Performance and nitrogen balance of laying hens fed increasing levels of digestible lysine and arginine

The objective of this experiment was to evaluate the effect of two digestible lysine levels and four digestible arginine levels on laying hens from 24 to 48 weeks of age. Three hundred and twenty Lohmann LSL laying hens were allotted in a completely randomized design in a 2 × 4 factorial arrangement, with two levels of digestible lysine (700 and 900 mg/kg of diet) and four digestible arginine levels (700, 800, 900 and 1000 mg/kg of diet). Results indicated requirement of 884 and 830 mg of digestible arginine/kg of diet, considering an average feed intake of 95 g/hen/day and an average hen weight of 1.5 kg, aiming at lesser feed intake and better nutritional balance of nitrogen, respectively. High digestible lysine levels in the diet require higher digestible arginine supplementation for a better performance of hens.


Introduction
Modern hen diets have been formulated according to the requirements of crude protein, which may unbalance the levels of amino acids as well as the relation between them.The ingestion and metabolism of amino acids increases the temperature of the body (Musharaf & Latshaw, 1999), uric acid excretion (Latshaw & Zhao, 2011), energy waste (Leclercq, 1998), and water intake, which may result in aqueous feces, leading to performance problems (Emadi et al., 2010).
Modern diet formulas for laying hens must consider the concept of ideal protein, based on the amount of amino acids, which would be more efficiently used by the hens.For this reason, it is important to establish the relations between the amino acids that are better adapted to the digestion and absorption capacity of hens, reducing the necessity of spending excessive excretion of amino acids, thus saving body energy (Parsons & Baker, 1994).The concept of ideal protein uses lysine as a reference amino acid due to its specific function in the animal organism, which is the protein deposition, and for the great amount of information about its concentration and digestibility (Parsons et al., 1992).Nevertheless, as the level of lysine is increased in diet, arginine becomes the limiting amino acid for the best hen performance.However, higher levels of lysine result in higher arginine requirements (Macari et al., 2002).
There is evidence of considerable antagonism between arginine and lysine (Austic & Scott, 1975).One reason for this antagonism is the urinary losses of arginine, caused by the lysine competition for urinary tubular reabsorption (Macari et al., 2002).Because of this, it is important to find the correct adjustment of amino acid ratios for the development of ideal protein formulas, and the achievement of the best production efficiency (Gadelha et al., 2003;Silva et al., 2005).
In hens, only 35 to 45% of the nitrogen from the protein intake turns into meat and eggs.The rest of the nitrogen is excreted, and becomes a source of environmental contamination (Penz Junior, 1993).Synthetic amino acid supplementation in laying hen diets allows better protein utilization, contributing to a reduction of excreted nitrogen and, consequently, improvement in the performance of hens.
The objective of this experiment was to evaluate the ratios of lysine and arginine supplementation for commercial Lohmann LSL laying hens at the starter production phase, and their effect on performance and nutritional balance of nitrogen.
LSL hens of 24 weeks of age were subjected to a performance trial.For the metabolic assays, 64 additional Lohmann LSL hens, at 34, 42 and 48 weeks of age, were used.The design was completely randomized, in a 2 × 4 factorial arrangement (digestible lysine vs. digestible arginine levels) with four replicates per treatment of 10 hens each.
A corn-soybean basal diet (Table 1) was formulated according to the feed composition and dietary requeriment, fulfilling the requirements established by Rostagno et al. (2000).In addition, 0.178% and 0.202% of L-lysine, as well as 0.0; 0.105; 0.211 and 0.316 of L-arginine were added to the diet in order to meet the suggested levels of 700 and 900 mg of digestible lysine/kg of diet, and of 700, 800, 900 and 1000 mg of digestible arginine/kg of diet, respectively.
At the end of each 28-day period, feed and nutrient intake, as well as the number of dead hens, were observed in order to correct consumption values.All the eggs produced were counted every day during all the experimental period, and the results were expressed in percentage.Feed conversion (kg/dozen and kg/kg egg mass) was obtained at each 28-day period.In the last three days of each period, 16 eggs per treatment per day were weighed and the egg mass was obtained.
Nitrogen balance was obtained during four days at 34, 42 and 48 weeks of age.Excreta was collected twice a day by the total excreta collection method, identified, stored and prepared for nitrogen analysis.The feed and the eggs weighed during the metabolic assay were used to calculate nitrogen balance and retention, expressed by milligrams of nitrogen per gram of egg mass produced.
Chemical analysis of diet and excreta were performed at the Laboratório de Nutrição Animal of Universidade Federal de Goias.Dry matter and total nitrogen were determined as proposed by Silva & Queiroz (2002).Nitrogen balance (NB) was determined as follows: NB (g): ingested N -excreted N; NB (g/g) = (ingested N -excreted N)/ ingested N * 100 and; NB per egg mass (mg/g) = (ingested N -excreted N)/ egg mass * 100 Results were analyzed by ANOVA using SAEG (Sistema para Análises Estatísticas e Genéticas, version 7.1), and submitted to polynomial regression for the levels of digestible arginine tested.

Results and Discussion
Egg production, average egg weight, egg mass, feed conversion per gram of egg produced, and feed conversion per dozen egg produced were not affected (Table 2).On the other hand, feed intake, lysine intake and arginine intake showed significantly effect nutritional treatments.
There was significant interaction (P<0.05) between the levels of digestible lysine and arginine for feed intake (Table 3).For the diet with 700 mg of digestible arginine/kg, there was lower feed intake the hens at the level of 700 mg of digestible lysine than at 900 mg/kg of diet.When the arginine level was incremented up to 900 mg/kg of diet, opposite results were observed.
Lysine intake (Table 3) occurred as expected, considering that the hens that were fed 900 mg of lysine/ kg of diet presented higher lysine intake when compared with the ones that were fed 700 mg of lysine/kg of diet.As regards arginine intake, the only difference observed occurred with diets at the levels of 700 and 800 mg of arginine/kg of diet, with higher arginine intake for hens that received 900 mg of digestible lysine/kg of diet than the ones that received 700 mg.
Responses to the increasing levels of digestible arginine/kg of diet showed a quadratic effect for the consumption of feed and digestible lysine for hens fed diets with 900 mg of digestible lysine/kg of diet (Table 3), whereas digestible arginine intake showed expected linear increase as the levels of digestible arginine per kg/diet were increased from 700 mg to 1000 mg at the two levels of digestible lysine tested.On the other hand, Araújo et al. (2005), in a study previously mentioned, did not find differences for feed intake.In a similar study with the same hen lineages, Lima et al. (2005) also did not find changes.
The reduction in lysine intake observed with digestible arginine supplementation, when high levels of digestible lysine are adopted, can be explained by the antagonistic relationship between these two amino acids.As both amino acids use the same renal tubular transportation system, lysine and arginine compete for renal re-absorption.This may cause arginine loss through the urine, and may decrease hepatic transaminase activity due to excessive lysine (Macari et al., 2002).Increase in arginine consumption confirms the suggestion by Leeson & Summers (2001), in which absence of a functional urea cycle requires arginine supply in the diet.The more lysine is added to the diet, the more arginine is required.
The levels of digestible arginine in diets fed to 34week old hens did not produce any changes in the nitrogen balance in grams, percentage, or per egg mass, in its intake or excretion (Table 4).Nitrogen intake was higher (P<0.05) in hens fed 900 mg digestible lysine/kg of diet.lysine resulted in a quadratic effect (P<0.05) for nitrogen balance in grams, with a peak at the level of 844 mg of digestible arginine/kg in diet.Linear effect for nitrogen balance in grams, in percentage, and per egg mass was observed at the level of 900 mg of lysine.When hens are fed diets containing excessive levels of either arginine or lysine, the activity of the enzyme arginase increases.Therefore, little excess of lysine in the diet may increase arginine degradation (Leeson & Summers, 2001).This antagonistic relationship may explain the increase seen in nitrogen balance in grams, in percentage, and per egg mass when the level of digestible arginine is increased in the diet.
Higher nitrogen intake was observed in the diet containing 900 mg of digestible lysine and 1000 mg of digestible arginine (Table 7); however, this did not occur for nitrogen excretion.A quadratic effect was observed for Rizzo et al. (2004) did not find differences in nitrogen intake, excretion and balance in the diet supplemented with 850 and 1000 mg of digestible lysine and fed to 51-week old Hisex White hens.When metabolic assays were conducted with 42-week old animals, statistical interaction was found between the two amino acids, for all the variables studied (P<0.05)(Table 5).
Hens fed supplementation with 1000 mg of digestible arginine (Table 6) showed higher nitrogen balance in g, in percentage, and per egg mass when 900 mg of digestible lysine were used (P<0.05).The same was not seen for the other supplementation levels of arginine/kg of the diet.Increase of arginine in the diet with 700 mg of digestible    nitrogen intake (P<0.05), for supplementation levels of digestible arginine in a diet with 700 mg of digestible lysine/kg.There was a peak at the level of 871 mg of digestible arginine.Positive linear effect (P<0.05) was observed for hens fed 900 mg of digestible lysine/kg of diet.The same was not observed for nitrogen excretion.Adding arginine when using 900 mg of digestible lysine resulted in an increase of nitrogen intake, but with no changes in nitrogen excretion.This result may be related to a greater biosynthesis of the nitric oxide, which is an arginine precursor (Bacila, 2003).
When hens reached 48 weeks of age, a statistical interaction (P<0.05) was found for levels of digestible lysine and arginine (Table 8).Supplementation with 800 mg of digestible arginine (Table 9) showed greater nitrogen balance in grams, in percentage and per egg mass for 700 mg of digestible lysine (P<0.05).However, when using 1000 mg of digestible arginine, nitrogen balance values in percentage and per egg mass were greater for 900 mg of digestible lysine/kg of diet.
Adding arginine to the diet containing 700 mg of digestible lysine resulted in a quadratic effect (P<0.05) for nitrogen balance in grams, in percentage and per egg mass, with peaks at the level of 821, 786 and 814 mg of digestible arginine/kg of diet, respectively (Table 9).No regression effect occurred for 900 mg of digestible lysine/kg of diet for the variables studied.
These values are similar to the 838 mg arginine/kg of diet suggested as the minimum amount required for digestible arginine for light hens fed 95 g of diet/hen/day and with average weight of 1.479 kg (Rostagno et al., 2005).
The values found for nitrogen balance (Table 9) showed higher supplementation levels of digestible arginine/kg of diet (786 and 826 mg) for 700 mg of digestible lysine.It is must be emphasized that the recommendation above was found based on an average intake of 95 g/hen/day for light hens with average weight of 1500 g.To improve hen performance, higher levels of digestible lysine should be accompanied by supplementation of digestible arginine.
For 700 mg of digestible arginine (Table 10), there was lower consumption of nitrogen for 700 mg of digestible lysine, if compared with 900 mg (P<0.05).The opposite was seen for 800 mg of digestible arginine in the diet.When supplementation of 1000 mg of digestible arginine was used, greater excretion of nitrogen was seen for the lowest level of digestible lysine (700 vs. 900).Supplementation of digestible arginine with 700 mg of digestible lysine/kg   generated a quadratic effect (P<0.05) with a peak at the level of 862 mg of digestible arginine.As for excretion, a linear effect was generated (P<0.05), with no difference for 900 mg of digestible lysine in the nitrogen intake or excretion.
For 700 mg of digestible lysine/kg of diet, nitrogen intake was maximum with 862 mg of digestible arginine, whereas greater nitrogen excretion was obtained with 1000 mg of digestible arginine, which is probably related to the balance of amino acids in the diet (Table 10).

Conclusions
As lysine is added to the diet, more arginine is required.Therefore, to improve performance of the hens and optimize nitrogen metabolism, increase in the levels of digestible lysine should be accompanied by digestible arginine supplementation.

Table 2 -
Performance of laying hens fed increasing levels of digestible lysine and arginine/kg of diet from 24 to 44 weeks of age

Table 4 -
Nitrogen metabolism of laying hens fed increasing levels of digestible lysine and arginine/kg of diet at 34 weeks of age Different letters in the column differ by the F test (0.05).CV -coefficient of variation; DM -dry matter.

Table 6 -
Effect of interaction for laying hens fed increasing levels of digestible lysine (mg/kg of diet) and arginine (mg/kg of diet) at 42 weeks of age

Table 5 -
Nitrogen metabolism of laying hens fed increasing levels of digestible lysine and arginine/kg of diet at 42 weeks of age CV -coefficient of variation; DM -dry matter.

Table 8 -
Nitrogen metabolism of laying hens fed increasing levels of digestible lysine and arginine/kg of diet at 48 weeks of age CV -coefficient of variation; DM -dry matter.