing the optimum dietarylipidin relation to growth, body composition, and physiobiochemical responses, a nutritionally balanced, cost-effective, and environmentally friendly diet can be formulated for the fish. Accordingly, based on WG% and SGR, second-order polynomial regression analysis optimized the dietary lipid requirement of GIFT juveniles reared condition of IGSW (15 ppt salinity) at the level of 99.1and 100.1 g/kg with the corresponding P : E value of 98.45and 97.46 mg protein/Kcal DE, respectively. Thus, the optimum dietary crude lipid requirement of GIFT juveniles in IGSW of 15 ppt salinity could range between 99.1 and 100.1 g/kg to maximize fish growth. This finding will help to develop nutritionally balanced diet for culturing GIFT in IGSW.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.