Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens

The objective of this study was to evaluate the process of L-methionine incorporation in the blood plasma, liver, breast muscle, and abdominal fat of 35to 59-d-old broiler chickens using the carbon stable isotope (12C and 13C) technique for the estimation of methionine requirements. In this experiment, 51 male broiler chickens orally received a solution of L-[C1] methionine (92 atm % 13C) at 29 μmol/kg live weight/h for 6 h. Three birds were sacrificed for tissue collection at times 0 h (control), 0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, 72, 96, 120, 144, 168, and 336 h after the administration of the first dose. Tissue L-[C1] methionine incorporation mass and percentage results were analyzed using Minitab 16 statistical software. Except for abdominal fat, tissue methionine levels gradually increased after the administration of the methionine solution. The calculated half-lives of methionine in the blood plasma, liver, and breast muscle were 2.52, 1.36, and 3.57 h, respectively, suggesting a greater rate of methionine incorporation in the liver, followed by blood plasma and breast muscle. The isotopic dilution showed that 2.81, 4.79, and 23.64% of the administered L-methionine were retained in the blood plasma, liver, and breast muscle, respectively. The methionine requirements of finisher broilers may be estimated using the carbon isotope technique, and approximately 3, 5, and 24% methionine is used for the synthesis of blood plasma, liver, and breast muscle, respectively, at the evaluated dose.


INTRODUCTION
Due to genetic improvement, modern broilers present greater potential for weight gain and premium cuts yield, particularly breast yield.Their management and feeding has become very complex, and synthetic essential amino acids are commonly added to the feed to supply their requirements.
Methionine, choline, betaine, and folic acid are methyl group donors, and therefore, essential for several metabolic routes.Labile methyl groups participate in the synthesis of amino acids, phospholipids, as well as of DNA and RNA (Saunderson & Mackinlay, 1990).Considering that poultry are not able to synthesize these substances, they must be supplemented in the diet.Methionine is the first limiting amino acid for feathering and body growth (Bertechini, 2006).Under field conditions, methionine is added to broiler diets to supply the specific requirements of this amino acid.
The stable isotope technique has been increasingly applied in animal metabolism and feed digestibility studies.Studies have shown that the isotopic composition of animal tissues depends mainly on ingested feed, water, and inhaled gases, and their associated isotope effects are linked to metabolic processes (Kennedy & Krouse, 1990).

Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens
Animal nutrition studies have focused on protein balance, nutrient partition and tissue turnover, and the use of markers.Poultry nutrition studies using stable isotopes in diet as markers produce estimates the rate with which tissue stable isotopes are replaced by those present in the diet (Denadai et al., 2007;Gottmann et al., 2008;Pelícia et al., 2011;Araujo et al., 2011;Sernagiotto et al., 2013).Labeled compounds can also be used as biological markers (Stradiotti et al., 2013).
Therefore, the objective of the present study was to evaluate the incorporation of L-[ 13 C 1 ]methionine in the blood plasma, liver, breast muscle, and abdominal fat of broiler chickens from 35 to 49 days of age, using the carbon stable isotope technique, and therefore, to obtain a better understanding of the dynamics of methyl groups derived from labeled methionine in the nutrient metabolism of broilers.

MATERIALS AND METHODS
This study was carried out at the facilities of the Poultry Nutrition Laboratory of the School of Veterinary Medicine and Animal Science, State University of São Paulo (UNESP), Botucatu campus, Brazil.
At the start of the experiment, 270 one-day-old male Cobb chicks were allocated to nine floor pens (2.5m 2 area), with 30 birds/pen, where they were reared until 49 days of age.Feed and water were offered ad libitum during the entire experimental period.
At 33 days of age, all birds were weighed, and 51 birds with 2,162.5 g ± 112.5 g average body weight were selected.On day 35, birds were orally fed a solution composed of enriched methionine (L-[ 13 C 1 ] methionine, with isotopic abundance of 92 atm % of 13 C; ISOTEC INC., Matheson, USA).The dose was 29 µmol (936,70 µg of 13 C) of L-[ 13 C 1 ] methionine/ kg live weight/h for 6 h (adapted from Muramatsu et al., 1987).For that purpose, enriched methionine was diluted in saline solution at 0.9% in order to obtain 10.874 mg of enriched methionine per 0.6-mL dose.

Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens
Due to the high velocity of carbon isotope dilution in tissues with fast metabolic rates, such as blood, blood plasma and liver, sampling times were concentrated around the first hours of the experimental period.
Breast muscle, liver, and abdominal fat samples were dried on a forced-ventilation oven at 56°C for 72 h.Both experimental diets and dried tissue samples were ground in a cryogenic mill to obtain a homogeneous material with fine particle size (smaller than 60 µm) (Ducatti, 2007).After grinding, fat was extracted from liver samples for 4 hours in ethyl ether using a Soxhlet extractor.
Isotopic analysis was carried out at the facilities of the Center of Stable Isotopes of the Biosciences Institute, UNESP, Botucatu campus, Brazil.
For the determination of isotopic composition, samples were weighed in tin capsules and submitted to an isotope ratio mass spectrometer (Delta S-Finnigan Mat, Bremen, Germany) coupled to an element analyzer (EA 1108 -CHN -Fisons Instruments, Rodano, Italy).Samples were quantitatively burnt to obtain CO 2 .
Minitab 16 Statistical Software ( 2010) was used to calculate the incorporation speed of L-[ 13 C 1 ] methionine represented by carbon substitution speed, applying an exponential time function in first-order exponential equations.Tissue incorporation percentage of labeled methionine was calculated using the isotopic dilution method.
Results were expressed in δ 13 C against the Peedee Belemnite (PDB) standard, with an analytical error of 0.2‰ (equation 1): (1) Where: δ 13 C = relative enrichment of the 13 C/ 12 C ratio of the sample relative to the PDB standard.Dimensionless.; R = isotopic ratio ( 13 C/ 12 C) of the sample and the standard.Dimensionless.
An exponential time function (equation 2) (Ducatti et al., 2002), obtained using the method of exponential equations of the first order, was used to quantitatively measure the velocity of tissue carbon substitution by orally administered carbon after a given period of time: (2) Where: δ 13 C (t) = tissue isotropic enrichment at any time (t).Dimensionless.; δ 13 C (f) = isotopic enrichment of tissue at equilibrium or final condition.Dimensionless.; δ 13 C (i) = isotopic enrichment of tissue at starter condition.Dimensionless.; k = turnover constant in units of time -1 ; t = time (in hours) elapsed since the administration of solutions containing L-[ 13 C 1 ] methionine.
The half-life (T 50% ) of 13 C in the tissues when 50% of the 13 C atoms were substituted was measured by (equation 3): (3) Where: T = half-life, time (hours); ln = Napierian logarithm; k = tissue turnover rate constant, time -1 , providing an estimate of the velocity of stable isotope exchange in the tissue (Ducatti et al., 2002;Ducatti, 2007).
Dry tissue mass and residual carbon percentage of each tissue were obtained for the calculation of tissue isotopic dilution.
The isotopic dilution method is based on the isotopic balance between isotopes of an element in a sample before and after the addition of a material enriched with one of the isotopes.Equations (4 and 5), adapted by Trivelin et al. (1994) and Sant Ana Filho (2011), show the isotopic mass balance under the conditions of the current experiment using L-[ 13 C 1 ] methionine: (4) (5) Where: f met and f nat = carbon fractions in L-[ 13 C 1 ] methionine and natural sources respectively; Ab met , Ab nat and Ab p = abundance (% of 13 C atoms) in the amino acid L[ 13 C 1 ]methionine, natural sources and tissues (breast muscle, liver, abdominal fat, or blood plasma), respectively.
The mass balance allows to differentiate the contributions of carbon from L-[ 13 C 1 ]methionine and from the natural source for isotope 13 C content in the final products.The 13 C incorporation rate in the tissues was calculated relative to the dose supplied.
The natural abundance (Ab nat ) of isotope 13 C was obtained for each fraction or product (blood plasma, liver, and breast muscle) evaluated in the present study.
The following equation is derived from equations 4 and 5: (6) In terms of percentage, the previous equation ( 6) may be written as: (7)

Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens
Where: % CPP met = percentage of carbon in the product from L-[ 13 C 1 ]methionine.
The amount of carbon in each fraction can be obtained based on the mass of the evaluated product (M p ) (equation 8): (8) Where CPP met = carbon in the product derived from L-[ 13 C 1 ]methionine (μg); M p = total product mass (μg); % Ct p = percentage of total carbon in the product.

RESULTS AND DISCUSSION
The carbon percentages determined in the breast muscle, liver, abdominal fat, and blood plasma were 41.56, 41.21, 69.44, and 2.71%, respectively.Dry mass weight, using as reference a 2.5-kg broiler, of the breast muscle, liver, and abdominal fat were determined as 133.22,9.37, and 37.93 g, respectively.
Considering that the blood plasma accounts for 5% of the live weight of a broiler (Macari & Luquetti, 2002), a 2.5-kg broiler has 125 g of blood plasma.
Applying those results in equation 8, the following amounts of carbon were determined in the breast muscle, liver, abdominal fat, and blood plasma: 55,365; 9,372; 37,926; and 2,667 mg of carbon, respectively.
Based on the average isotopic values (δ 13 C, ‰) of each tissue, obtained by mass spectrometry, Table 2 shows the relationship between isotopic enrichment (δ 13 C, ‰) of the blood plasma, breast muscle, liver, and abdominal fat and time.
These results shows the ( 13 C) tissue enrichment 336 few hours after the administration of the last dose of the solution containing L-[ 13 C 1 ]methionine to the birds.The isotope data presented in Table 2 shows a gradual change of δ 13 C values with time.The peak of blood plasma and liver enrichment was observed 12 hours after of the first administration of the solution containing L-[ 13 C 1 ] methionine.However, in the breast muscle, maximum 13 C enrichment was obtained 72 hours after of administration of the first dose, indicating lower carbon isotope exchange compared with the blood plasma and the liver.No changes in abdominal fat isotope signals were detected, indicating that 13 C from methionine was not incorporated in this tissue.
After the time of tissue enrichment peak, and depending on the continuity of tissue metabolic synthesis and turnover, a dilution of the isotopic signal was observed, i.e., the 13 C concentration from L-[ 13 C 1 ] methionine was reduced.This is completely normal given the discontinuation of the administration of enriched amino acid.
The results of tissue enrichment as a function of time are shown in Figures 1, 2, and 3 for the blood plasma, liver, and breast muscle, respectively.These graphs were built according to the following equations: δ 13 C = -13.01-6.51e -0.5094t (R 2 = 0.998) with a carbon half-life of 2.52 hours for blood plasma (Figure 1); δ 13 C = -13.01-6.51e -0.5094t (R 2 = 0.937) with a carbon halflife of 1.36 hours for the liver (Figure 2); and δ 13 C = -17.62-1.86e -0.1942t (R 2 = 0.965) with a carbon half-life of 3.57 hours for the breast muscle (Figure 3).

Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens
The percentage of carbon-13 mass derived from L-[ 13 C 1 ] methionine (Table 3) was calculated, and represent the amount of methionine incorporated in each tissue at each time analyzed.
At the time of enrichment peak, 2.81, 4.79, and 23.64% of the dose of L-[ 13 C 1 ] methionine administered were retained in the blood plasma, liver and breast muscle, respectively.
These results indicate that, out of all the enriched methionine retained in the broiler body, about 23.64% is used for breast muscle synthesis, demonstrating the importance of methionine for the formation of this tissue.No incorporation of 13 C was detected in abdominal fat, which was expected because the main    The present study showed that the process of L-methionine incorporation in the tissues of finisher broilers can be determined by the technique of carbon stable isotopes.The velocity and percentage of 13 C incorporation are different among tissues.The liver and blood plasma incorporate 13 C faster, but at a lower percentage compared with the breast muscle.
Further studies with marked methionine and amino acids are warranted to estimate their incorporation percentages in different broiler tissues and during different rearing phases to evaluate the importance of each amino acid for tissue synthesis, thereby supporting broiler nutrition research.

Figure 1 -
Figure 1 -Exponential model of the period of isotopic 13 C enrichment of the blood plasma of 35-to 49-d-old broilers orally receiving a solution containing L-[ 13 C 1 ] methionine.

Figure 2 -
Figure 2 -Exponential model of the period of isotopic 13 C enrichment of the liver of 35-to 49-d-old broilers orally receiving a solution containing L-[ 13 C 1 ] methionine.

Figure 3 -
Figure 3 -Exponential model of the period of isotopic 13 C enrichment of the breast muscle plasma of 35-to 49-d-old broilers orally receiving a solution containing L-[ 13 C 1 ] methionine.

Table 1 -
Feedstuff composition and calculated nutritional values.

Table 2 -
Average δ 13 C values (mean ± standard deviation) expressed as ‰ of blood plasma (BP), liver (LI), breast muscle (PM), and abdominal fat (AF) of 35-to 49-d-old broilers orally fed a solution containing L-[ 13 C 1 ]methionine, as a function of time.

Table 3 -
Mass (µg)and percentage (%) of 13 C incorporated into the blood plasma (BP), liver (LI), and breast muscle (PM) of 35-to 49-d-old broilers as a function of the dose of a solution containing L-[ 13 C 1 ]methionine and time.
*Actual amount of 13 C in each dose of the orally-administered L-[ 13 C 1 ] methionine solution.