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Brazilian Journal of Poultry Science

Print version ISSN 1516-635XOn-line version ISSN 1806-9061

Rev. Bras. Cienc. Avic. vol.18 no.4 Campinas Oct./Dec. 2016

http://dx.doi.org/10.1590/1516-635x1804719-724 

ARTICLES

Incorporation of Labeled Methionine as a Tissue Tracer in Broiler Chickens

AC StradiottiI 

JA BendassolliII 

C DucattiIII 

JR SartoriIV 

VC PelíciaI 

PC AraujoI 

MK MarunoI 

AC PezzatoIV 

IPost-graduation students in Animal Sciences, FMVZ/UNESP, Botucatu, SP, Brazil

IIAss. Prof., PhD, Center of Nuclear Energy in Agriculture, CENA/USP, Piracicaba, SP, Brazil

IIIAss. Prof., PhD, Department of Physics and Biophysics, IBB/UNESP, Botucatu, SP, Brazil

IVAss. Prof., PhD, Department of Genetic Improvement and Animal Nutrition, FMVZ/UNESP, Botucatu, SP, Brazil


ABSTRACT

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 35- to 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-[13C1] 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-[13C1] 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.

Keywords: Isotopic dilution; labeled isotopes; turnover

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).

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-[13C1]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 (2.5-m2 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.

The chemical composition of the feedstuffs was analyzed at Bromatology Laboratory of UNESP, Botucatu campus, according to AOAC standards (2006) before diet formulation. The experimental feeds were formulated to supply the nutritional requirements of male broilers as recommended by Rostagno et al. (2005). The feeding program was divided into four phases: pre-starter (1-7 d), starter (8-21 d), grower I (22-35 d), and grower II (36-49 d). In order to maintain the dietary isotopic signal during the sampling period (35 to 49 days), the grower II diet was formulated to contain similar nutritional levels as those of the grower I diet, except for methionine (Table 1).

Table 1 Feedstuff composition and calculated nutritional values. 

Phases
Ingredients (%) Pre-starter Starter Grower I Grower II
Corn 56.220 59.320 62.140 62.130
Soybean meal 37.240 34.430 30.840 30.860
Soybean oil 2.070 2.300 3.260 3.270
Dicalcium phosphate 1.950 1.800 1.670 1.670
Limestone 0.940 0.900 0.840 0.840
DL-Methionine 0.217 0.155 0.155 0.134
L-Lysine HCl 0.335 0.185 0.203 0.203
L-Threonine 0.135 0.050 0.050 0.050
Choline chloride 0.060 0.050 0.050 0.050
Anticoccidial agent1 0.025 0.025 0.025 0.025
Sodium chloride 0.515 0.495 0.470 0.470
Vit/min supplement2 0.300 0.300 - -
Vit/min supplement3 - - 0.300 0.300
Nutritional Levels
Metabolizable energy, kcal/kg 2,950 3,000 3,100 3,100
Crude protein, % 22.040 20.790 19.410 19.410
Calcium, % 0.940 0.880 0.820 0.820
Available phosphorus, % 0.470 0.440 0.410 0.410
Methionine, % 0.519 0.447 0.429 0.407
Methionine+Cystine, % 0.940 0.810 0.770 0.770
Lysine, % 1.330 1.150 1.070 1.070
Threonine, % 0.870 0.750 0.700 0.700
K, % 0.590 0.590 0.590 0.590
Na, % 0.220 0.210 0.210 0.210
Cl, % 0.200 0.190 0.180 0.180
Mean Isotopic Values4
d13C, ‰ -18,8 -18,4 -18,1 -18,1

1Monenpac mc 400®: Sodium monensin (40%).

2Starter vitamin/mineral supplement TORTUGA® (per kg feed): manganese 559.86 mg, iron 434.37 mg, zinc 433.56 mg, copper 85.65 mg, iodine 5.61 mg, selenium 3.39 mg, vitamin A 106,560 IU, vitamin D3 25,537.5 IU, vitamin E 148.77 mg, vitamin K3 18 mg, vitamin B1 20.1 mg, vitamin B2 45 mg, vitamin B6 24.9 mg, vitamin B12 120 mcg, niacin 300 mg, folic acid 117.45 mg, pantothenic acid 7.5 mg, biotin 0.99 mg, antioxidant 42 mg.

3Finisher vitamin/mineral supplement TORTUGA® (per kg feed): manganese 559.85 mg, iron 434.37 mg, zinc 433.56 mg, copper 85.65 mg, iodine 5.61 mg, selenium 3.39 mg, vitamin A 88,054.5 IU, vitamin D3 21,264.23 IU, vitamin E 124.17 mg, vitamin K3 15 mg, vitamin B1 16.74 mg, vitamin B2 37,47 mg, vitamin B6 20,73 mg, vitamin B12 99.9 mcg, niacin 249.75 mg, folic acid 97.74 mg, pantothenic acid 6.27 mg, biotin 0.84 mg, antioxidant 42 mg.

4Isotopic mean values expressed as δ13C, ‰ relative to the PeeDee Belemnite (PDB) standard.

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-[13C1]methionine, with isotopic abundance of 92 atm % of 13C; ISOTEC INC., Matheson, USA). The dose was 29 µmol (936,70 µg of 13C) of L-[13C1] 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.

One dose was administered every hour up to six hours after the first dose was given (time 0). At times 0 (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, three birds were electrically stunned and killed. Blood plasma, liver, breast muscle (Pectoralis major), and abdominal fat samples were collected to analyze the incorporation labeled methionine in these tissues.

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 CO2.

Minitab 16 Statistical Software (2010) was used to calculate the incorporation speed of L-[13C1] 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 d13C against the Peedee Belemnite (PDB) standard, with an analytical error of 0.2‰ (equation 1):

(1)

Where: δ13C = relative enrichment of the 13C/12C ratio of the sample relative to the PDB standard. Dimensionless.; R = isotopic ratio (13C/12C) 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: δ13C (t) = tissue isotropic enrichment at any time (t). Dimensionless.; δ13C (f) = isotopic enrichment of tissue at equilibrium or final condition. Dimensionless.; δ13C (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-[13C1] methionine.

The half-life (T50%) of 13C in the tissues when 50% of the 13C 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-[13C1] methionine:

(4)

(5)

Where: fmet and fnat = carbon fractions in L-[13C1]methionine and natural sources respectively; Abmet, Abnat and Abp = abundance (% of 13C atoms) in the amino acid L[13C1]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-[13C1]methionine and from the natural source for isotope 13C content in the final products. The 13C incorporation rate in the tissues was calculated relative to the dose supplied.

The natural abundance (Abnat) of isotope 13C 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)

Where: % CPPmet = percentage of carbon in the product from L-[13C1]methionine.

The amount of carbon in each fraction can be obtained based on the mass of the evaluated product (Mp) (equation 8):

(8)

Where CPPmet = carbon in the product derived from L-[13C1]methionine (µg); Mp = total product mass (µg); % Ctp = 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 13C, ‰) of each tissue, obtained by mass spectrometry, Table 2 shows the relationship between isotopic enrichment (δ13C, ‰) of the blood plasma, breast muscle, liver, and abdominal fat and time.

Table 2 Average δ13C 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-[13C1]methionine, as a function of time. 

Sampling time (h) BP LI PM AF
0 -19.3 ±0.1 -18.7 ±0.4 -19.5 ±0.2 -21.5 ±0.5
0.5 -18.5 ±0.3 -17.6 ±0.3 -19.3 ±0.2 -21.3 ±0.3
1 -17.9 ±0.2 -17.4 ±0.2 -19.0 ±0.2 -22.2 ±0.3
2 -18.1 ±0.6 -16.8 ±0.5 -19.2 ±0.2 -21.9 ±0.4
3 -15.9 ±0.4 -15.4 ±0.5 -18.6 ±0.1 -21.6 ±0.2
4 -15.0 ±0.3 -14.1 ±0.2 -18.4 ±0.2 -21.5 ±0.1
5 -14.8 ±0.9 -13.8 ±0.1 -18.4 ±0.4 -21.6 ±0.3
6 -13.4 ±0.4 -13.4 ±0.3 -18.1 ±0.4 -21.7 ±0.4
12 -13.3 ±0.1 -13.3 ±0.1 -17.9 ±0.5 -22.0 ±0.1
24 -15.2 ±0.5 -15.0 ±0.5 -17.8 ±0.4 -22.3 ±0.2
48 -16.2 ±0.3 -16.0 ±0.5 -17.8 ±0.2 -21.5 ±0.5
72 -17.1 ±0.2 -16.7 ±0.2 -17.7 ±0.2 -21.8 ±0.1
96 -17.7 ±0.4 -17.4 ±0.8 -18.0 ±0.2 -22.5 ±0.3
120 -18.0 ±0.4 -17.4 ±0.2 -18.0 ±0.3 -21.9 ±0.4
144 -18.1 ±0.4 -17.5 ±0.2 -18.1 ±0.4 -21.7 ±0.1
168 -18.1 ±0.2 -17.1 ±0.1 -18.0 ±0.2 -21.7 ±0.1
336 -18.8 ±0.2 -18.5 ±0.3 -18.5 ±0.6 -22.7 ±0.4

These results shows the (13C) tissue enrichment 336 few hours after the administration of the last dose of the solution containing L-[13C1]methionine to the birds. The isotope data presented in Table 2 shows a gradual change of δ 13C 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-[13C1] methionine. However, in the breast muscle, maximum 13C 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 13C 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 13C concentration from L-[13C1]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: δ13C = -13.01-6.51e-0.5094t (R2 = 0.998) with a carbon half-life of 2.52 hours for blood plasma (Figure 1); δ13C = -13.01-6.51e-0.5094t (R2 = 0.937) with a carbon half-life of 1.36 hours for the liver (Figure 2); and δ13C = -17.62-1.86e-0.1942t (R2 = 0.965) with a carbon half-life of 3.57 hours for the breast muscle (Figure 3).

Figure 1 Exponential model of the period of isotopic 13C enrichment of the blood plasma of 35- to 49-d-old broilers orally receiving a solution containing L-[13C1] methionine. 

Figure 2 Exponential model of the period of isotopic 13C enrichment of the liver of 35- to 49-d-old broilers orally receiving a solution containing L-[13C1] methionine. 

Figure 3 Exponential model of the period of isotopic 13C enrichment of the breast muscle plasma of 35- to 49-d-old broilers orally receiving a solution containing L-[13C1] methionine. 

The percentage of carbon-13 mass derived from L-[13C1] methionine (Table 3) was calculated, and represent the amount of methionine incorporated in each tissue at each time analyzed.

Table 3 Mass (µg) and percentage (%) of 13C 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-[13C1]methionine and time. 

Sampling time (h) Dose* (µg 13C) 13C recovery (µg) 13C incorporation (%)
BP LI PM BP LI PM
0.5 936.70 22.82 46.41 35.04 2.44 4.95 3.74
1 936.70 38.21 53.90 266.15 4.08 5.75 28.41
2 1873.40 37.15 83.40 209.77 1.98 4.45 11.20
3 2810.10 79.57 132.82 611.50 2.83 4.73 21.76
4 3746.80 110.43 210.95 779.29 2.95 5.63 20.80
5 4683.50 106.35 261.58 914.61 2.27 5.59 19.53
6 5620.20 157.04 262.05 1166.51 2.79 4.66 20.76
12 5620.20 158.62 270.64 1241.58 2.81 4.79 21.97
24 5620.20 98.85 176.38 1205.93 1.75 3.12 21.34
48 5620.20 78.91 152.25 1288.50 1.40 2.69 22.80
72 5620.20 59.01 115.99 1336.09 1.04 2.05 23.64
96 5620.20 50.93 71.98 1196.88 0.90 1.27 21.18
120 5620.20 40.88 70.44 1124.73 0.72 1.25 19.90
144 5620.20 39.05 74.78 1070.51 0.69 1.32 18.95
168 5620.20 39.93 94.92 1080.92 0.71 1.68 19.13
336 5620.20 27.51 30.66 791.75 0.49 0.54 14.01

*Actual amount of 13C in each dose of the orally-administered L-[13C1] methionine solution.

At the time of enrichment peak, 2.81, 4.79, and 23.64% of the dose of L-[13C1] 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 13C was detected in abdominal fat, which was expected because the main utilization of the ingested amino acids is protein tissue synthesis.

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 13C incorporation are different among tissues. The liver and blood plasma incorporate 13C 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.

ACKNOWLEDGMENTS

We would like to thank Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESP (Process n. 07/57776-0), for grant given to the first author.

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ETHICS AND BIOSECURITY COMMITTEE All the procedures in this study followed the guidelines of the Ethics and Research Committee of the School of Veterinary Medicine and Animal Science, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu campus, SP, Brazil (Protocol n. 139/2007).

Received: July 2016; Accepted: September 2016

Corresponding author e-mail address Ana Cristina Stradiotti Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Melhoramento e Nutrição Animal. Distrito de Rubião Júnior, s/n. Caixa Postal 560. Botucatu, São Paulo, Brazil. Zip code: 18.618-000. Tel: +55-14-3880-2998 Email:ana.stradiotti@hotmail.com

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