The Nile tilapia ( Oreochromis niloticus ) is a teleost fish belonging to the family Cichlidae. It is one of the most cultivated species, mainly in intensive fish far­­­­­­­­­­ming. In this culture system, fish require com­plete di­ets, sin­­ce the natural food available in the a­quatic envi­ron­­ment does not meet their nutritional re­quirements.

Fish feed represents the highest production cost in commercial aquaculture. According to Wilson (2002) , protein is the main and most costly component in formulated diets for aquatic animals. Thus, it is essen­tial to maximize the efficiency of dietary protein use by the fish and present the amino acids in balanced quantities ( Wilson, 1985 ).

Valine is described as hydrophobic and belongs to the group of branched-chain amino acids (BCAA) to­gether with leucine and isoleucine ( NRC, 2011 ). This group contains approximately 35% of the amino acids e­ssential for muscle protein and 40% of the amino acids required for mammals ( Harper et al ., 1984 ), consti­tu­ting 18-20% of the total amino acids present in plant and animal proteins ( Li et al ., 2009 ). These ami­no acids are involved in a series of metabolic reactions as in the neurotransmitter synthesis and energy production ( Fernstrom, 2005 ), which are essential for the synthe­sis of glutamine and alanine ( Wu, 2009 ), muscle syn­thesis ( Shimomura et al ., 2006 ), maintenance of im­mune parameters ( Calder, 2006 ), and recently Feng et al . (2015) found that valine has a series of benefits the integrity maintainer of gills, in the expression of cytokines among other enzymes and antioxidants.

Santiago & Lovell (1988) determined a requirement of 0.78% valine for Nile tilapia; however, they used pu­ri­­­fied ration, which is not commonly used in a tilapia cul­ture system and evaluated the effects of valine on mus­cle growth and blood parameters that provide im­portant information for the assessment of fish health status.

Therefore, the present study evaluated the effects of valine in diets for juvenile Nile tilapia, at perfor­mance, body composition, blood hematological and bio­chemical profiles, and muscle fiber development.

There were significant differences ( p <0.05) for the variables weight gain, daily weight gain and apparent feed conversion. Not significant differences were observed ( p >0.05) for the parameters condition factor, specific growth rate, protein efficiency ratio, survival, protein retention efficiency, visceral fat, hepatosomatic index, and fish uniformity (Table 3).

Weight gain and daily weight gain were higher in the fish fed diets containing 0.81% valine; however, did not significantly differ from the fish that received the 0.90% and 0.99% valine. Fish fed diets containing 0.54% valine demonstrated the worst results; however, they did not differ significantly from the fish fed diets containing 0.63% and 0.72% valine. There was better apparent feed conversion in the fish that received 0.54%, 0.81%, 0.90% and 0.99% valine in the diet; however, the results did not differ from the treatment with 0.63% valine, which in turn did not differ from the treatment with 0.72% valine.

The quadratic regression equation applied to the weight gain data showed a good level of valine 0.86% inclusion ( R ² = 0.55) (Fig. 1).

There were no significant differences ( p >0.05) for the chemical composition variables moisture content, crude protein, ether extract, or mineral matter (Table 4).

The hemoglobin showed a difference ( p <0.05), with the highest value of 12.3 g/dL observed in the treat­ment containing 0.54% valine in the diet; however, did not differ from the treatments containing 0.81%, 0.99% and 0.99% valine. The lowest hemoglobin (9.69 g/dL) was observed in the fish fed 0.63% vali­ne (Table 5).

Fish fed 0.81 and 0.99% valine in the diet exhibi­ted higher rates of triglyceride in the blood (171.71 and 170.25 g/dL, respectively), however, they did not differ from the treatments containing 0.63% and 0.90% valine. Fish fed with diets containing 0.54% and 0.72% valine had the lowest triglyceride levels. Tila­pia fed diets containing 0.81% and 0.90% valine showed higher levels of cholesterol in the blood, differing from the other treatments. Tilapia fed diets containing 0.81% and 0.90% valine showed higher levels of cholesterol in the blood, differing from the other treatments (Fig. 2).

There were no significant differences ( p >0.05) for muscle fiber distribution in the diameter classes between below 20 µm, 20-50 µm and above 50 µm among the different treatments (Fig. 3).

The results of growth performance of Nile tilapia juveniles were influenced by the use of valine in the diet. This amino acid deserves greater attention becau­se it is involved in various metabolic processes and is considered essential for adequate animal producti­ve performance. The only study in the scientific lite­rature for Nile tilapia was conducted by Santiago & Lovell (1988) , who used younger animals, purifi­ed ra­tion, breeding conditions and variables evaluated in the study different from our work. The authors found vali­ne requirement 0.78% for Nile tilapia lar­vae through the supply of purified feed. The nutritional requirement of fish is affected by several factors, including the physiological state of the animals, the en­vironmental conditions, and the animal size and age ( Wilson, 2002 ).

The growth performance results demonstrated that an increased valine supplement level provided better performance, with weight gain, daily weight gain and apparent feed conversion showing better results in the fish fed a diet containing 0.81-0.99% valine. The valine levels in the diet did not significantly affect the other parameters of performance. The regression e­quation applied to the gain data by weight showed that the level of inclusion of valine that gave the best growth to the animals was 0.86%. It was observed that the re­­quirement of valine obtained by Santiago & Lovell (1988) of 0.78% for tilapia larvae fed with purified ration was lower than that found in our current stu­dy of 0.86% for juveniles of tilapia fed with practical diets.

We believe that the development phase may have influenced this observed difference since older and larger fish have a greater contribution of growth due to muscular hypertrophy in relation to younger fish that depend more on the hyperplasia process. This may explain the higher (0.86%) requirement of the animals in our study since valine acts in conjunction with other branched-chain amino acids in muscle deposition and growth. In addition, in a purified diet the nutrients are readily available for absorption by the body of the fish, unlike the practical diets, where the body needs to digest the ingredients to later be able to absorb the nutrients. Thus, the higher requirement observed in the current study is expected because the nutrients were not readily available for absorption as in the purified diet, in which case it is expected that a greater % inclusion of the nutrient will be necessary, due to the loss of nutrients during the digestion process.

Although it is not clear how different levels of dietary valine influenced the weight gain of fish, some studies indicate possible mechanisms of action of this amino acid in fish growth, such as increased secretion of digestive enzymes and also in balancing with other amino acids of branched chain, since the excess or deficiency of one of these amino acids can hinder the use of others. Dong et al . (2012) suggest that increased valine in the diet increases the activity of digestive enzymes such as trypsin, chymotrypsin, lipase and amylase, thus providing better digestion and, consequently, better utilization of nutrients by the fish organism. Some studies indicate that the balance between the three branched-chain amino acids is es­sential because antagonism may occur between them, that is, the excess or deficiency of one of the amino a­­­cids may impair the use of another. This is because the three amino acids compete for the same metabo­lic path­way of absorption and active transport ( Hughes et al ., 1984 ).

In contrast to the results in the present study, the protein efficiency ratio and protein retention efficiency are thought to be affected by different valine levels in the diet because branched-chain amino acids (leucine, isoleucine, and valine) are essentially anabolic and exhibit translation regulation and the onset of protein synthesis in various tissues as their main function ( Shimomura et al ., 2006 ).

The body composition of the fish remained unaf­fected by the different valine levels in the diets, corroborated with Han et al . (2014) in a study with Paralichthys olivaceus . In contrast, Dong et al . (2012) found differences in the body composition of Cyprinus carpio var. Jian, Hughes et al . (1984) found an effect in Salvelinus namaycush , Rahimnejad & Lee (2013) found an effect in Pagrus major . In the current study, the diets were most likely prepared for isoproteic and iso­caloric maintenance to avoid the occurrence of chan­ges in the fish chemical composition.

The hematological analysis of the fish is very important because it is indicative of the fish's state of health and physiological normality. Little information is available in the scientific literature regarding the effect of valine on the hematological parameters of fish ( Rahimnejad & Lee, 2013 ). In the present study, only the hemoglobin differed significantly among the treatments, with the fish that received feed containing 0.54% valine exhibiting the highest hemoglobin. Abi­­di & Khan (2007) reported that branched-chain amino acids were important for hemoglobin production, glu­cose regulation in the blood, and resistance to stress. It is difficult to state the influence of valine on hemoglo­bin levels since this amino acid apparently does not act di­rectly on iron metabolism. So, differences obtained could be related to intrinsic variations among animals.

The white blood cells of the juvenile Nile tilapia remained unaffected by the different valine levels in the diet. The leukocyte values were within the range considered normal for Nile tilapia ( Hrubec et al ., 2000 ). Similarly, the number of total thrombocytes remained within the range considered adequate for healthy fish ( Tavares-Dias & Moraes, 2004 ).

The triglyceride and cholesterol levels differed in the tilapia fed increasing valine levels in the diet; however, these values were within the range considered normal for the species ( Hrubec et al ., 2000 ). The total plasma protein and plasma glucose did not differ a­mong the treatments. The fish fed diets containing 0.81% and 0.99% valine exhibited the highest triglyceride levels. No information in the literature that supports a possible relationship between the weights of the ani­mals with triglyceride levels has been described for fish diets with low lipid levels. Plasma proteins include albumins, globulins, and fibrinogens; a reduction in their levels may indicate liver alterations or that the feed is unbalanced. Thus, the levels of these proteins can be used to evaluate the health conditions of the fish. The total plasma protein concentration in this study ranged from 2.96 to 3.30 g/dL; these values were considered normal for healthy tilapia according to Chen et al . (2003) . In contrast, Rahimnejad & Lee (2013) found that increasing valine (1.22 to 2.04%) in the diet increased the plasma protein level and total cholesterol in the blood of Pagrus major and explained this result as the hypercholesterolemic effect conferred by this amino acid. This last claim corroborated the results in the present study because the feed containing 0.81% and 0.90% valine resulted in high blood cholesterol values.

Increasing levels of valine have no effect on ske­le­tal muscle growth in juvenile tilapia. Muscle fi­bers with diameters smaller than 20 µm suggest the presence of hyperplasia, and muscle fibers with dia­meters larger than 50 µm indicate the presence of hy­pertrophy ( Valente et al ., 1999 ). In the present study, muscle growth due to hypertrophy and hyper­plasia was observed in fish from all treatment groups. The fiber diameter did not differ among the treatments, indicating that the different valine levels in the diet provided similar contributions in muscle development.

Muscle growth can be affected by several factors, including nutrition ( Koumans & Akster, 1995 ). Although valine is a branched-chain amino acid with important physiological functions, muscle tissue maintenance and the growth and regulation of protein synthesis ( Suryawan et al ., 2011 ), the levels included in the diets were sufficient for normal muscle growth. More studies on nutrition related to muscle growth are necessary (especially those linked to amino acids) because they uniquely contribute to the development of myofibrils and are key elements for muscle deposition in fish.

Based on the information obtained in the present study, we concluded that diets containing 0.81% vali­­ne (2.90% of dietary protein) to 0.99% (3.55% of die­tary protein) provided better performance results for juvenile Nile tilapia. It is recommended the inclusi­on of 0.86% of valine in the diet of juveniles of Nile tila­­pia because it provides greater weight gain.

The authors thank the GEMAq (Study Group on Management in Aquaculture) for the technical support for research execution and the funding agencies that finance the project.