Valine belongs to the group of branched-chain amino acids, has an important structural role and is primarily deposited as body protein. The present study evaluated the effects of valine in diets of juvenile Nile tilapia. A total of 216 juveniles with weight of 21.40 ± 0.42 g and length of 10.07 ± 1.00 cm were distributed into 18 aquarium in a completely randomized design with six treatments and three replicates. Six diets containing 24.30% digestible protein, 3,100 kcal/kg digestible energy, and increasing valine levels (0.54; 0.63; 0.72; 0.81; 0.90; 0.99%) were prepared. The following factors were evaluated: performance, chemical composition, blood hematological and biochemical, and skeletal muscle development. Differences were observed in productive performance between treatments for weight gain, daily weight gain and apparent feed conversion, where inclusion levels from 0.81% to 0.99% provide better performances. According to the equation of quadratic regression, the inclusion of 0.86% of valine provided greater weight gain. Regarding the hematological and biochemical parameters, there were differences among the treatments for hemoglobin, triglycerides, and cholesterol. Not difference was observed for the chemical composition and muscle fiber growth. It is recommended the inclusion of 0.86% of valine in the diet of juveniles of Nile tilapia because it provides greater weight gain.
Additional key words:animal health;aquaculture;branched-chain amino acids;essential amino acid;fish nutrition;hematology;histology.Araucária Foundation / Fundação Araucária; Brazilian Federal Agency; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (notice 12/2013); National Council for Scientific and Technological Development / Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (process 457.186/2014-3).
Author's contributions:
Conceived and designed the experiments: RBR, DHN, FB and WRB. Coordinating the research project and supervising the work: FB and WRB. Performed the experiments: RBR, MZH and IWAM. Analyzed the data: RBR, DHN and FB. Contributed reagents/materials/analysis tools: RBR, DHN, FB and WRB. All authors wrote and approved the final manuscript.
Citation
Rodrigues, R. B.; Hassemer, M. Z.; Melo, I. W. A.; Neu, D. H.; Bittencourt, F.; Boscolo, W. R. (2019). Valine in diets for juvenile Nile tilapia (
Oreochromis niloticus
): growth performance, chemical composition, blood parameters and skeletal muscle development. Spanish Journal of Agricultural Research, Volume 17, Issue 2, e0602.
https://doi.org/10.5424/sjar/2019172-13953
Competing interests:
The authors have declared that no competing interests exist.
Introduction
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 methodsEthical 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 ˚C), 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.
Ingredients of the experimental diets.
Chemical composition of the experimental diets.
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 ˚C) 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 ˚C for 72 h, followed by drying at 105 ˚C 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 ˚C 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.
Results
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).
Growth performance of juvenile Nile tilapia fed diets with increasing levels of valine (Means values with their
standard deviation).
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).
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).
Chemical composition of juvenile Nile tilapia fed diets with increasing levels of valine (Means
values with their standard deviation).
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).
Hematological variables of juvenile Nile tilapia fed diets with increasing levels of valine (means values with
their standard deviation).
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).
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).
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).
Discussion
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.
Acknowledgements
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.
ReferencesAbidiSF
,
KhanMA
,
2007
.
Dietary leucine requirement of fingerling Indian major carp, Labeo rohita (Hamilton).38
:
478
-
486
.
https://doi.org/10.1111/j.1365-2109.2007.01687.xAlmeidaFLA
,
CarvalhoRF
,
PinhalD
,
PadovaniCR
,
MartinsC
,
Dal Pai-SilvaM
,
2008
.
Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases.39
:
1306
-
1311
.
https://doi.org/10.1016/j.micron.2008.02.011AOAC2005Official methods of analysis of association of official analytical chemists.CalderPC
,
2006
.
Branched-chain amino acids and immunity.136
:
288
-
293
.
https://doi.org/10.1093/jn/136.1.288SChenCY
,
WoosterGA
,
GetchellRG
,
BowserPR
,
TimmonsMB
,
2003
.
Blood chemistry of healthy, nephrocalcinosis affected and ozone treated tilapia in a recirculation system, with application of discriminant analysis.21889102https://doi.org/10.1016/S0044-8486(02)00499-4CollierHB
,
1944
.
The standardization of blood haemoglobin determinations.50
:
550
-
552
.
DeriggiGF
,
InoueLAKA
,
MoraesG
,
2006
.
Stress responses to handling in Nile tilapia (Oreochromis niloticus Linnaeus): assessment of eugenol as an alternative anesthetic.28
:
269
-
274
.
DongM
,
FengL
,
KuangSY
,
LiuY
,
JiangJ
,
HuK
,
JiangWD
,
LiSH
,
TangL
,
ZhouXQ
,
2012
.
Growth, body composition, intestinal enzyme activities and microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary valine.19
:
1
-
14
.
https://doi.org/10.1111/j.1365-2095.2011.00926.xFengL
,
LuoJB
,
JiangWD
,
LiuY
,
WuP
,
JiangJ
,
KuangSY
,
TangL
,
ZhangYA
,
ZhouXQ
,
2015
.
Changes in barrier health status of the gill for grass carp (Ctenopharyngodon idella) during valine deficiency: Regulation of tight junction protein transcript, antioxidant status and apoptosis-related gene expression.45
:
239
-
249
.
https://doi.org/10.1016/j.fsi.2015.04.023FernstromJD
,
2005
.
4th amino acid assessment workshop: Branched chain amino acids and brain function.135
:
1539
-
1546
.
https://doi.org/10.1093/jn/135.6.1539SFuruyaWM
,
2010
.
Tabelas brasileiras para a nutrição de tilápias101GoldenfarbPB
,
BowyerFP
,
HallE
,
BrosiusE
,
1971
.
Reproducibility in the hematology laboratory: the microhematocrit determinations.56
:
35
-
39
.
https://doi.org/10.1093/ajcp/56.1.35HanY
,
HanR
,
KoshioS
,
IshikawaM
,
YokoyamaS
,
GaoJ
,
2014
.
Interactive effects of dietary valine and leucine on two sizes of Japanese flounder Paralichthys olivaceus.432
:
130
-
138
.
https://doi.org/10.1016/j.aquaculture.2014.05.004HarperAE
,
MillerRH
,
BlockKP
,
1984
.
Branched-chain amino acid metabolism.4
:
409
-
454
.
https://doi.org/10.1146/annurev.nu.04.070184.002205HrubecTC
,
CardinaleJL
,
SmithSA
,
2000
.
Haematology and plasma chemistry reference intervals for cultured tilapia (Oreochromis hybrid).29
:
7
-
12
.
https://doi.org/10.1111/j.1939-165X.2000.tb00389.xHughesSG
,
RumseyLJ
,
NesheimMC
,
1984
.
Effects of dietary excesses of branched-chain amino acids on the metabolism and tissue composition of lake trout (Salvelinus namaycush).78
:
413
-
418
.
https://doi.org/10.1016/0300-9629(84)90569-3KoumansJTM
,
AksterHA
,
1995
.
Myogenic cells in development and growth of fish.110
:
3
-
20
.
https://doi.org/10.1016/0300-9629(94)00150-RLiP
,
MaiKS
,
TrushenskiJ
,
WuGY
,
2009
.
New developments in fish amino acid nutrition: towards functional and environmentally oriented aquafeeds.37
:
43
-
53
.
https://doi.org/10.1007/s00726-008-0171-1NRC2011Nutrient requirements of fish and shrimp.390RahimnejadS
,
LeeKJ
,
2013
.
Dietary valine requirement of juvenile red sea bream Pagrus major. Aquaculture 416-417
:
212
-
218
.
https://doi.org/10.1016/j.aquaculture.2013.09.026Ranzani-PaivaMJT
,
Benites de PáduaS
,
Tavares-DiasM
,
EgamiMI
,
2013
.
Métodos para análise hematológica em peixes.140https://doi.org/10.7476/9788576286530RosenfeldG
,
1947
.
Corante pancrômico para hematologia e citologia clínica. Nova combinação dos componentes do May-Grünwald e do Giemsa num só corante de emprego rápido.20
:
329
-
334
.
SantiagoCB
,
LovellRT
,
1988
.
Amino acid requirement for growth of Nile tilapia.118
:
1540
-
1546
.
https://doi.org/10.1093/jn/118.12.1540ShimomuraY
,
YamamotoY
,
BajottoGS
,
2006
.
Nutraceutical effects of branched-chain amino acids on skeletal muscle.36529S532Shttps://doi.org/10.1093/jn/136.2.529SSuryawanA
,
OrellanaRA
,
FiorottoML
,
DavisTA
,
2011
.
Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs.89
:
2004
-
2016
.
https://doi.org/10.2527/jas.2010-3400Tavares-DiasM
,
MoraesFR
,
2004
.
Hematologia de peixes teleósteos.144ValenteLMP
,
RochaE
,
GomesEFS
,
SilvaMW
,
OliveiraMH
,
MonteiroRAF
,
FauconneauB
,
1999
.
Growth and dynamics of white and red muscle fibres in fast- and slow-growing strains of rainbow trout.55
:
675
-
691
.
https://doi.org/10.1111/j.1095-8649.1999.tb00710.xWilsonRP
,
1985
.
Amino acid and protein requirements of fish.116In: Nutrition and feeding fish; Cowey CB, Mackie AM, Bell JG (eds.).WilsonRP
,
2002
.
Amino acids and proteins.143179In: Fish Nutrition; Halver JE, Hardy RW (eds.).https://doi.org/10.1016/B978-012319652-1/50004-5WuG
,
2009
.
Amino acids: metabolism, functions, and nutrition.37
:
1
-
17
.
https://doi.org/10.1007/s00726-009-0269-0