Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition

The study was conducted to evaluate carcass yield and chemical composition of thigh and breast muscles of broilers fed three different lysine requirement levels: high lysine (+10% NRC), standard (NRC) and low lysine (-10% NRC) and two ways of expressing amino acids in feedstuffs (total or digestible amino acids). The results of this study showed that diet formulation based on digestible amino acids significantly influenced breast yield and abdominal fat deposition. The study showed that increasing lysine level (+10% NRC) of diet significantly increased both carcass and breast percentage and also carcass, breast and thigh weight. The interaction between digestible amino acids and lysine requirement levels affected carcass and breast percentage. Feeding broilers high lysine diets (+10% NRC) significantly increased lysine content in breast and thigh. The response to formulation based on digestible amino acids was maximized when broilers received the high lysine level diet.


INTRODUCTION
Amino acids (AA) requirements of broilers have been extensively studied, as well as factors that influence such requirements, such as sex, age, genetic strain, heat stress, dietary energy concentration and its interaction with crude protein level (Acer et al. 1991, Baker & Han 1994, Vazquez & Pesti 1997, Garcia et al. 2006, Sterling et al. 2006).The wide variation in the composition and digestibility of AA present in feedstuffs is of great concern in diet formulation.The importance of feeding the correct amount of balanced dietary protein and AA for poultry is a priority for two reasons.First, protein and AA are some of the most expensive nutrients in feeds per weight unit .Second, there are environmental concerns as to nitrogen excretion in poultry waste.
The concentration of protein and AA in broiler diets has a large impact on breast meat yield, feed to gain ratio, and number of days required to achieve the appropriate body weight for each type of market.Depending upon genetic strain and market objectives of each broiler complex, a broiler integrator will probably utilize several different protein and AA dietary programs (Acer et al., 1991).In commercial practice, formulating diets to supply adequate AA minimum levels is critical to optimize live production and meat yield of broiler chickens.During the last 10 years, the demand for breast fillets and value-added products has led to increasing broiler market weights.Market weight, product mix, live cost, and genetic strain may determine AA supplementation.Amino acids are critical for muscle development (Tessseraud et al., 1996) and lysine (Lys) content in the breast muscle is relatively higher than other AA (Munks et al., 1945).Lysine represents approximately 7% of the protein in breast meat (Munks et al., 1945).Dietary Lys inadequacy

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
has been shown to reduce breast meat yield compared with other muscles (Tessseraud et al.,1996).Therefore, defining dietary AA needs for optimal growth and meat yield is of utmost importance.
Lysine, one the key AA for protein synthesis and muscle deposition has also been demonstrated to be involved in the synthesis of cytokines, proliferation of lymphocytes and thus in the optimal functioning of immune system in response to infection.Inadequate Lys supply reduces antibody response and cell-mediated immunity in chickens (Geraert & Mercier, 2010).
Lysine needed for optimizing breast meat yield may be higher than the amount needed for optimal body weight gain and feed efficiency (Moran & Bilgili, 1990, Acar et al., 1991, Gorman & Balnave, 1995).Holsheimer & Ruesink (1998) showed that breast meat yield was increased in male broilers fed a diet containing increasing Lys levels from 1-14 days of age; however, their performance was not affected by dietary Lys from 15 to 49 days of age.It Is well known that protein and Lys and their interaction are important factors that affect performance and carcass quality of growing chickens and therefore, dietary protein requirement is actually the requirement of Lys contained in the protein.
The objective of the nutritionists has long been to optimize growth and tissue accretion by increasing nutrient density such as AA.The question remains about the potential benefits of AA beyond protein synthesis for muscle development.The overall efficiency of lysine used for protein accretion in young growing chicks and pigs is between 70 and 80% (Edwards et al., 1999;Mohn et al., 2000).Essential amino acid recommendations for broilers in the NRC (1994) are largely based on experimentation conducted several decades ago.
Therefore the objective of this study was to evaluate the effects of three different lysine requirement levels -high lysine (+10% NRC), standard (NRC) and low lysine (-10% NRC) -and two ways of expressing amino acid levels in feedstuffs -total amino acids (TAA) or digestible amino acids (DAA) -on the yield and chemical composition of thigh and breast meat of Arian broilers.

MATERIAL AND METHODS
An experiment with Arian male broilers was conducted from 1 to 6 weeks of age.On day 1, 300 male chicks were placed in 30 floor pens (10 chicks per pen and 0.1m 2 floor space/chick).Water and feed were supplied ad libitum.A continuous lighting regimen was adopted, with 24 hours of light daily, throughout the experimental periods.
The basic chemical composition of the feedstuffs and of breast and thigh muscles was determined according to AOAC (1990).
The total amino acid values of the ingredients were assayed by high-pressure liquid chromatography analysis (Moore, 2004).In order to determine digestibility, TAA levels determined in the analysis were multiplied by their respective digestibility coefficients, as determined by Yaghobfar (2002Yaghobfar ( , 2003)).
A completely randomized experimental design in a factorial scheme (2 × 3) was applied.The first factor included three different lysine requirement levels -high lysine (+10% NRC), standard (NRC) and low lysine (-10% NRC) -and the second factor included formulation based on total amino acid (TAA) or digestible amino acid (DAA) levels.Therefore, the following treatments were applied: Feeds were provided in mash form and were milled with a 3-mm screen to obtain a similar particle size in all diets.Diets wereformulated, according to NRC (1994) recommendations, to contain 23% CP and 3,100 kcal of ME/kg in starter period and 19% CP and 3,200 kcal ME/kg in grower period.Diets were formulated to contain equal energy and nitrogen levels and were based on corn and soybean meal (ileal digestibility coefficients of 0.81-0.81), in which AA are highly digestible (Ravindran et al., 2005).Diet composition is shown in Tables 1 and 2.
At the end of the experimental period (42 days of age), in order to evaluate carcass quality, three birds per replicate, with body weights as close as possible to the average body weight of the experimental unit, were slaughtered.Birds were weighed, de-feathered, eviscerated, and weighed again to obtain carcass weight (without head and feet), and breast, thigh, liver and abdominal fat weights.Breast, thigh and carcass

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
yields were determined as carcass weight relative to body weight and expressed as a percentage of body weight (%).
Parameters were tested for normal distributions before analyses.Data were analyzed per factor (GLM procedure, ANOVA, SAS Institute, 2001) and where significance was detected, means were compared by Duncan's multiple range tests.Output data are expressed as means and SEM.

RESULTS AND DISCUSSION
The main effects of diet formulation based on DAA or TAA did not influence carcass, thigh percentage and liver weight (Table 3).These results are consistent with those of Rostagno et al. (1995) and Dari & Penz (1996), who found no differences in carcass yield.Diets formulated on DAA, however, significantly influenced breast percentage and abdominal fat deposition (Table 3).Breast percentage and abdominal fat were

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
significantly higher when the diet was formulated on DAA basis (p<0.05).This may explained by a close relationship between abdominal fat weight and dietary energy:protein ratio.Excessive AA intake is related to the energy: protein ratio in the diet, and consequently, to carcass composition.In the present study, diets were formulated to contain equal amino acid (isonitrogenous) and energy (iso-energetic) levels, but based either on different formulation criteria (total or digestible amino acids) and different lysine requirement levels.Therefore, the AA intake of broilers fed the diets formulated based on DAA was higher than of those fed the TAA-based diet.When crude protein intake increases, the energy: protein ratio is reduced, resulting in fatter carcasses (Summers et al., 1965, Griffiths et al., 1977, Rosebrough & Steele, 1985).Postnatal protein accretion results from an increase in protein synthesis or a decrease in protein degradation.Diets containing low Lys may limit breast meat development early in development as protein synthesis and RNA content are reduced (Tesseraud et al., 1992(Tesseraud et al., , 1996)).

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
Dietary lysine requirement levels had no influence on thigh percentage and liver weight.The results confirmed the findings of previous studies that demonstrated that Lys requirements for growing chickens are higher than the NRC's (1994) recommendation for maximal growth (Kidd et al.,1998;Kerr et al., 1999).It is also confirmed that increasing dietary Lys level increases breast meat yield (Kerr et al., 1999).
Dietary lysine levels significantly influenced carcass and breast percentage (Table 3), as well as carcass, breast and thigh weights (Figure 2).Abdominal fat weight significantly increased when Lys levels were above or below the requirements.This higher abdominal fat deposition is probably related to AA imbalance of these diets.This study showed that higher dietary Lys level (+10% NRC) significantlyincreased breast meat yield, as shown in other research studies (Bilgili et al.,1992;Gorman & Balnave, 1995;Han & Baker, 1994;Kidd et al., 1998).Although feeding high Lys diets throughout the rearing period optimizes breast meat yield (Kidd et al., 1998;Kerr et al., 1999), it may not always be economically justified.However, evidence in literature suggests that feeding Lys diets during the starter period impacts subsequent breast meat yield at marketing (Holsheimer & Ruesink, 1993;Kerr et al., 1999).Dietary Lys concentration can significantly influence breast meat yield because the breast has high Lys content (Table 4) and it represents a large portion of carcass meat.Breast muscle development is also affected by sex, age, breed and genetics strain (Moran & Bilgili, 1990;Acar et al., 1991;Bilgili et al., 1992, Gorman & Balnave, 1995;Han & Baker, 1991), and these studies also showed that an additional dietary Lys increases breast meat accretion.
Treatment 4 (H Lys × DAA) promoted significantly higher carcass, breast and thigh weights than the

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
other treatments (Figure1).This study showed the higher efficiency of these diets as they allow a better utilization of dietary AA for tissue synthesis and accretion.This is possibly related to a higher AA availability to synthesize muscle.Diets formulated with high Lys levels promoted a better conversion of DAA into carcass and breast yield.It was also verified that the H Lys diet formulated on DAA basis promoted better conversion of AA into carcass and breast percentage (Table 3) and carcass, breast and thigh weight (Figure 1) compared with other treatments.The combination of DAA and standard Lys level possibly resulted in less AA available for fat deposition.
Genetic differences have been observed for breast meat yield, abdominal fat pad percentage, and yields of other parts (Acar et al., 1991;Holsheimer & Veerkamp, 1992;Smith & Pesti, 1998;Smith et al., 1998).Leclercq (1998) stated that the required level of lysine is highest for minimizing abdominal fat pad percentage followed by maximizing breast meat yield and body weight gain.Acar et al. (1991) found significant interactions of genotype with lysine for abdominal fat, breast fillet, and breast tender yields.However, dietary protein level has been found to affect the lysine requirement (expressed as a percentage of the diet) of chicks (Morris et al., 1987;Hurwitz et al., 1998;Sterling et al., 2002).Feeding high-Lys and AA-dense diets to broilers increases breast meat yield (Dozier et al., 2007;Eits et al., 2003).Dietary AA responses influencing breast meat yield may be additive among AA (De Leon, 2006;Hickling et al., 1990;Kerr et al., 1999), but other studies found no interactions between Lys and Met (Si et al., 2004).Differences in dietary AA density responses in literature (Corzo et al., 2004 and2005;Kidd et al., 2004) may be related to genetic lines.The response to dietary AA/CP density  (Smith & Pesti, 1998;Smith et al., 1998;Sterling et al., 2006) and dietary Lys (Bilgili et al., 1992;Acar et al., 1991;Han & Baker, 1991;Pesti et al., 1994) differs among genetic lines.A high-yielding strain was shown to contain more breast muscle total RNA and protein on a weight basis and total DNA content than a lowyielding strain (Acar et al., 1993).Muscle growth is largely related to the number of nuclei or total DNA (Kang et al., 1985).Hence, strains exhibiting rapid muscle growth should have balanced high dietary AA requirements for muscle accretion.In some previous studies, feeding low protein diets to broilers impaired growth performance (Ferguson et al., 1998;Jacob et al., 1994;Jensen, 1991).It has been suggested that amino acid requirements of broilers linearly increase with dietary CP (Garu, 1984;Boomgaardt & Baker, 1973;Morris et al., 1987;Morris & Abbeb, 1990;Morris et al., 1992).

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
Although in the present study treatment 4 (H Lys × DAA) promoted the highest carcass, breast and thigh weights (Figure 1), poor-quality dietary proteins and AA may cause major problems in broiler production.The reason dietary protein and AA cause high heat increment from metabolism is the inefficient process of incorporating feed protein and AA into body proteins.The formation of new body proteins both from endogenous and dietary AA can be inefficient with to using available metabolic energy.The nitrogen that is not utilized by the body need be converted into a non-toxic metabolite called uric acid and eliminated from the body.The production of the nitrogen waste product, uric acid, requires a significant amount of metabolic energy, which is taken away from the energy needed for growth.This was shown in the present study, where the diet fomulated based on DAA and with high H Lys resulted in significantly higher carcass and breast yield, whereas the broilers fed the diet fomulated on TAA basis and low Lys level presented the lowest carcass and breast yield.
Main effects of diet formulation based on DAA or TAA and lysine requirements levels did not affect breast and thigh meat protein or lipid contents.The breast contains a higher percentage of lysine and methionine than other body components (6.5 and 1.96%, respectively) and the thigh contains a 5.86 and 1.93 % of lysine and methionine, respectively, in Ross broilers (Coon, 2000).The overall increase in carcass lysine and methionine in amino acid composition

Effects of Lysine Levels of Diets Formulated Based on Total or Digestible Amino Acids on Broiler Carcass Composition
in older broilers because breast meat percentage increases in older broilers is probably the reason why these AA have been shown to increase breast meat yield (Table 3, 4).The amino acid requirements for different genetic broiler lines are partially dependent on the amino acid content of each body component (i.e., breast, thigh, drumstick) and on the extent to which the carcass components change as a percentage of the whole bird.In present study, however, breast and thigh lysine percentages were 4.742 and 4.133, respectively, which were lower than in Ross broilers.Lysine represents approximately 7% of the protein in breast meat (Munks et al., 1945).
Amino acids effects beyond their roles of building blocks of the protein accretion have been largely demonstrated, from a better gut functioning to an enhanced immune system.More research is necessary to determine the optimal AA requirements to improve not only muscle development, but also meat quality.It has been determined that optimal AA levels are not the same for maximization weight gain and breast muscle development (Pesti & Miller, 1997), but in the present study, the high dietary Lys level promoted the highest body, breast and thigh weights (Figure 2).

CONCLUSION
Feeding broilers high lysine diets (+10% NRC) increases carcass and breast percentages by 4.4% and 1.81%, respectively, compared with broilers fed low lysine diets.Diet formulation based on digestible amino acids levels in feedstuffs and high lysine dietary levels influence broiler carcass and breast percentage.The interaction between digestible amino acids and high lysine level allows the full expression of the genetic potential for growth of Arian broilers.Formulating broiler diets based on digestible amino acids provides better prediction of dietary protein quality and bird performance than total amino acids.Feeding broilers high lysine density diets (+10% NRC) significantly increase percentage of lysine in breast and thigh meat.

Figure 1 -
Figure 1 -Effects of treatments on mean body weight, breast weight and thigh weight (42 d).

Table 3 -
Effects of amino acids of feedstuffs and lysine requirement levels on carcass composition.

Table 4 -
Effects of amino acids of feedstuffs and lysine requirement levels on chimical composition