IntroductionTop

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.

Material and methodsTop

Ethical considerations

The present study was approved by the Ethics Committee for Animal Experiments of UNIOESTE - Universidade Estadual do Oeste do Paraná (Brazil) under protocol no. 11/2015 in July, 2015.

Fish and experimental conditions

The fish were acquired in a commercial fish, loca­ted in the city of Toledo-PR, Brazil. A total of 216 juvenile Nile tilapia with a mean weight of 21.40 ± 0.42 g and total length of 10.07 ± 1.00 cm were used. The fish were distributed in 18,500-L glass fi­ber tanks with an experimental unit comprising one tank containing twelve fish. Each tank was coupled to a water recirculation system (4 L of water/min per tank) with a central biological filter, constant aeration, and controlled temperature.

The water quality was controlled by monitoring the tem­perature (27.5 ± 0.08 ºC), dissolved oxygen (5.85 ± 0.15 mg/L), pH (7.05 ± 0.22), and conducti­vity (110.05 ± 1.85 μS/cm) using YSI Professional Plus Multi­pa­rameter Water Quality Meter (YSI, Pro Plus, Yellow Springs, OH, USA) apparatus. Daily siphoning was performed to remove the leftover feed and fish feces.

Experimental diets and feeding management

Six isoproteic (24.30% digestible protein) and isocaloric (3100.00 kcal digestible energy) rations we­re prepared (Tables 1 and 2) that contained increa­sing valine levels (0.54; 0.63; 0.72; 0.81; 0.90; 0.99%) following the recommendation to meet the nutritional requirements of juvenile Nile tilapia (Furuya, 2010). The ingredients were ground in hammer mill (Vieira, MCS 280, Tatuí-SP, Brazil) with a 0.3 mm sieve and the feed was processed by extrusion (Extec, Ex-Micro, Ribeirão Preto, SP, Brazil) with a 2 mm sieve. The fish were fed four times daily (8:00 am, 11:00 am, 2:00 pm, and 5:00 pm) until apparent satiation, over 15 min, for 77 days.

Performance

After the 77-day experimental period, the fish we­re fasted for 24 h to empty the gastrointestinal tract and then stunned in 80.0 mg/L eugenol (Deriggi et al., 2006) to record the individual weight (g) and total length (cm) measurements. Three fish from each tank were euthanized in 300 mg/L eugenol and then pla­ced on ice to remove the visceral fat and liver. These sa­­me animals were freezer (-80 ºC) prior to the perfor­man­ce of the following analyses.

The following productive performance data were evaluated: weight gain (g) (final body weight – initial body weight); daily weight gain (g) (weight gain/day of the experiment); apparent feed conversion (feed consumed/weight gain); visceral fat (%) [visceral fat weight (g) * 100/final body weight (g)]; hepatosoma­­tic index (%) [liver weight (g) * 100 /final body weight (g)]; condition factor (%) [(body weight (g)/total length (cm)³) *100]; specific growth rate (%/day) [(ln (final weight) – ln (initial weight))/time) * 100]; prote­in efficiency ratio (weight gain/protein consumed); protein retention efficiency (%) ((final carcass protein content * final biomass) – (initial carcass protein con­tent * initial biomass))/protein consumed); survival (%) [(final number of fish/initial number of fish) * 100]; and fish uniformity (%) (quantity of fish with body weight within the mean ± SD/(total number of fish * 100).

Feed and fish chemical composition

Three fish from each experimental unit (9 fish per treatment) were used to determine the body composi­­tion. The determination of the fish chemical composi­­­tion followed the method suggested by the AOAC (2005) for the analysis of the moisture content (pre-drying at 55 ºC for 72 h, followed by drying at 105 ºC for 8 h) (Method 950.46), crude protein (Kjeldahl method; MA-036 model, Piracicaba, SP, Brazil) (Method 981.10), ether extraction (Soxhlet extractor with ether as the solvent; TE-0.44 model, Piracica­ba, SP, Brazil), and mineral matter (calcination of the sam­ples at 550 ºC for 6 h; 2000B model, Belo Hori­zon­te, MG, Brazil) (Method 920.153). All analyses we­re performed in triplicate.

The determination of the centesimal composition of the experimental diets followed the method suggested by AOAC (2005): Moisture (method 950.46); Cru­de protein (method 981.10); Ash (method 920.153); Ethereal extract (method 920.39); Crude fiber (method 962.09). The composition analyzes of the experimen­tal diets were performed in triplicates.

Hematological and biochemical analyses

For blood collection, three fish were randomly cap­tured from each experimental unit to sample a 2.0 mL aliquot by caudal puncture using a syringe contai­ning 10% EDTA. The total erythrocyte count was perfor­med in whole blood using a Neubauer chamber. The hemoglobin was evaluated using the method descri­bed by Collier (1944), and the hematocrit percentage was evaluated following the method described by Goldenfarb et al. (1971).

Blood smears were prepared on frosted glass slides, air-dried, and stained using the method of Rosenfeld (1947). The reading was performed under an optical microscope with 100x magnification using immer­sion oil. Total leukocytes and thrombocytes were counted using the indirect method (Ranzani-Paiva et al., 2013). A total of 100 leukocytes were counted un­der an ordinary light microscope with 100x immer­­­si­­­on increase to determine the ratio of different leuko­­cy­­­­tes: lymphocytes, neutrophils and monocytes. The num­­ber of each element is expressed as a percentage.

Biochemical analyses of the total plasma proteins (g/dL), triglycerides (mg/dL), total cholesterol (mg/ dL), and plasma glucose (mg/dL) were perfor­­med. The analyses were performed using specific kits (Gold Ana­lisa Diagnóstica, Belo Horizonte-MG, Brazil) and processed according to the manufacturer's instructi­ons. The results were read in a spectrophotometer.

Muscle fiber development

A white muscle sample was removed from the left side of each fish (12 fish per treatment) above the late­­ral line using a scalpel. These samples were placed in 10% buffered formalin for 24 h and then preserved in 70% alcohol. The samples were subjected to the para­­ffin embedding process and then cut from the para­­f­fin blocks using a microto­me (MICROM, Internatio­nal GmbH 69190, Walldorf, Germany). Cross-sections (5 μm) were subjected to hematoxylin-eosin (HE) stai­ning. An image analysis system was used for morpho­me­­try (Image-Pro Plus® v. 4.5.0.29). The smallest dia­me­ter (μm) of 200 muscle fibers was determined per fish; then, the fish were grouped into diameter classes (<20 μm, 20-50 μm, and >50 μm) to evaluate the contribu­tion of hyperplasia and hypertrophy to mus­cle growth (Almeida et al., 2008).

Experimental design and statistical analysis

The experiment was a completely randomized design with six treatments and three replicates. The data in the tables are shown as mean ± SD. The data were submitted to the Kolmogorov-Smirnov normality and the Levene’s homogeneity tests and subjected to analysis of variance (ANOVA). When significant differences were observed, Duncan’s mean comparison test at a 5% significance level was applied. Quadratic regression analysis was applied on weight gain data to obtain a better inclusion level of valine. The analyses were performed using the Statistical Analysis System 9.4 and GraphPad Prism 7.0.

ResultsTop

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 (R² = 0.55) (Fig. 1).

Figure 1. Weight gain of Nile tilapia juveniles fed diets containing increasing levels of valine.

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).

Figure 2. Blood biochemical values found in juveniles of Nile tilapia fed diets containing increasing levels of valine. A: Triglycerides; B: Total cholesterol; C: Total plasma protein; D: Plasma glucose. Different letters indicate significant differences by the Duncan’s test (p<0.05); n = 9 fish per treatment.

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).

Figure 3. Muscle fiber distribution into three diameter classes (<20 μm, 20-50 μm and >50 μm) in juvenile Nile tilapia fed diets with increasing levels of valine. Not significant by analysis of variance (p>0.05); n = 12 fish per treatment. Muscle fiber diameters (μm) were measured, using the Image-Pro Plus® v. 4.5.0.29 software. Stain: Hematoxylin and eosin (HE).

DiscussionTop

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.

AcknowledgementsTop

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.