Digestible methionine + cystine requirement for Nile tilapia from 550 to 700 g

This trial was conducted to determine the dietary digestible methionine + cystine requirement of Nile tilapia (550 to 700 g) based on the ideal protein concept. Six hundred fish were distributed in a completely randomized design with five treatments and four replicates, with 30 fish per experimental unit. The fish were fed diets containing approximately 262 g of digestible protein/kg, 3,040 kcal of digestible energy/kg and 7.90, 9.40, 10.90, 12.40 or 13.90 g of methionine + cystine/kg. The fish were hand-fed three times a day until apparent satiation for 30 days. No effects of dietary methionine + cystine on feed conversion ratio, daily protein deposition, whole body moisture, fillet moisture, crude protein, ether extract and ash, plasmatic HDL and LDL cholesterol were observed. Dietary methionine resulted in a linear increase in whole body protein and linear reduction in lipid deposition rate, hepatosomatic index, whole body ether extract and ash, plasmatic total cholesterol, plasmatic total lipids and plasmatic triglycerides. According to the Linear Response Plateau, the daily weight gain and fillet yield increased up to a level of 9.00 and 9.90 g methionine + cystine/kg of diet, respectively. The digestible methionine + cystine requirement of Nile tilapia is 9.00 g/kg for weight gain and 9.90 g/kg for fillet yield, corresponding to methionine + cystine:lysine ratios of 0.60 and 0.66, respectively.


Introduction
Tilapia is one of the most important freshwater farmraised fish and has had an increasing role in the global aquatic food trade.The Nile tilapia (Oreochromis niloticus) is an economically important freshwater fish in Brazil.
The dietary methionine requirements have been estimated for several species of fish, ranging from 1.80 to 4.00 g/kg of dietary protein (Wilson, 2002).Because of the interrelation between methionine and cystine, it is more appropriate to show their requirements as methionine plus cystine.The fingerlings of tilapia mossambica (Oreochromis mossambicus) require a diet with 10.10 g of methionine + cystine/kg (Jackson & Capper, 1982), whereas Nile tilapia require 9.00 g/kg and 10.00 g/kg according to the recommendations of the NRC (1993) and Furuya et al. (2004), respectively.Based on the ideal protein concept, the methionine + cystine:lysine ratio for Nile tilapia varies from 0.57 to 0.6 (Nguyen & Davis, 2009;Furuya et al., 2001).
There is no information about the dietary methionine + cystine requirement for Nile tilapia weighing more than 500 g considering growth, feed efficiency, body composition, economical analysis and fillet yield.Due to the high cost of the feed during the final phase, it is important to determine the methionine + cystine requirements to develop cost-effective diets considering not only the fish growth, but the fillet yield and the economic analysis.The present study was conducted to determine the dietary digestible methionine + cystine requirement of Nile tilapia from 550 to 700 g.

Material and Methods
The experiment was conducted in Rio do Corvo, Diamante do Norte, Paraná State, Brazil, from December 2009 to January 2010, during 30 days.Six hundred (563.40 ±10.50 g) of all-male population of Nile tilapia obtained by hormonal induction during the larval phase were distributed into 20 cages with a unit volume of 1 m3 (1.00 × 1.00 × 1.20 m high) in a completely randomized design with five treatments and four replicates.
Five diets were formulated to contain approximately 3,040 kcal of digestible energy/kg and 262 g of crude protein/kg and crystalline amino acids were added to keep the amino acid requirement for the Nile tilapia, except for methionine + cystine (NRC, 1993), and 7.90, 9.40, 10.90, 12.40 or 13.90 g of methionine + cystine/kg (Table 1).The values of digestible protein and amino acids, digestible energy and available phosphorus were obtained from Furuya et al. (2001b), Pezzato et al. (2002) and Guimarães et al. (2008a, b), in studies conducted with the Nile tilapia.
The diets were extruded at Universidade do Oeste do Paraná -UNIOESTE, Paraná, Brazil, using a single screw extruder which obtained granules of approximately 5.50 mm of diameter.All ingredients were previously ground in a hammer mill with a sieve of 0.80 mm of diameter.
The fish were hand-fed until apparent satiation three times a day (9h00, 13h00 and 17h00), seven days per week.Each diet was randomly assigned to triplicate cages.Fish were slowly hand-fed little by little until apparent satiation on the basis of visual observation of the fish feeding behavior.
During the experiment, the water quality was checked every week.The average values of temperature, dissolved oxygen and pH of the water cages were 27.50±1.20ºC, 5.70±0.12mg L -1 and 7.37±0.57,respectively.The data obtained are within the range recommended by Sipauba-Tavares (1994) for tropical freshwater fish.
At the beginning of the experimental period, 30 fish were homogenized, ground in an electric meat grinder and store frozen (-20 o C) of initial whole body composition.At the end of the experimental period, fish were feeddeprived for 24 hours prior to sampling, then anesthetized with clove oil at 300 mg/L (Vidal et al., 2008), counted and weighed to determine survival rate, final body weight, daily weight gain and feed conversion ratio.Eight fish from each cage were randomly collected for determination of the hepatosomatic index and fillet yield.Five fish were collected for determination of the whole body composition and three fish were collected for plasmatic lipid pattern.
Samples of whole fish body and fillets were ground in an electric meat grinder to obtain homogeneous samples.All samples were dried at 55 °C for 62 hours.The protein deposition ratio and lipid deposition ratio were determined using the methodology described by Fraga (2002).Blood samples were taken from the caudal vein of three fish per cage.The plasma was obtained by centrifugation at 1200 g for 8 minutes at room temperature.After centrifugation, the plasma was used for analysis of total cholesterol, total lipids, triglycerides, LDL and HDL cholesterol.The levels of plasma triglycerides and cholesterol were determined using colorimetric enzymatic method and photometric enzymatic method, respectively.The HDL and LDL were determined according to Friedewald et al. (1972).
The chemical analyses of diets, whole body and fish fillet samples were determined following the methodology described by Silva & Queiroz (2002).All amino acid analyses of the experimental diets were performed at Ajinomoto Animal Nutrition Laboratory (São Paulo, Brazil).
The statistical analysis was performed through program SAEG (Sistema para Análises Estatísticas e Genéticas, version 8.0).The quantitative dietary methionine + cystine requirement was determined with linear regression, quadratic or discontinuous Linear Response Plateau (LRP) analysis according to the best fit obtained for each variable, based on the significance of regression coefficients for the F test, the coefficient of determination, the sum of squared deviation and the phenomenon under study.

Results and Discussion
The levels of digestible methionine + cystine did not affect (P>0.05) the feed conversion ratio and protein deposition rate of fish, but they affected the daily lipid deposition and hepatosomatic index, which decreased linearly, and fillet yield, which increased linearly.The regression analysis of daily weight gain and fillet yield data using the Linear Response Plateau model indicated that daily weight gain and fillet yield increased up to 9.00 and 9.90 g of methionine + cystine/kg, respectively (Table 2).
The digestible methionine + cystine influenced (P>0.05)daily weight gain in the fish of the present study, close to the values obtained by Santiago & Lovell (1988) and Furuya et al. (2001a) for Nile tilapia, of 9.00 and 10.50 g methionine + cystine/kg, respectively.On the other hand, Jackson & Capper (1982) determined the requirement of 12.70 g of methionine + cystine/kg for tilapia mossambica (Oreochromis mossambicus) for maximum weight gain.Likewise, values higher than those found in this study were obtained by Burtle & Cai (1995) and Schwarz et al. (1998) for channel catfish (13.40 g/kg) and common carp (12.40 g/kg).
The proportion of methionine:cystine can also affect the weight gain of fish, as described by Nguyen & Davies (2009), who observed the best weight gain of Nile tilapia fed diets containing 0.50:0.50,0.60:0.40 or 0.70:0.30 of methionine:cystine ratios.In the present study, the proportion of methionine to cystine estimated for weight gain and fillet yield production were approximately 0.60:0.40 and 0.70:0.30,respectively.
The result obtained for the feed conversion ratio differs from those found by several authors, who determined the effects of dietary methionine + cystine on feed conversion for juvenile rainbow trout (Kim et al., 1992), channel catfish (Burtle & Cai, 1995), yellow perch (Twibell et al., 2000) and Nile tilapia (Furuya et al., 2004).Discrepancies among values also may be due to differences in basal diet composition, size or age, genetic differences, feeding regime and culture conditions of the fish.
There was no effect (P>0.05) of the dietary digestible methionine + cystine levels on the protein deposition rate.On the contrary, Burtle & Cai (1995) estimated the requirement of 9.90 g/kg of methionine + cystine (5.80 g/kg of methionine) in the diet for maximal utilization of the dietary protein for channel catfish, while Kim et al. (1992), determined the requirement of 7.80 g/kg for maximum utilization of the dietary protein by rainbow trout.The differences in the results compared with this study may be related to the levels of protein, energy and amino acids balance, since the previous studies did not consider the balance of energy:protein and essential amino acids:lysine ratios.
There was no effect (P>0.05) of the dietary digestible methionine + cystine levels on fillet composition.The levels of digestible methionine + cystine affected (P<0.05)fillet yield, which increased up to 9.90 g of methionine + cystine /kg.
The higher fillet yield may be related to a lower hepatosomatic index value with increasing levels of dietary methionine + cystine (Table 2), in addition to a higher protein and lower ether extract and ash depositions in the whole body (Table 3).
It is important to consider that the requirements of methionine + cystine also have to be assessed separately in fish diets, as well as other non-ruminants that require a minimum of dietary methionine, considering that methionine can not be obtained from cystine (trans-sulfidation).Thus, based on the results of this study, a minimum of 5.30 and 5.70 g/kg of methionine is required for weight gain and fillet yield, respectively, in diets for the Nile tilapia.In this study, the highest levels of protein in the whole body are in accordance with the results obtained by Yan et al. (2007).However, the authors did not observe reduction in the whole body fat.
The highest concentration of protein in the whole body, in addition to lower levels of fat, are probably related to the functions of methionine on the synthesis of body protein and lipid metabolism.Methionine is an essential amino acid for normal growth of fish, because it participates in the protein synthesis and other metabolic functions (Teshima et al., 2002;Alam et al., 2001).
The increasing levels of methionine + cystine did not affect (P>0.05) the serum HDL and LDL cholesterol levels, but resulted in a linear reduction (P<0.05) in the values of total cholesterol, total lipids and serum triglycerides (Table 4).
The reduction of the total cholesterol, total lipids and plasma triglycerides may be related to the different functions of methionine.Methionine affects the profile of plasma amino acids in many species of fish (Twibell et al., 2000;Mai et al., 2006) as well as the metabolism of sulfur amino acids (Espe et al., 2008).The S-adenosylmethionine, synthesized from methionine, originates compounds such as carnitine (Swenson & Reece, 1996) and choline (Swenson & Reece, 1996;Kasper et al., 2000).Choline also acts as a lipotropic factor, enhances the synthesis of lipoproteins and prevents fatty liver (Swenson & Reece, 1996), which may explain the hepatosomatic index reduction of fish fed adequate levels of methionine + cystine (Table 2).
The choline molecule has three methyl groups (-CH3), and reacts with acetyl coenzyme A, acting as a precursor of acetylcholine, a neurotransmitter (Zeisel, 2000) and phosphatidylcholine, which is a structural component of cell membranes acting also in the metabolism and transport of lipid-cholesterol (Bender, 2003).It also acts as a lipotropic factor, and improves the synthesis of lipoproteins and prevents fatty liver (Swenson & Reece, 1996).This can resulted lower levels of whole body fat.

Conclusions
The dietary digestible methionine + cystine requirements for weight gain and fillet yield of Nile tilapia from 550 to 700 g are 9.00 and 9.90 g/kg, respectively.

Table 1 -
Composition of the experimental diets

Table 3 -
Whole body and fillet compositions of the Nile tilapia fed diets containing increasing levels of digestible methionine + cystine CV -coefficient of variation; NS -not significant.Linear effect (P<0.05).