Dietary valine improved growth, immunity, enzymatic activities and expression of TOR signaling cascade genes in rainbow trout, Oncorhynchus mykiss fingerlings

This study was conducted to determine the effects of dietary valine (Val) on growth, hemato-biochemical parameters, immunity, enzymatic activities, antioxidant status and expression of target of rapamycin (TOR) and 4E-BP genes in rainbow trout, Oncorhynchus mykiss (1.57 ± 0.03 g; 5.10 ± 0.34 cm). Six isonitrogenous (450 g kg−1) and isoenergetic (20.90 kJ 100 g−1, gross energy) diets were designed to represent varied Val levels (10.5, 13.0, 15.5, 18.0, 20.5 and 23.0 g kg−1 dry diet basis). Growth parameters improved significantly (P < 0.05) with the amelioration of dietary Val level up to 18.0 g kg−1. Highest (P < 0.05) body protein content was noted at 18.0 g kg−1 dietary Val. Significant differences in hematological, intestinal enzymatic activities and antioxidant parameters were noted. However, plasma variables did not show any significant differences except aspartate transaminase and uric acid. Total protein content increased significantly, while the albumin and globulin content did not show any significant (P > 0.05) difference. Moreover expression of TOR mRNA and elF4E-binding protein (4E-BP) was observed higher (P < 0.05) at 18.0 g kg−1 Val. On the basis of results, optimum dietary Val requirement for maximal growth of rainbow trout was determined to be 18.19 g kg−1 of dry diet, corresponding to 40.42 g kg−1 of dietary protein.

Protein is yet to be considered as an expensive dietary supplement that supporting the growth performance of fish. However, an understanding of fish protein requirement is of inadequate value devoid of essential amino acids (EAAs) requirement data 1 . Amino acids are considered as vital biological compounds of animals including fish, as they are treated not only as building blocks of proteins, but also participate in various metabolic pathways, nutritional physiology, behavior, sexual metamorphosis of fish etc. [2][3][4] . Supplement feeding is considered as one of the critical factors influencing growth, well-being and physiology of fish, while the commercial viability of fish culture mainly depends upon marketability and value of production. The major portion of the production cost (40-70%) mainly lies on the fish feed [5][6][7] . The success of fish farming primarily relies upon the accessibility of satisfactory amount of nutritionally balanced supplemented feeds in the form which is appropriate for the fish 8 . The advancement of nutritionally competent and cost-effective diets for cultured fish species is of immense concern to the commercial success of aquaculture 9,10 . However, the challenges faced in aquaculture are additionally for incorporation of compatible, digestible, less wasteful and cost-effective diets. The development of such diets must subject to meet the nutritional requirements of species specific based balanced feed and its appropriate feeding practices 11,12 .
Valine (Val), isoleucine and leucine represents the family branched chain amino acids (BCAAs). Val is known to have a vital role in several physiological and metabolic reactions and in the growth of various preruminant and monogastric terrestrial animals 9,13 , besides take part in the synthesis of protein and amine neurotransmitters serotonin 14 . In addition to this, Val is also involved in several processes such as protein synthesis, repairement of tissues and nitrogen balance in fish 1,15 . Since, fish cannot synthesize these amino acids including Val, therefore it must be supplied through the diet 16  Hematological parameters. In the present study, dietary Val levels affected hematological parameters of fingerling O. mykiss as indicated in Table 3. Hemoglobin (Hb), hematocrit (Hct) and red blood cell (RBC) count significantly (P < 0.05) improved with increasing concentrations of dietary Val up to 18.0 g kg −1 diet, thereafter significant decrease in these three parameters were seen. Likewise, significant (P < 0.05) differences in total leukocyte (WBC) counts were also observed in all the Val fed groups with highest WBC (3.15 × 10 4 mm −3 ) count was occurred at lowest Val fed group i.e. 10.5 g kg −1 . Erythrocyte sedimentation (ESR) rate was found lowest (1.67 mm h −1 ) at fish fed 20.5 g kg −1 Val diet, while highest ESR content (3.08 and 2.68 mm h −1 ) was registered in those groups that fed lower dietary Val quantities i.e. 10.5 and 13.0 g kg −1 , respectively. Significant (P < 0.05) differences in mean corpuscular value (MCV) was also recorded with highest (175.06 fl) MCV was noted with fish fed higher Val level, while lowest MCV value was found for fish fed least Val containing diet i.e. 10.5 g kg −1 . However, no significant (P > 0.05) differences in MCHC and MCH values were seen among all the treatments.
Plasma indices. The plasma indices of rainbow trout are shown in Fig. 2, which indicated no significant (P > 0.05) differences in plasma cholesterol (CHO), triglycerides (TG), alanine transaminase (ALT), glucose and urea contents. While, significant reduction in aspartate transaminase (AST) was seen with increasing Val diet up to 18.0 g kg −1 and afterwards an increase in AST content trend was noted. However, uric acid content increased constantly with increasing Val levels and reached its maximum value at 23.0 g kg −1 Val fed diet.
Non-specific immune response. During the present study, effect of Val on non-specific immune response parameters was also analyzed and the findings are presented in Fig. 3. Plasma total protein showed significantly (P < 0.05) elevated trend with increasing dietary Val level up to 18.0 g kg −1 diet, thereafter decrease in plasma total protein was noted, whereas as alkaline phosphatase (ALP) content showed a decreasing trend with each incremental Val concentrations. Contrary to this, the albumin and globulin contents showed no significant (P > 0.05) differences among all the treatments.
Intestinal enzyme activities. The results of digestive intestinal enzyme activities of rainbow trout fed varied levels of Val are presented in Fig. 4. Significant (P < 0.05) differences were noted in trypsin, chymotrypsin, Intestinal antioxidant status. Intestinal antioxidant variables were also carried out and the observations are presented in Table 4. The contents of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase

Discussion
The branched chain amino acid Val along with leucine and isoleucine plays a significant role in protein synthesis and optimal growth of the fish. The main function of Val is to produce propionyl-CoA, the glycogenic precursor of succinyl-CoA 9 . It has been used as a supplement for fish growth along with leucine and isoleucine, besides also involved in the body to produce several biochemical compounds which mainly help in energy production 9 .The findings of the present study based on LWG%, FCR and PER indicated that the optimum dietary Val requirement was estimated to be 18.19 g kg −1 dry diet, corresponding to 40.42 g kg −1 of dietary protein, which is very close to the values reported on other fish species such as Cirrhinus mrigala 9 38.0 g kg −1 , rainbow trout 13 38.5-41.0 g kg −1 , Jian carp 23 40.0 g kg −1 and blunt snout bream 38.8 g kg −1 , but higher than the values reported for red sea bream 1 20.0 g kg −1 , Catla catla 56 30.9 g kg −1 , red drum 21 32.0-35.0 g kg −1 and juvenile hybrid grouper 17 31.6 g kg −1 . However, the Val requirement on trout in the present study is lower than that reported for grass carp 26 47.7-48.2 g kg −1 and golden pompano 25 46.0-47.0 g kg −1 . These data revealed substantial disparity in the optimal Val requirement among fish species. The huge variations recorded in the dietary Val requirement among various fish species could be attributed to various factors such as experimental designs, intact source of protein 57 , feed formulation, composition of test diets and utilization of different mathematical approaches 9,16,34,58,59 . Besides these above factors, various other attributes are also responsible for variations in optimum requirement of fish like water temperature, dissolved oxygen, source of food, size, age of the fish, assimilation rate, amino acids form and the energy utilized from feed-stuff and stocking density [60][61][62] . Moreover, several other physiological and metabolic needs for particular amino acid could also differ among different species, which may lead to differences in essential amino acid requirements 63,64 . Bureau et al. 65 suggested that different environmental factors can cause a significant impact on nutritional requirement of fish.
In the present study, the lower LWG% observed in fish fed 10.5 or 23.0 g kg −1 dietary Val levels compared to that fed 18.0 g kg −1 dietary Val indicated that either deficient or excessive dietary Val levels could induce a decrease in growth performance and feed utilization. Similar observations were also recorded for C. mrigala 9 ; C. catla 66 ; Oreochromis niloticus 24 ; Epinephelus fuscoguttatus x E. lanceolatus 17 . The reduction in growth performance due to above facts might be attributed to imbalance of amino acids, the loss of appetite caused by Val and poor feed utilization efficiency 9,23 . The another reason that could affect growth performance of fish is the presence of excess amount of specific amino acid, that is being consumed by the fish, which resulted demand of extra energy for the deamination or excess accumulation of extra nitrogen in the body as well as oxidation of ketones and harmful metabolites [66][67][68] . For C. mrigala, Ahmed and Khan 9 reported that the reduced growth and feed efficiency induced by an excess of one of the BCAAs was due to the extra energy expenditure directed towards the deamination and excretion of the BCAAs in excess. In the similar manner, some authors reported that the reduction in growth of fish fed excess valine may be due to the antagonistic effects between valine and other BCAAs in different fish species including rainbow trout 69,70 .
The whole-body composition of fish is always treated as an indicator of fish quality. Several internal and external markers like fish size, rearing conditions, strain, season including growth, feeding rate and nature of the diet  82,83 . Similar results have also been reported for Indian major carp, C. mrigala 8 . Whole body protein also elevated with increasing Val concentrations up to 18.0 g kg −1 Val fed diet and thereafter decreased in protein content was noted. Our result on fluctuation in body protein with respect to the elevation of each testing amino acid is in accordance with the finding on other fish species such as C. mrigala 9 ; L. rohita 66 and C. catla 84 . This rise in whole body protein content could be because of interest of several proteins or due to amino acid transamination responses occurring in fish body 9 . Body ash content of rainbow trout did not showed any difference with respect to the elevation of Val in the diet and remained constant among all the doses, except at lower Val fed diets i.e. 10.5 and 13.0 g kg −1 . Higher ash contents reported at lowest Val level diets may be due to lower muscle deposition, which proportionally increases organic and inorganic content in the fish body 85 . Moreover, HSI is an important indicator applied to studies on nutritional requirements 56,[86][87][88] , as it indicates the nutritional and physiological status of fish, besides provide an information pertaining to energy reserves in  www.nature.com/scientificreports/  www.nature.com/scientificreports/ the body 89 . In the present research work, the lowest dietary Val concentrations resulted in higher HSI values in fish compared to the fish fed high levels of dietary Val, which could resultant in higher hepatic activity due to Val deficiency that resulted in the deamination of unused amino acid or their transamination into intermediate metabolic products for use as an energy source 84 . Increased HSI content in Val and isoleucine deficient diets have previously been documented in C. catla 84 and Channa punctatus 68 , respectively. Hematological variables are treated as an important markers in response to dietary manipulations 28,[90][91][92] . These variables in fish can also be a critical indicator to assess alterations in circulatory system by toxic substances 93 . In the current research work, hematological parameters of rainbow trout fed with varied levels of Val showed significant differences. Among them, hematocrit assay is a reliable index in the aquaculture and fishery management for checking the anemic condition as well as fish health relative to nutrition, disease and stress status 94 . Highest Hb, Hct and RBC values were noted in the fish fed 18.0 g kg −1 Val diet, thereafter reduction in these three variables were recorded. The higher values for these variables at optimal dietary Val level might be ascribed to expansion in fish growth gave a capable degree of blood oxygen transport framework from respiratory systems to tissues 95 , as well as due to higher metabolic demand. A diminished trend in ESR was noted with increasing concentrations of dietary Val and was least in the fish fed 20.5 g kg −1 Val diet, which could be attributed to high blood consistency, resultant an elevation in erythrocytes count. Erythrocyte indices (MCV, MCH and MCHC) have a particular importance in the diagnosis of anemia in most animals including fish 96 . In the present study, fish fed lower and higher dietary Val containing diets i.e. 10.5 and 23.0 g kg −1 actuated macrocytic anemia, because higher values were noted in these levels. Neverthless, no significant differences in MCH and MCHC data were acquired, which is accordance with our previous findings on another amino acids i.e. leucine 55 and isoleucine (unpublished).
The blood plasma variables are generally treated as health condition indicators of fish and in response to nutritional supplements 33 . In the present study, plasma CHO, TG, ALT, glucose and urea contents were not influenced with increased Val concentrations, which is in agreement with the findings for other fish species for example, red sea bream 1 ; blunt snout bream 22 and Nile tilapia 24 . In the study, AST parameter demonstrated a decreased pattern with augmentation of dietary Val up to 18.0 g kg −1 diet indicating an enhancement in fish health status, which is in agreement with the study for O. niloticus 24 . Moreover, uric acid contents in the current research work constantly increased with increasing Val concentration and reached maximum at 23.0 g kg −1 Val diet.
In addition, branched-chain amino acids are associated in different immune functions in fish and also upregulate critical metabolic pathways in response to infectious pathogens 69,97,98 . The total protein concentration in plasma has been used as a health indicator, stress marker and to check nutritional condition in fish 99 . In the present study, significant increase in plasma total protein level was noted by increasing Val levels up to the fish fed 18.0 g kg −1 Val diet. Xiao et al. 24 also suggested that appropriate supplementation of Val can positively influence the non-specific immune reaction of juvenile Nile tilapia. Similar perceptions were reported for juvenile hybrid grouper and red sea bream fed with varied concentrations of Val diet 1,17 . While ALP as a regulative enzyme is associated with many vital functions and take part in the process of nutrients like fat, glucose, calcium and inorganic phosphatase absorption 100 . We noticed decreased ALP pattern with fish fed elevated Val in the diets, which supports the finding of Dong et al. 23 for juvenile Jian carp. However, in the present research work, we did not found any significant differences in plasma albumin and globulin contents of the fish fed varied concentrations of Val.
Fish have a high adaptability in their digestive processes, where intestine is treated as the main site at which fish easily digest and absorb nutrients, and its structural integrity plays a vital role in the maintenance of effective digestion and absorption 16,101 . Several studies pertaining to the activities of digestive enzymes in fish showed that enzymatic activity is influenced by the diet ingested or by feeding habits [102][103][104] . In the present study, there were significant improvement in trypsin chymotrypsin, amylase and lipase activities up to the fish fed 18.0 g kg −1 Val diet, showing that suitable dietary Val could improve digestion capacity in fish. Similar perceptions were accounted for juvenile jian carp, C. carpio in hepatopancreas and intestine 23,35 . Moreover, GOT and GPT are mainly liver enzymes, but they are also found in several organs or tissues. In the present study, the activities of GOT and GPT were decreased with increasing concentrations of dietary Val up to a certain point, thereafter reduction in these parameters were observed. To the best of our knowledge, no work has so far been carried out on intestinal GOT and GPT activities when fed with varied levels of dietary Val, however, some studies pertaining to these activities in hepatopancreas and muscle have been established on other fish species, where they observe significant significant increment in these parameters in hepatopancrease and muscle of Cyprinus carpio fed threonine and tryptophan diet, respectively 51,52 .
The intestinal epithelial integrity in fish plays a crucial role in maintaining the normal function of physical barriers, but intestinal epithelial cells are very sensitive to oxidative damage 105 . To fight oxidative damage, fish possess several antioxidant enzymes, which includes SOD), CAT, GPx, GR and MDA 106 . Among antioxidant enzymes, the activity of SOD indirectly reflected the ability of removing the oxygen free radicals in the fish body by regulating superoxide radical's dismutation to hydrogen peroxide, which can be eliminated by GPx 41 . SOD can efficiently eject the reactive oxygen species (ROS) in the fish body, results in generating hydrogen peroxide, which may in turn be detached by the activities of CAT. SOD and CAT also take part in several reactions independently 24 . In the present research work, SOD, CAT, GPx and GR activities were positively influenced with each incremental dietary Val levels up to 18.0 g kg −1 , which indicated an enhancement in the ability of scavenging free radical in the intestine of rainbow trout at optimum dietary Val concentration. Similar results were also noted in other fish species such as juvenile hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂ 17 , golden pompano, T. ovatus 25 and Nile tilapia, O. niloticus 24 . MDA is treated as an important biomarker for protein oxidation and lipid peroxidation 107 , and it has been reported that MDA levels indirectly reflected the severity of fish body cell from free radicals attack 41  www.nature.com/scientificreports/ the lipid per-oxidation of rainbow trout. Overall, the above data indicated that appropriate supplementation of Val levels could improve non-specific immune response variables of rainbow trout fingerling. In fish, the activation of TOR signaling cascade by dietary amino acids is reported, thereby promoting protein synthesis [108][109][110] . Besides acting as a substrate for protein synthesis, Val has also been considered as a key regulator of several biological and physiological processes in fish. For instance, Val plays a crucial role in regulating the TOR signaling pathway in response to nutritional status, resultant to protein synthesis and growth of fish 17 . In the TOR signaling pathway two downstream effectors, 4E-BP and S6K1, are the most important rate determining factors in protein synthesis 111,112 . Many amino acids, including tryptophan 52 , leucine 22,113,114 and arginine [115][116][117] , have revealed to up-regulate fish growth performance and promote protein synthesis through the TOR signaling pathway. However, there are only few studies reported on the effect of Val on fish protein synthesis and its inter-relationship with TOR signaling pathway. There the present study aimed to explore the influence of dietary Val on TOR signaling pathway, which showed that relative expression of TOR gene was positively progressed by dietary Val concentrations up to 18.0 g kg −1 dry diet, and thereafter a reduction pattern was noted. Similar trends have been previously observed in our laboratory on rainbow trout fed with graded dietary leucine 55 and isoleucine levels (under publication), and also in other fish species such as jian carp 29 , blunt snout bream 57 and hybrid grouper 44 . Wacyk et al. 118 also reported decreased trend in rainbow trout fed deficient branched chain amino acids diet. However, in this study the fish fed 13.0 and 15.5 g kg −1 Val containing diets had significantly lower relative 4E-BP mRNA expression levels compared to other groups and both TOR and 4E-BP levels were highest at 18.0 g kg −1 Val diet. These resulted is in supportive with the study in hybrid grouper 44 fed varied levels of Val diet. The current results suggest that Val probably acts on the growth performance of fish by modulating the transcriptional regulation of its downstream effectors 4E-BP and S6K1. However, whether Val affects phosphorylation of the target proteins in TOR signaling pathway needs further exploration.
Our findings suggest that diets containing 18.19 g kg −1 Val in the diet would be useful for the improvement of growth performance, hemato-biochemical parameters and enhanced non-specific immune response, antioxidant status and digestive enzymatic activities in rainbow trout fingerlings. Besides, it has also been observed that optimal dietary Val also up-regulate an expression of TOR signaling pathway through its downstream effector i.e. 4E-BP, which can promote protein synthesis. Based on the quadratic regression analysis for growth parameters, the requirement of Val for rainbow trout was estimated to be 18.19 g kg −1 dietary Val, corresponding to

Materials and methods
Experimental diets. Six isonitrogenous (450 g kg −1 ) and isoenergetic (20.90 kJ 100 g −1 , gross energy (GE)) diets having a elevated content of Val 10.5, 13.0, 15.5, 18.0, 20.5, and 23.0 g kg −1 were supplied in six diet containing casein (fat-free), gelatin and L-crystalline amino acid premix ( Table 5). The level of protein content in all the diets was fixed at 450 g kg −1 , which was estimated optimum for maximum growth in our previous study 45 . L-crystalline amino acids in all the diets were used to adjust the amino acid content of the diets similar to that of 40% whole egg protein, excluding Val. The quantity of L-Val in all the diet was increased with the increment of 2.5 g kg −1 diet. The ratio of casein and gelatin was selected in all the diets on the basis of criteria that provide the lowest quantity of Val. In each incremental level, Val was ameliorated with the replacement of non-essential amino acids i.e. aspartic acid, serine and glycine, so as to maintain the uniform nitrogen level in all the diets. The doses of Val in all the diets were designed on the basis of previous studies on Val requirement of fish 119 .
The procedure for preparation of experimental diets in this feeding experiment was same as adopted in earlier experiment 80 . The premixes of vitamin and mineral used in the study were prepared as per Halver 120 . Feed pellets were made with the help of pelletizer connected with a 2 mm die and were then dried in oven at 40 °C in order to decrease the moisture less than 100 g kg −1 . Finally, the dry feed pellets were crushed, sealed in air tight bags at 4 °C until used.

Experimental design and feeding trial.
Fingerling rainbow trout of equal size and good health from the same lot were obtained from local government hatchery unit to experimental station at University of Kashmir. The fish were first mild treated with potassium permanganate (KMnO 4 ) to rule out any contamination, and were stocked under continuous flow through system for a fortnight by feeding a combination of practical diet in pellet form. Required number of fish was taken from the lot and was further acclimated to synthetic diet for 2 weeks 120 .
After the period of acclimatization, 360 fingerlings (average initial body weight: 1.66 ± 0.02 g/fish; average initial length: 5.25 ± 0.34 cm/fish) were then randomly distributed in 70 L circular tanks (water volume 60 L) connected with water flow through system (2-2.5 Lmin −1 ) at the rate of 20 fish per tank for each diets in triplicates. The diets were fed at the rate of 5% BW day −1 on dry to wet-weight basis to each group 80 . The experimental period was lengthening up to 8 weeks and diets were offered at 09:00 and 17:00 h. Initial and weekly body weights were recorded on a top loading balance (Sartorius AG Germany, CPA224S). Troughs were siphoned off to remove fecal matters before feeding on daily basis. Accumulation of the diet at the bottom of the trough was avoided. Uneaten food, if any was siphoned off immediately, dried in a hot air oven and reweighed to measure the amount of food consumed. On the day of weekly measurements, fish were not offered any feed. Troughs were scrubbed and disinfected thoroughly with water and KMnO 4 solution on the day they were batch weighed. Mortality, if any, was recorded.
Water quality analysis. During the entire feeding trial, various physico-chemical parameters were estimated on alternate day basis. The water samples were taken early in the morning before routine feeding. Water temperature (12.7-16.2 °C) was monitored by using a mercury thermometer, while other parameters like dissolved oxygen (7.1-8.2 mg L −1 ), free carbon dioxide (6.1-15.2 mg L −1 ), and total alkalinity (70-83 mg L −1 ) were analyzed by standard methods of APHA 121 . The pH (6.9-7.5) was recorded with the help of digital pH meter (pH ep-HI 98,107, USA).
Chemical analysis. At the beginning of the feeding trial, 30 fish specimens were taken up for initial whole-body proximate composition. After the completion of 8-week feeding trial, final weight of each tank was recorded. Eight fish were taken from each replicate groups and three sub-samples of each replicate (n = 3 × 3) were analyzed for final whole-body composition. Erythrocyte sedimentation rate (ESR). ESR was measured by using Wintrobe tube method 126 . The anticoagulated blood was filled in a Wintrobe tube up to the zero mark on top and kept undisturbed in vertical position in a rack. This allows the sedimentation of erythrocytes. After one hour level of fall of the column of sediment was noted as ESR and expressed in mm per hour.

Erythocyte indices (MCH, MCHC and MCV). The erythrocyte indices including MCH, MCHC and
MCV were calculated using standard formulae 127 . After blood collection, the liver was removed from the same sample and weighed subsequently nearby 0.1 mg (Sartorius AG Germany CPA224S) for HSI analysis through following formula: HSI (%) = (Liver weight / Total body weight) × 100 (Rajaguru 128 ).
Plasma analysis and non-specific immune response. Before the start of the experiment (n = 10) and after the completion of feeding trial, pooled blood (n = 2 × 3) samples were collected. Clotted blood in eppendrof tubes was centrifuged for 10 min using a high-speed centrifuge (REMI-12C) 4100 × g for 10 min at 4 °C. The separated plasma was then analyzed for plasma parameters and non-specific immune responses such as ALP, ALT, AST, glucose, urea, uric acid, total protein, albumin, globulin, cholesterol and triglycerides by using veterinary biochemistry analyzer (Vetscan VS2 Abaxis, USA).
Intestinal enzyme activities. After dissection, intestine samples (n = 2 × 3) were collected immediately and homogenized in a 10 volumes of saline solution and then centrifuged at 6000 × g for 20 min at 4 °C to obtain a supernatant and was then freezed at -20 °C for further examination. Trypsin and chymotrypsin activities were analyzed as per Hummel 129 , while for assaying the activities of amylase and lipase, the technique given by Furné et al. 130 was employed. GOT and GPT activities were assayed by the method given by 131,132 .
Intestinal antioxidant status. The activities of SOD, CAT, GPx, content of GR and MDA were analyzed with the help of kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and were assayed as per the protocol described by Zhang et al. 133 .
Expression of TOR and 4E-BP genes by Real-Time PCR. For establishing the appropriate Val level, TOR signaling pathway mRNA gene expression was analyzed by assessing through real-time 52 . The extraction of total RNA from fish muscle was carried out by Trizol method (Thermo Fisher Scientific, Darmstadt, Germany) as per manufacturer´s guidelines followed by the quantification and purification of total RNA by spectrophotometer (GENESYS 10S UV-VIS Thermo Scientific). Then, 2 µl total RNA was taken to synthesize complementary DNA (cDNA) by use of cDNA synthesis kit (iScript™cDNA Synthesis Kit Biorad, Hercules, CA). The identified genes (TOR, 4E-BP and β-actin) were synthesized by using specific primers (Table 6). PCR confirmation of identified genes was done by running 2% agarose gel. The real-time PCR was employed for above genes (LightCycler 480 Roche, USA). PCR amplication was carried out for these genes with the help of Chromo 4TM www.nature.com/scientificreports/ fluorescence detector (Bio-Rad, Hercules, CA, USA) under different thermo cycling conditions. Melting curve analysis was performed by running a gradient from 95 to 50 0 C in order to confirm the presence of single PCR products. The 2 −∆∆ Ct method was carried out to find out the TOR and 4E-BP signaling gene expression comparative to those for β-actin as per the method given by 134 .
Statistical analysis. The data obtained with respect to various Val fed diets in the form of LWG%, FCR, PER, SGR% and body composition parameters were analyzed by one-way analysis of variance (ANOVA) 135,136 .
To predict the significant differences (P ˂ 0.05) among the groups, polynomial contrast method i.e. cubic regression 137 was applied to analyze the growth data. Second-degree polynomial regression (Y = a + bx + cx 2 ) analysis was also employed in growth data to determine the appropriate breaking points in response to dietary Val levels 137 . Statistical analyses were made with the help of origin software (version 8.5.1; San Clemente, CA).
Ethical statement. During the present research work, all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All the protocols used have been approved by the Institutional Animal Ethics Committee ARRIVE guidelines. The present study has been carried out in accordance with ARRIVE guidelines.

Data availability
The data used in this is including within the manuscript. www.nature.com/scientificreports/