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14 N)-stable isotopes
Poultry offal meal in chicken: Traceability using the technique of carbon (13C/12C)- and nitrogen (15N/14N)-stable isotopes V. C. Cruz,*1 P. C. Araújo,† J. R. Sartori,† A. C. Pezzato,† J. C. Denadai,‡ G. V. Polycarpo,† L. H. Zanetti,* and C. Ducatti‡ *Univ Estadual Paulista (UNESP), Campus de Dracena, Curso de Zootecnia, Dracena, SP, 17900-000 Brazil; †Univ Estadual Paulista (UNESP), Campus de Botucatu, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Melhoramento e Nutrição Animal, and ‡Instituto de Biociências, centro de Isótopos Estáveis, Botucatu, SP, 18618-000 Brazil
Key words: animal origin ingredient, carbon-13, certification, nitrogen-15, poultry 2012 Poultry Science 91:478–486 doi:10.3382/ps.2011-01512
INTRODUCTION
been increasing with concern for food safety. This change in the consumer trend is one of the main stimulating factors to propose and adopt methods that allow for the certification of the origin and quality of animal and vegetable products in the world. A research approach aiming to trace the inclusion of animal-origin ingredients in the broiler diet was developed because of the need to find a technology that was, at first, independent in order to certify the vegetable feeding pattern of these birds and their products, and then to meet the demands of consumers who wish for transparency of the conditions and methods for food production, besides guaranteeing fair competition among companies from competing countries. Traceability of the meat production system is a guarantee to consumers because it makes them sure that a product is controlled in all production stages: from the farm to the final consumer. The term traceability refers to the capacity to trace the history, application, or localization of an item through previously registered information (Rastreabilidade, 2004).
Brazil is the world’s leading chicken meat exporter and its exportation volume has been growing—3,819.7 thousand tonnes in the last year. It is the world’s second leading chicken meat producer and currently has an estimated herd of 473.3 million chicks produced per month (Avisite, 2011). Although there have been some decreases in production and global exportation as well as in intake and importation in some countries due to consumer’s fear of possible risks from the avian flu, which occurred some years ago, poultry production in Brazil has shown great dynamism and efficiency. The consumer trend to acquire healthy foods of which the origin, nutritional value, and quality is known has
©2012 Poultry Science Association Inc. Received March 29, 2011. Accepted October 2, 2011. 1 Corresponding author: [email protected]
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and nitrogen (15N/14N) by mass spectrometry. In total, 720 one-d-old chicks were distributed into 6 groups: vegetable diet (VD) from 1 to 42 d; 8% poultry offal meal (OM) diet from 1 to 42 d; VD from 1 to 21 d and 8% OM diet from 22 to 42 d; VD from 1 to 35 d and 8% OM diet from 36 to 42 d; 8% OM diet from 1 to 21 d and VD from 22 to 42 d; and 8% OM diet from 1 to 35 d and VD from 36 to 42 d. Through the analysis of C and N, it is possible to trace the use of OM in broiler feeding when this ingredient is part of the feeding throughout the breeding phase or when it substitutes a strictly VD even up to 35 d. When an OM diet is substituted by a VD, the animal ingredient has to be part of the feeding for 21 d or longer to be detected by this method.
ABSTRACT Studies on the detection of animal byproducts in poultry meat are rare and practically nonexistent in chicken meat. With the development of the technique of stable isotopes for traceability purposes and the certification of broiler diet patterns, it has been necessary to know the behavior of the isotopic signature of different tissues in birds, in case of a potential replacement of a diet containing animal ingredients with a strictly vegetable one and vice versa. Thus, this study, carried out at the São Paulo State University, Botucatu Campus, Brazil, aimed to evaluate meat from the breast, thigh, drumstick, and wings to trace the presence of poultry offal meal (OM) in broiler feed using the analysis of stable isotopes of carbon (13C/12C)
OFFAL MEAL TRACEABILITY WITH ISOTOPES
broiler chickens, it is necessary to know the behavior of the isotopic signature of different poultry tissues to detect an eventual substitution of an animal diet for a strictly vegetable diet and vice versa. Therefore, this study aimed to analyze meat from the breast, drumstick, thigh, and wings to trace the presence of poultry offal meal in broiler feed using the analysis of carbon (13C/12C) and nitrogen (15N/14N) stable isotopes through mass spectrometry.
MATERIALS AND METHODS Birds and Diets In total, 720 one-day-old male Cobb chicks from a flock of 43-wk-old hens were used. The chicks were previously vaccinated against infectious bursal disease, Marek’s disease, and Bouba’s disease, and they were raised until 42 d of age. During the experiment, the animals were vaccinated against coccidiosis and infectious bursal disease via drinking water. Then, the birds were distributed in a completely random block design with 6 treatments and 4 replications, and they were raised and kept in an experimental rearing house divided into twenty-four 2.5-m2 cages, with 30 birds/cage, in a density of 12 birds/m2. Water and feed were offered ad libitum using a trough drinker and feeder that were substituted gradually by a hanging drinker and a tubular feeder. For each cage, initial brooding for the chicks was done using a 250-W infrared light bulb that was removed when the chicks were 8 d old. Temperature and ventilation were controlled manually by managing the rearing house lateral curtains. The light program was constant using 60-W incandescent light bulbs. The experimental treatments were established according to the following groups (G): • G1, vegetable diet with corn and soybean meal without animal-origin by-product (basal diet) from 1 to 42 d old; • G2, 8% poultry offal meal (OM) diet from 1 to 42 d old; • G3, basal diet from 1 to 21 d old and 8% OM diet from 22 to 42 d old; • G4, basal diet from 1 to 35 d old and 8% OM diet from 36 to 42 d old; • G5, 8% OM diet from 1 to 21 d old and basal diet from 22 to 42 d old; and • G6, 8% OM diet from 1 to 35 d old and basal diet from 36 to 42 d old. The percentage of OM inclusion in the diet was calculated at 8%, according to the information on adopted practice levels in broiler producing companies (Frangoeste Avicultura Ltda, Tietê, São Paulo, Brazil). Initially, G3 and G4 birds were submitted to a strictly vegetable diet (0% OM) and then changing to a poultry
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Not only is the traceability process a requirement to aggregate value to a specific product, but it is also a market requirement that arises due to the increase in the consumer’s demand and food safety that resulted from several crises and episodes related to human and animal health. This process should be adopted by companies involved in the national poultry production if they wish to keep important market shares, such as the European market, in the near future. Numerous methods have been proposed to identify the presence of animal-origin by-products in animal feeding, such as DNA hybridization, ELISA, and PCR (Bloch, 2002). On the other hand, although there is no official traceability system in the poultry production chain, mass spectrometry has been used successfully, through the carbon-13 isotopic ratio analysis, to test the authenticity, quality, and geographical origin of several products, such as fruit juice (Bricout and Koziet, 1987; Koziet et al., 1993), wine (Martin et al., 1988), honey (Brookes et al., 1991; White et al., 1998), dairy products (Rossmann et al., 2000; Manca et al., 2001), vegetal oils (Kelly et al., 1997), and the characterization and differentiation of the diet regimen of Iberian swines (González-Martin et al., 1999), certification of geographical origin, types of sheep feeding (Piasentier et al., 2003), traceability of bovine meat and bone meal in broiler feed (Carrijo et al., 2006), poultry offal meal in quail and broiler feed (Móri et al., 2007, 2008; Oliveira et al., 2010), poultry offal meal with yeast and wheat meal in broiler feed (Gottmann et al., 2008), and bovine meat and bone meal in the feed of laying hens (Denadai et al., 2009). This allows for the classification of animals according to the type of feed during the breeding period. The stable isotope technique is based on the fact that in the photosynthesis process, 13C of atmospheric CO2 undergoes isotopic differentiation in relation to the total carbon when integrated into plant tissues. The stable isotopes were initially used in geological and archeological studies. Given that the isotopic compositions of animal tissues are frequently related to the isotopic compositions of their diets (DeNiro and Epstein, 1978), in the last decades, they have been used increasingly and continuously in research on agriculture, environment, and animal and human nutrition and metabolism studies (Cruz et al., 2004). According to the concept that an animal reflects the isotopic signal of its diet (DeNiro and Epstein, 1978) after a determined time interval, broiler chickens, as well as other production animals, can eventually be submitted to a diet containing animal ingredients in the starter phase with posterior substitution for a vegetable diet in an attempt to imprint the isotopic signal of the second diet in their tissues at slaughter age. Besides, the improvement of this technique can certify poultry destined for the international market as if they were exclusively fed with a vegetable diet. Due to the development of the stable-isotope technique to trace and certify the dietary standard of
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Sample Collection and Analytical Determination At 42 d old, 4 birds/treatment were randomly chosen and killed by neck dislocation to obtain samples of the breast, drumstick, thigh, and wings for isotopic analyses. All tissue samples were identified and frozen at −20°C. Later, the samples were unfrozen, washed in distilled water, and dried in a forced-ventilation oven (Marconi model MA 035, Greifensee, Switzerland) at 56°C for 48 h. Then, all of the samples were ground in a cryogenic mill (Spex, model 6700 freezer/mill, Metuchen, NJ) at −196°C for 3 min (tissues) and 5 min (diets) at maximum frequency to obtain a very thin granulometric homogeneous material with microscopic aspect (Licatti, 1997; Ducatti, 2004). The isotopic analyses of tissue samples and diets were carried out at the Stable Isotopes Center of the Institute of Biosciences of UNESP, Botucatu, SP, Brazil. Approximately 50 to 60 μg and 500 to 600 μg of samples were used to measure the isotopic ratios of 13C/12C and 15N/14N, respectively. They were weighed inside tin capsules, and then they were coupled to an elemental analyzer (EA 1108, CHN, Fisons Instruments, Rodano, Italy). An isotopic ration mass spectrometer (DELTA-S, Finnigan Mat, Bremen, Germany) was used
Table 1. Ingredient percentage of composition, calculated nutritional values, and average isotopic values of broiler diets Starter diet Item Ingredient (%) Corn, grain Soybean, meal Poultry, offal meal Soybean, oil Limestone Dicalcium phosphate dl-Methionine l-Lysine Choline chloride, 70% Salt Vitamin-mineral supplement1 Total Calculated composition AMEn (kcal/kg) CP (%) Crude fiber (%) Ca (%) Available P (%) Methionine (%) Methionine + cysteine (%) Lysine (%) Average isotopic value2 δ13C (‰) δ15N (‰)
Grower diet
0%
8%
0%
8%
57.70 35.56 — 2.64 0.98 1.83 0.23 0.16 0.04 0.46 0.40 100
65.16 23.90 8.00 0.10 0.82 0.80 0.20 0.22 0.04 0.36 0.40 100
63.25 29.82 — 3.11 0.94 1.63 0.22 0.22 0.04 0.38 0.40 100
70.66 18.18 8.00 0.59 0.77 0.60 0.18 0.28 0.04 0.30 0.40 100
3,000 21.40 3.23 0.96 0.45 0.56 0.90 1.26 −17.72 ± 0.25 1.75 ± 0.18
3,000 21.40 2.80 0.96 0.45 0.56 0.90 1.26 −16.22 ± 0.10 2.67 ± 0.11
3,100 19.30 3.00 0.88 0.41 0.51 0.83 1.16 −17.09 ± 0.16 1.85 ± 0.24
3,100 19.30 2.57 0.88 0.41 0.51 0.83 1.16 −15.76 ± 0.07 3.26 ± 0.10
1Starter vitamin-mineral supplement Vaccinar (levels per kilogram of diet, Vaccinar Nutrição and Saúde Animal, Belo Horizonte, Minas Gerais, Brazil): vitamin A, 14,000 IU; vitamin D3, 2,500 IU; vitamin E, 25 mg; vitamin K3, 3 mg; thiamine, 2 mg; riboflavin, 5 mg; pyridoxine, 4 mg; vitamin B12, 25 µg; niacin, 35 mg; pantothenic acid, 12 mg; biotin, 0.10 mg; folic acid, 1 mg; choline, 800 mg; butylated hydroxytoluene antioxidant, 2 mg; selenium, 0.18 mg; iron, 50.10 mg; manganese, 78 mg; iodine, 0.70 mg; copper, 10 mg; and zinc, 55 mg. Grower vitamin-mineral supplement Vaccinar (levels per kilogram of diet): vitamin A, 10,000 IU; vitamin D3, 2,000 IU; vitamin K3, 2 mg; thiamine, 2 mg; riboflavin, 4 mg; pyridoxine, 4 mg; vitamin B12, 20 µg; niacin, 30 mg; pantothenic acid, 10 mg; biotin, 0.06 mg; folic acid, 1 mg; choline, 600 mg; antioxidant, 2 mg; selenium, 0.18 mg; iron, 50.10 mg; manganese, 78 mg; iodine, 0.70 mg; copper, 10 mg; and zinc, 55 mg. 2Average isotopic values of δ13C and δ15N relative to the international standard Peedee Belemnite and atmospheric nitrogen (N ), respectively. 2
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offal meal (8% OM) diet after a certain age. The opposite was done to G5 and G6 birds that started the rearing phase with an OM diet and then were changed to a vegetable diet. The G2 birds received the OM diet throughout the experimental period (OM up to 42 d). Tissues from all broiler groups were compared with the respective standard tissues of green chicken (G1, the control group), without animal ingredients in the diet throughout the rearing period (vegetable diet up to 42 d old). The diets were formulated to meet the nutritional requirements by Rostagno et al. (2000) of a feeding program with 2 nutritional levels from 1 to 21 d old (starter diet) and from 22 to 42 d old (grower diet). The nutritional contents of both diets were equal for energy, protein, calcium, phosphorus, methionine, methionine + cystine, and lysine. Table 1 shows the percentage of composition, calculated nutritional contents, and isotopic values of the experimental diets. Each ingredient used in the diet came from the same batch. The poultry OM used in this experiment was from a poultry slaughterhouse located in the city of Tietê, State of São Paulo, Brazil, and had the following average values: δ13C = −16.85 ± 0.13‰ and δ15N = 4.07 ± 0.10‰. All procedures involving animals were approved by the São Paulo State University Committee for Animal Care and Use.
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(13C/12C)
(15N/14N)
to determine carbon and nitrogen isotopic ratios according to the method described by Ducatti et al. (1979). The results of the analyses were expressed in parts per thousand (‰) in relation to Peedee Belemnite international standard and atmospheric nitrogen (N2) for carbon and nitrogen elements, respectively, according to the expression: δ‰ (sample, standard) = [(Rsample – Rstandard)/Rstandard] × 1,000,
Statistical Analyses The data obtained in the carbon and nitrogen isotopic analysis were submitted to multivariate ANOVA with the help of GLM of the SAS statistical program (SAS Institute, 2000). From the data generated by the error matrices for each tissue, regions (ellipses) were defined within 95% confidence to verify differences among the averages of the experimental treatments and the average of the control group (a strictly vegetable diet).
RESULTS AND DISCUSSION The results of the isotopic analyses (δ13C and δ15N) of the diets used in this study are in Table 1. It was observed that the values of δ15N enriched both the vegetable diet (0%) as well as the OM diet (8%) like the starter diet (1–21 d old) and the grower diet (22–42 d old) within the same diet. This probably occurred due to the variation in the percentage of composition of the ingredients in the diet. The feed of the OM diet had less soybean meal and more corn and OM when compared with that of the vegetable diet feed. A similar enrichment observed in the starter diet is also verified in the grower diet and this may be due to the same fact, there was a smaller percentage of soybean meal in the grower diet. Corn (δ13C = −12.15 ± 0.04‰ and δ15n = 6.81 ± 0.04‰), soybean meal (δ13C = −26.15 ± 0.10‰ and δ15n = 0.55 ± 0.04‰), and poultry OM (δ13C = −16.85 ± 0.13‰ and δ15n = 4.07 ± 0.10‰) used to make the feed were also isotopically analyzed for nitrogen and carbon. It is known that soybean meal has a value of δ15N that is close to the standard value of atmospheric N2 (δ15N ≅ 0.0 ± 1.0‰) because the soy plants fix the air nitrogen and present a fractioning factor of δ15N around one unity, generally lower than 1.003‰ (Kohl and Shearer, 1980; Handley and Raven, 1992; Werner and Schmidt, 2002). The value of δ15N presented by plants that cannot fix atmospheric nitrogen depends
greatly on the abundance of this isotope in the managed soil and fertilizers, like in corn (Choi et al., 2002). Sleiman et al. (2004), assessing corn samples from some Brazilian states, found average values of 4.77 ± 1.16‰ for δ15N and −11.74 ± 0.40‰ for δ13C, respectively. Similar to the enrichment that occurred for the values of δ15N described above, the same was observed for the values of δ13C in the experimental diets because when OM is included in the diet formulation, a smaller quantity of soybean meal and oil was required to balance it in relation to the corresponding isocaloric and isonitrogenous vegetable diet. Besides that, the inclusion of an energetic ingredient (i.e., corn) was increased. This happens because plants from the C3 photosynthetic cycle (e.g., soybean meal, rice, and wheat) have a modal value of δ13C = −27.6‰ in relation to the Peedee Belemnite international standard, and in plants from the C4 photosynthetic cycle (e.g., corn, sorghum, and sugarcane) this modal value is δ13C = −12.6‰ (Vogel, 1993). Grower diets would be richer in 13C when compared with that of starter diets because they are nutritionally more energetic and less proteic, making the percentage of composition of the ingredients change within the same diet, although these values may not present significant differences. Therefore, grower diets would have more corn and less soybean meal. These figures related to the values of δ13C and δ15N in broiler diets, as well as in other species’ diets, are fundamental to identifying the presence of animal byproducts in food once the isotopic signature of the diet is reflected in the animals’ organism. According to DeNiro and Epstein (1976, 1978), an animal is what it consumes isotopically, up to 2.0‰ for δ13C and up to 3.0‰ for δ15N, although each tissue of the same animal may present a particular isotopic signature, fractioning factor (Hobson and Clark, 1992b), and isotopic turnover (Hobson and Clark, 1992a). The average values of δ13C and δ15N for broiler breast, drumstick, thigh, and wing meat in the end of the experimental period are presented in Table 2, and the results of the statistical analysis for isotopic pairs (δ13C and δ15N) for the tissues of the birds from groups G2, G3, G4, G5, and G6 when compared with that of the control group (G1) are illustrated in Figure 1. In the G3 and G4 treatments, the birds were fed with a vegetable diet at first, and then after a determined age (21 and 35 d, respectively) 8% OM was included in the diet until slaughtering age (42 d). In all analyzed tissues, it was observed that these treatments differed from the control group (G1) because their confidence regions did not overlap any axis of the graph (Figure 1). Likewise, when analyzing the data from the breast (Figure 1A), drumstick (Figure 1B), thigh (Figure 1C), and wing (Figure 1D), besides treatments G5 and G6 where birds had an 8% OM diet in the first breeding phase followed by a vegetable diet after a specific age (21 and 35 d, respectively), treatment G2 also differed from the control group showing the biggest distance
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where R represents the ratio between the heaviest and the lightest isotope, specifically 13C/12C and 15N/14N. Each sample was analyzed twice to obtain average values. The measurements were replicated when the analytical SD was greater than 0.2‰ for δ‰13C.
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482 1G1 = vegetable diet with corn and soybean meal without animal-origin by-product (basal diet) from 1 to 42 d old; G2 = 8% poultry offal meal (OM) diet from 1 to 42 d old; G3 = basal diet from 1 to 21 d old and 8% OM diet from 22 to 42 d old; G4 = basal diet from 1 to 35 d old and 8% OM diet from 36 to 42 d old; G5 = 8% OM diet from 1 to 21 d old and basal diet from 22 to 42 d old; and G6 = 8% OM diet from 1 to 35 d old and basal diet from 36 to 42 d old.
0.09 0.09 0.12 0.08 0.03 0.14 ± ± ± ± ± ± 2.75 3.57 3.47 3.01 3.01 3.47 ± ± ± ± ± ± −18.99 −17.00 −17.42 −18.43 −18.46 −17.49 0.09 0.13 0.19 0.13 0.21 0.18 ± ± ± ± ± ± 3.25 4.30 4.13 3.62 3.58 4.15 ± ± ± ± ± ± −19.18 −17.19 −17.50 −18.52 −18.66 −17.62 0.12 0.32 0.27 0.05 0.17 0.26 ± ± ± ± ± ± ± ± ± ± ± ± −19.06 −16.94 −17.32 −18.38 −18.56 −17.56 0.11 0.05 0.13 0.06 0.16 0.08 ± ± ± ± ± ± ± ± ± ± ± ± −19.28 −17.18 −17.57 −18.56 −18.69 −17.69 G1 G2 G3 G4 G5 G6
Diet1
δ13C
0.15 0.14 0.08 0.19 0.17 0.09
2.36 3.26 3.15 2.74 2.55 3.05
δ15N
δ13C
0.13 0.09 0.06 0.20 0.13 0.12
3.30 4.36 4.25 3.78 3.72 4.26
δ15N
δ13C
0.14 0.11 0.11 0.15 0.07 0.09
Thigh (‰) Drumstick (‰) Breast (‰)
Table 2. Average δ13C and δ15N values and their respective SD in different tissues of 42-d-old broilers
between the x- and y-axes among the analyzed groups. This is possibly because G2 consisted of birds having an OM diet throughout the experimental period. Overall, the differences among the treatment δ15N averages in relation to the control group are smaller than the differences among the δ13C averages when comparing all tissues, making δ15N important for the detection of OM utilization in the broiler diet. Thus, the confidence regions in the presented graphics become more distant along the carbon axis than along the nitrogen one. This probably resulted from the carbon and nitrogen isotopic differences between the vegetable diet and the OM diet where the latter is, on average, 1.41‰ richer for δ13C and 1.16‰ richer for δ15N. Carrijo et al. (2006) verified that the 8% meat and bone meal diet was 1.17‰ richer for δ13C and 1.20‰ richer for δ15N, on average, when compared with the respective vegetable diet. Oliveira et al. (2010), when feeding broilers an 8% poultry offal meal diet, observed that it was 2.27‰ richer for δ13C and 1.05‰ richer for δ15N. The differences of nitrogen isotopic ratios observed among the analyzed tissues must be greatly related to the composition of these tissues in muscle fibers. Broiler chickens’ muscle, when transformed into meat during the postmortem period, is an important source of highquality protein for human nutrition. Regardless of not performing the muscle functions anymore, it is important to consider that the meat is still a live substance whose metabolic processes are partly dependent on the originating muscle. The skeletal muscle presents striated fibers and an important physiological function in providing movement to the living bird. Its varied fiber composition allows the adaptation to different environments or selection realities and may be reflected in the also varied quality of the meat (Scheuermann, 2008). When the ellipsis formed by the data from the drumstick (Figure 1B) and thigh (Figure 1C) were observed, a very similar behavior among them was noticed. This is probably due to the composition of their muscles that are red, richly vascularized, and formed by oxidative and oxidative-glycolytic fibers. Simultaneously, when the ellipsis formed by data from the breast (Figure 1A) are observed, they behave differently from the previous ones. This is probably because this tissue is formed predominantly by glycolytic fibers and oxidative-glycolytic fibers, which is different from the muscle constitution of drumsticks and thighs. On the other hand, the breast ellipses were similar to the ellipses from the wing data (Figure 1D); it was possibly because the function of this muscle in broilers does not have the same function as in flying poultry once they have lost their flying capacity along the species evolution, showing a similar behavior to the breast. Dubowitz and Brooke (1984) reported that the difference in metabolic and contractile behaviors among muscle fibers can be shown by histochemical techniques, classifying them into 3 muscle fibers: white, red, and intermediate ones. The white fibers, rich in glycolytic enzymes, are adapted to the anaerobic metabolism and
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δ15N
δ13C
0.15 0.04 0.07 0.06 0.11 0.06
Wing (‰)
δ15N
Cruz et al.
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Figure 1. A) Confidence regions formed by the difference among the 42-d-old broiler breast isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). B) Confidence regions formed by the difference among the 42-d-old broiler drumstick isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). C) Confidence regions formed by the difference among the 42-d-old broiler thigh isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). D) Confidence regions formed by the difference among the 42-d-old broiler wing isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing).
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in the percentage of composition of their biochemical fractions, such as lipids, carbohydrates, and proteins, comparatively in which the lipid fraction can present depletion of 13C (DeNiro and Epstein, 1978). Analyzing the composition of CP, ether extract, and mineral matter of the breast, legs (drumstick and thigh), and wings of 49-d-old broilers, Faria Filho et al. (2006) found higher values of protein in the breast (72.6%), followed by the legs (50.5%) and wings (45.3%); higher values of ether extract were found for the wing (42.9%), followed by the legs (37.8%) and breast (21.2%). The latter had significantly lower values of mineral matter (6.2%) compared with that of the legs (11.7%) and wing (11.8%), which did not differ among them. In addition to what has been reported until now, it is probable that the management of diets richer in 15N and 13C to animals that have a larger contrast in the isotopic signatures in relation to the vegetable diet allows for an easier determination of significant differences between the treatments and the basal diet. This is because, at first, the isotopic enrichment of nitrogen and carbon in tissues is related to the isotopic abundance of the diet and the analyzed tissue type (DeNiro and Epstein, 1978; Tieszen et al., 1983; Shoeninger and DeNiro, 1984). The results found in this study seem to be important for the technological improvement involved in the identification process of broiler carcasses of birds fed without poultry OM in their diet to meet the demands of specific markets. However, complementary studies have to be done to evaluate the practical potential of this technique in the poultry segment, helping the traceability process that is an important tool for the certification of poultry products. This is because the stable isotopes serve as natural tracers in the investigation of food programs (Jones et al., 1979; Tyrrell et al., 1984; DeNiro, 1987; Peterson and Fry, 1987; Carrijo et al., 2006; Móri et al., 2007, 2008; Gottmann et al., 2008; Denadai et al., 2009; Oliveira et al., 2010), using only the natural isotopic variation of ingredients without the need for using specifically marked or radioactive compounds. Besides, other tissues deserve to be studied for this purpose. Still, it is worthwhile to note that the analyses were carried out in 42-d-old broilers. The application of this technique can be viable for the same kind of study in younger birds because they would have the isotopic signal of a stabilized diet in their tissues when they are around 2 wk old (Oliveira et al., 2010). Therefore, if this technique were validated in practice in the poultry industry, it would be suggested that even before the slaughtering age, the birds would be sampled for isotopic analysis still in the poultry farms, rather than in the slaughter house or in the exportation container. Thus, the use of the technique of carbon- and nitrogen-stable isotopes is promising for the detection of animal ingredient use in a broiler diet through the isotopic analyses of their tissues.
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use glycogen as an energetic substrate. The red fibers, rich in oxidative enzymes, have aerobic metabolism and use fat as the main energetic substrate. The white fibers, also called fast glycolytic (FG), presented a larger area and fast contraction, whereas the red fibers are called slow oxidative (SO) and have a small area and slow contraction. The intermediate fibers are called fast oxidative glycolytic (FOG) and present an area size that is intermediate to the white and red ones, aerobic and glycolytic metabolism and fast contraction. They also have red color (Peter et al., 1972). According to the metabolic characteristics, demonstrated by histochemical techniques, the fibers are classified as type I with low contraction and oxidative metabolism; type IIA with fast contraction and oxidative-glycolytic metabolism; and type IIB with fast contraction and gycolytic metabolism. These types correspond to SO, FOG, and FG fibers, respectively. Type I or SO fibers are small, have numerous mitochondria and abundant myoglobin that gives them a red color. Mitochondria are large and have several crysts; therefore, the oxygen storage by myoglobin in these fibers avoids fatigue. Type II fibers are large muscle cells that have a small amount of myoglobin and mitochondria. Type IIA (FOG) fibers are resistant to fatigue, whereas IIB (FG) fibers are easily fatigued and latic acid accumulates in them easily. Type IIA fibers are considered intermediate between I and IIB and adaptable, becoming more glycolytic or more oxidative according to the demand of the muscle activity. Type I and IIA fibers are aerobic and depend on a high rate of oxygen and metabolic exchanges. However, anaerobic fibers (IIB) have a lower rate of metabolic exchanges and oxygen and present a larger area, and therefore, a greater hypertrophic process. In the presence of one or more types of fibers, their distribution and subtype frequency is what determines the metabolic and contractile characteristics of the skeletal muscle tissue, showing their biochemical and physiological properties (Macari et al., 2002). In laying hens and broilers, the breast muscle has predominantly FG and FOG fibers and, histologically, presents a small density of blood capillaries; their cells have a small number of mitochondria. The red muscle of the drumstick and thigh, on the other hand, is richly vascularized with many mitochondria in their fibers, mainly SO and FOG types (Macari et al., 2002). The muscle fiber distribution in a determined muscle in different animal species and individuals is a functional adaptation to their multiple types of activity. Thus, the rate of different muscle fiber types is directly correlated to its function in the animal organism. The definitive phenotype of adult skeletal muscle fibers is the result of events that start in the embryo and are continuously modulated and refined throughout the organism’s life. The observed variations among the tissues for the δ13C values can be partly related to the differences
OFFAL MEAL TRACEABILITY WITH ISOTOPES
Under experimental conditions, it can be concluded that the application of the technique of carbon- and nitrogen-stable isotopes can trace poultry OM use in a broilers’ diet during breeding or when OM substitutes a strictly vegetable diet, even up to 35 d old. When a poultry OM diet is substituted by a strictly vegetable diet, the animal-origin ingredient must be part of the diet for 21 d or longer so that its detection can be possible using this method.
ACKNOWLEDGMENTS
REFERENCES Avisite. 2011. Estatísticas e preços. WebMD. Accessed Aug. 2011. http://www.avisite.com.br/economia/estatistica.asp?acao= producaopintos. Bloch, C., Jr. 2002. Monitoramento da qualidade de rações brasileiras para ruminantes por espectrometria de massa. Pages 251–252 in Simpósio sobre manejo e nutrição de aves e suínos e tecnologia da produção de rações. Campinas: Colégio Brasileiro de Nutrição Animal. Bricout, J., and J. Koziet. 1987. Control of the authenticity of orange juice by isotopic analysis. J. Agric. Food Chem. 35:758–760. Brookes, S. T., A. Barrie, and J. E. Davies. 1991. A rapid 13C/12C test for determination of corn-syrups in honey. J. Assoc. Anal. Chem. 74:627–629. Carrijo, A. S., A. C. Pezzato, C. Ducatti, J. R. Sartori, L. Trinca, and E. T. Silva. 2006. Traceability of bovine meat and bone meal in poultry by stable isotope analysis. Braz. J. Poult. Sci. 8:37–42. Choi, W. J., S. M. Lee, H. M. Ro, K. C. Kim, and S. H. Yoo. 2002. Natural 15N abundances of maize and soil amended with urea and composted pig manure. Plant Soil 245:223–232. Cruz, V. C., A. C. Pezzato, C. Ducatti, D. F. Pinheiro, J. R. Sartori, and J. C. Gonçalves. 2004. Tracing metabolic routes of feed ingredients in tissues of broiler chickens using stable isotopes. Poult. Sci. 83:1376–1381. Denadai, J. C., C. Ducatti, J. R. Sartori, A. C. Pezzato, C. Móri, R. Gottmann, and M. A. O. Mituo. 2009. Rastreabilidade da farinha de carne e ossos bovinos em ovos de poedeiras alimentadas com ingredientes alternativos. Pesquisa Agropecu. Bras. 44:1–7. DeNiro, M. J. 1987. Stable isotopes and archaelogy. Am. Sci. 75:182–191. DeNiro, M. J., and S. Epstein. 1976. You are what you eat (plus a few ‰) the carbon isotope cycle in food chains. Geol. Soc. Am. 6:834. (Abstr.) DeNiro, M. J., and S. Epstein. 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochim. Cosmochim. Acta 42:495–506. Dubowitz, V., and M. Brooke. 1984. Page 472 in Muscle Biopsy: A Modern Approach. W. B. Saunders Co., London, UK.
Ducatti, C. 2004. Page 65 in Isótopos estáveis ambientais. Instituto de Biociências, Universidade Estadual Paulista, Botucatu, Brazil. Ducatti, C., E. Salati, and E. Matsui. 1979. Método de análise da razão 13C/12C em matéria orgânica e das razões 13C/12C e 18O/16O em carbonatos. Proc. Acad. Bras. Cienc. 51:275–286. Faria Filho, D. E., P. S. Rosa, D. F. Figueiredo, F. Dahlke, M. Macari, and R. L. Furlan. 2006. Dietas de baixa proteína no desempenho de frangos criados em diferentes temperaturas. Pesquisa Agropecu. Bras. 41:101–106. González-Martin, I., C. González-Pérez, J. Hernández-Méndez, E. Marqués-Macias, and F. Sanz Poveda. 1999. Use of isotope analysis to characterize meat from Iberian-breed swine. Meat Sci. 52:437–441. Gottmann, R., A. C. Pezzato, C. Ducatti, J. C. Denadai, C. Mori, M. A. O. Mituo, and J. R. Sartori. 2008. Rastreabilidade de subprodutos de origem animal em dietas com levedura e trigo para frangos. Pesquisa Agropecu. Bras. 43:1641–1647. Handley, L. L., and J. A. Raven. 1992. The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ. 15:965–985. Hobson, K. A., and R. G. Clark. 1992a. Assessing avian diets using stable isotopes. I: Turnover of 13C in tissues. Condor 94:181–188. Hobson, K. A., and R. G. Clark. 1992b. Assessing avian diets using stable isotopes. II: Factors influencing diet-tissue fractionation. Condor 94:189–197. Jones, R., M. Ludlow, and J. Troughton. 1979. Estimation of the proportion of C3 and C4 plant species in diet of animals from the ratio of natural 12C and 13C isotopes in the feces. J. Agric. Sci. 92:91–100. Kelly, S., I. Parker, M. Sharman, J. Dennis, and I. Goodall. 1997. Assessing the authenticity of single-seed vegetable oils using fatty acid stable carbon isotope ratios (13C/12C). Food Chem. 59:181–186. Kohl, D. H., and G. Shearer. 1980. Isotopic fractionation associated with symbiotic N2 fixation and uptake of NO3– by plants. Plant Physiol. 66:51–56. Koziet, J., A. Rossmann, G. J. Martin, and P. R. Ashurst. 1993. Determination of carbon-13 content of sugars of fruit and vegetable juices. Anal. Chim. Acta 271:31–38. Licatti, F. 1997. Page 40 in Isótopos estáveis do carbono (13C/12C) em plantas do ciclo bioquímico C3 e C4. Monografia (Trabalho de Graduação em Biologia)—Instituto de Biociências, Universidade Estadual Paulista, Botucatu, Brazil. Macari, M., R. L. Furlan, and E. Gonzales. 2002. Page 375 in Fisiologia aviária aplicada a frangos de corte. FUNEP, Jaboticabal, Brazil. Manca, G., F. Camin, G. Coloru, A. Del Caro, D. Detentori, M. A. Franco, and G. Versini. 2001. Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope (13C/12C and 15N/14N) ratios and free amino acid ratios. J. Agric. Food Chem. 49:1404–1409. Martin, G. J., C. Guillou, M. L. Martin, M. T. Cabanis, X. Tep, and J. Aerny. 1988. Natural factors of isotope fractionation and the characterization of wines. J. Agric. Food Chem. 36:316–322. Móri, C., E. A. Garcia, C. Ducatti, J. C. Denadai, R. Gottmann, and M. A. O. Mituo. 2008. Poultry offal meal traceability in meat quail tissues using the technique of stable carbon (13C/12C) and nitrogen (15N/14N) isotopes. Braz. J. Poult. Sci. 10:45–52. Móri, C., E. A. Garcia, C. Ducatti, J. C. Denadai, K. Pelícia, R. Gottmann, M. A. O. Mituo, and A. M. Bordinhon. 2007. Traceability of animal by-products in quail (Coturnix coturnix japonica) tissues using carbon (13C/12C) and nitrogen (15N/14N) stable isotopes. Braz. J. Poult. Sci. 9:263–269. Oliveira, R. P., C. Ducatti, A. C. Pezzato, J. C. Denadai, V. C. Cruz, J. R. Sartori, A. S. Carrijo, and F. R. Caldara. 2010. Traceability of poultry offal meal in broiler feeding using isotopic analysis (δ13C and δ15N) of different tissues. Braz. J. Poult. Sci. 12:13–20. Peter, J. B., R. J. Barnard, V. R. Edgerton, C. A. Gillespie, and K. E. Stempel. 1972. Metabolic profiles of three fiber types of skeletal muscle in Guinea pig and rabbits. Biochem. 11:2627–2633. Peterson, B. J., and B. Fry. 1987. Stable isotope in ecosystem studies. Annu. Rev. Ecol. Syst. 18:293–320.
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The authors thank the National Council for Scientific and Technological Development (CNPq, Brasília, Distrito Federal, Brazil) for their financial support. We also thank the postdoc fellowship [Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, Brazil] for the publication financial support, the company Frangoeste (Tietê, São Paulo, Brazil) for the donation of the poultry offal meal, the Laboratory of Poultry Nutrition (Botucatu, São Paulo, Brazil) and The Center of Stable Isotopes of UNESP—Campus of Botucatu (Botucatu, São Paulo, Brazil) for allowing the development of this study and the samples and data processing.
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Piasentier, E., R. Valusso, F. Camin, and G. Versini. 2003. Stable isotope ratio analysis for authentication of lamb meat. Meat Sci. 64:239–247. Rastreabilidade. 2004.Rastreabilidade e certificação: Um modelo de etapas para implantação de um sistema nacional para rastreabilidade na cadeia de produção de aves. Revista Avicultura Industrial, Porto Feliz, ano 95, n. 03, ed. 1121. WebMD. Accessed Oct. 2004. http://www.aviculturaindustrial.com.br. Rossmann, A., G. Haberhauer, S. Holzl, P. Horn, F. Pichlmayer, and S. Voerkelius. 2000. The potential of multielement stable isotope analysis for regional origin assignment of butter. Eur. Food Res. Technol. 211:32–40. Rostagno, H. S., L. F. T. Albino, and J. L. Donzele. 2000. Tabelas brasileiras para aves e suínos: Composição de alimentos e exigências nutricionais. H. S. Rostagno, ed. UFV, Viçosa, Brazil. SAS Institute. 2000. User’s Guide. SAS Inst. Inc., Cary, NC. Scheuermann, G. N. 2008. Alteração na quantidade e qualidade da carne de aves através da manipulação das fibras musculares. WebMD. Accessed Jul. 2010. http://www.avisite.com.br/cet/trabalhos.asp?codigo=69. Shoeninger, M. J., and M. J. DeNiro. 1984. Nitrogen and carbon isotopic composition of bone collagen from marine and terrestrial animals. Geochim. Cosmochim. Acta 48:625–639.
Key words: animal origin ingredient, carbon-13, certification, nitrogen-15, poultry 2012 Poultry Science 91:478–486 doi:10.3382/ps.2011-01512
INTRODUCTION
been increasing with concern for food safety. This change in the consumer trend is one of the main stimulating factors to propose and adopt methods that allow for the certification of the origin and quality of animal and vegetable products in the world. A research approach aiming to trace the inclusion of animal-origin ingredients in the broiler diet was developed because of the need to find a technology that was, at first, independent in order to certify the vegetable feeding pattern of these birds and their products, and then to meet the demands of consumers who wish for transparency of the conditions and methods for food production, besides guaranteeing fair competition among companies from competing countries. Traceability of the meat production system is a guarantee to consumers because it makes them sure that a product is controlled in all production stages: from the farm to the final consumer. The term traceability refers to the capacity to trace the history, application, or localization of an item through previously registered information (Rastreabilidade, 2004).
Brazil is the world’s leading chicken meat exporter and its exportation volume has been growing—3,819.7 thousand tonnes in the last year. It is the world’s second leading chicken meat producer and currently has an estimated herd of 473.3 million chicks produced per month (Avisite, 2011). Although there have been some decreases in production and global exportation as well as in intake and importation in some countries due to consumer’s fear of possible risks from the avian flu, which occurred some years ago, poultry production in Brazil has shown great dynamism and efficiency. The consumer trend to acquire healthy foods of which the origin, nutritional value, and quality is known has
©2012 Poultry Science Association Inc. Received March 29, 2011. Accepted October 2, 2011. 1 Corresponding author: [email protected]
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and nitrogen (15N/14N) by mass spectrometry. In total, 720 one-d-old chicks were distributed into 6 groups: vegetable diet (VD) from 1 to 42 d; 8% poultry offal meal (OM) diet from 1 to 42 d; VD from 1 to 21 d and 8% OM diet from 22 to 42 d; VD from 1 to 35 d and 8% OM diet from 36 to 42 d; 8% OM diet from 1 to 21 d and VD from 22 to 42 d; and 8% OM diet from 1 to 35 d and VD from 36 to 42 d. Through the analysis of C and N, it is possible to trace the use of OM in broiler feeding when this ingredient is part of the feeding throughout the breeding phase or when it substitutes a strictly VD even up to 35 d. When an OM diet is substituted by a VD, the animal ingredient has to be part of the feeding for 21 d or longer to be detected by this method.
ABSTRACT Studies on the detection of animal byproducts in poultry meat are rare and practically nonexistent in chicken meat. With the development of the technique of stable isotopes for traceability purposes and the certification of broiler diet patterns, it has been necessary to know the behavior of the isotopic signature of different tissues in birds, in case of a potential replacement of a diet containing animal ingredients with a strictly vegetable one and vice versa. Thus, this study, carried out at the São Paulo State University, Botucatu Campus, Brazil, aimed to evaluate meat from the breast, thigh, drumstick, and wings to trace the presence of poultry offal meal (OM) in broiler feed using the analysis of stable isotopes of carbon (13C/12C)
OFFAL MEAL TRACEABILITY WITH ISOTOPES
broiler chickens, it is necessary to know the behavior of the isotopic signature of different poultry tissues to detect an eventual substitution of an animal diet for a strictly vegetable diet and vice versa. Therefore, this study aimed to analyze meat from the breast, drumstick, thigh, and wings to trace the presence of poultry offal meal in broiler feed using the analysis of carbon (13C/12C) and nitrogen (15N/14N) stable isotopes through mass spectrometry.
MATERIALS AND METHODS Birds and Diets In total, 720 one-day-old male Cobb chicks from a flock of 43-wk-old hens were used. The chicks were previously vaccinated against infectious bursal disease, Marek’s disease, and Bouba’s disease, and they were raised until 42 d of age. During the experiment, the animals were vaccinated against coccidiosis and infectious bursal disease via drinking water. Then, the birds were distributed in a completely random block design with 6 treatments and 4 replications, and they were raised and kept in an experimental rearing house divided into twenty-four 2.5-m2 cages, with 30 birds/cage, in a density of 12 birds/m2. Water and feed were offered ad libitum using a trough drinker and feeder that were substituted gradually by a hanging drinker and a tubular feeder. For each cage, initial brooding for the chicks was done using a 250-W infrared light bulb that was removed when the chicks were 8 d old. Temperature and ventilation were controlled manually by managing the rearing house lateral curtains. The light program was constant using 60-W incandescent light bulbs. The experimental treatments were established according to the following groups (G): • G1, vegetable diet with corn and soybean meal without animal-origin by-product (basal diet) from 1 to 42 d old; • G2, 8% poultry offal meal (OM) diet from 1 to 42 d old; • G3, basal diet from 1 to 21 d old and 8% OM diet from 22 to 42 d old; • G4, basal diet from 1 to 35 d old and 8% OM diet from 36 to 42 d old; • G5, 8% OM diet from 1 to 21 d old and basal diet from 22 to 42 d old; and • G6, 8% OM diet from 1 to 35 d old and basal diet from 36 to 42 d old. The percentage of OM inclusion in the diet was calculated at 8%, according to the information on adopted practice levels in broiler producing companies (Frangoeste Avicultura Ltda, Tietê, São Paulo, Brazil). Initially, G3 and G4 birds were submitted to a strictly vegetable diet (0% OM) and then changing to a poultry
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Not only is the traceability process a requirement to aggregate value to a specific product, but it is also a market requirement that arises due to the increase in the consumer’s demand and food safety that resulted from several crises and episodes related to human and animal health. This process should be adopted by companies involved in the national poultry production if they wish to keep important market shares, such as the European market, in the near future. Numerous methods have been proposed to identify the presence of animal-origin by-products in animal feeding, such as DNA hybridization, ELISA, and PCR (Bloch, 2002). On the other hand, although there is no official traceability system in the poultry production chain, mass spectrometry has been used successfully, through the carbon-13 isotopic ratio analysis, to test the authenticity, quality, and geographical origin of several products, such as fruit juice (Bricout and Koziet, 1987; Koziet et al., 1993), wine (Martin et al., 1988), honey (Brookes et al., 1991; White et al., 1998), dairy products (Rossmann et al., 2000; Manca et al., 2001), vegetal oils (Kelly et al., 1997), and the characterization and differentiation of the diet regimen of Iberian swines (González-Martin et al., 1999), certification of geographical origin, types of sheep feeding (Piasentier et al., 2003), traceability of bovine meat and bone meal in broiler feed (Carrijo et al., 2006), poultry offal meal in quail and broiler feed (Móri et al., 2007, 2008; Oliveira et al., 2010), poultry offal meal with yeast and wheat meal in broiler feed (Gottmann et al., 2008), and bovine meat and bone meal in the feed of laying hens (Denadai et al., 2009). This allows for the classification of animals according to the type of feed during the breeding period. The stable isotope technique is based on the fact that in the photosynthesis process, 13C of atmospheric CO2 undergoes isotopic differentiation in relation to the total carbon when integrated into plant tissues. The stable isotopes were initially used in geological and archeological studies. Given that the isotopic compositions of animal tissues are frequently related to the isotopic compositions of their diets (DeNiro and Epstein, 1978), in the last decades, they have been used increasingly and continuously in research on agriculture, environment, and animal and human nutrition and metabolism studies (Cruz et al., 2004). According to the concept that an animal reflects the isotopic signal of its diet (DeNiro and Epstein, 1978) after a determined time interval, broiler chickens, as well as other production animals, can eventually be submitted to a diet containing animal ingredients in the starter phase with posterior substitution for a vegetable diet in an attempt to imprint the isotopic signal of the second diet in their tissues at slaughter age. Besides, the improvement of this technique can certify poultry destined for the international market as if they were exclusively fed with a vegetable diet. Due to the development of the stable-isotope technique to trace and certify the dietary standard of
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Sample Collection and Analytical Determination At 42 d old, 4 birds/treatment were randomly chosen and killed by neck dislocation to obtain samples of the breast, drumstick, thigh, and wings for isotopic analyses. All tissue samples were identified and frozen at −20°C. Later, the samples were unfrozen, washed in distilled water, and dried in a forced-ventilation oven (Marconi model MA 035, Greifensee, Switzerland) at 56°C for 48 h. Then, all of the samples were ground in a cryogenic mill (Spex, model 6700 freezer/mill, Metuchen, NJ) at −196°C for 3 min (tissues) and 5 min (diets) at maximum frequency to obtain a very thin granulometric homogeneous material with microscopic aspect (Licatti, 1997; Ducatti, 2004). The isotopic analyses of tissue samples and diets were carried out at the Stable Isotopes Center of the Institute of Biosciences of UNESP, Botucatu, SP, Brazil. Approximately 50 to 60 μg and 500 to 600 μg of samples were used to measure the isotopic ratios of 13C/12C and 15N/14N, respectively. They were weighed inside tin capsules, and then they were coupled to an elemental analyzer (EA 1108, CHN, Fisons Instruments, Rodano, Italy). An isotopic ration mass spectrometer (DELTA-S, Finnigan Mat, Bremen, Germany) was used
Table 1. Ingredient percentage of composition, calculated nutritional values, and average isotopic values of broiler diets Starter diet Item Ingredient (%) Corn, grain Soybean, meal Poultry, offal meal Soybean, oil Limestone Dicalcium phosphate dl-Methionine l-Lysine Choline chloride, 70% Salt Vitamin-mineral supplement1 Total Calculated composition AMEn (kcal/kg) CP (%) Crude fiber (%) Ca (%) Available P (%) Methionine (%) Methionine + cysteine (%) Lysine (%) Average isotopic value2 δ13C (‰) δ15N (‰)
Grower diet
0%
8%
0%
8%
57.70 35.56 — 2.64 0.98 1.83 0.23 0.16 0.04 0.46 0.40 100
65.16 23.90 8.00 0.10 0.82 0.80 0.20 0.22 0.04 0.36 0.40 100
63.25 29.82 — 3.11 0.94 1.63 0.22 0.22 0.04 0.38 0.40 100
70.66 18.18 8.00 0.59 0.77 0.60 0.18 0.28 0.04 0.30 0.40 100
3,000 21.40 3.23 0.96 0.45 0.56 0.90 1.26 −17.72 ± 0.25 1.75 ± 0.18
3,000 21.40 2.80 0.96 0.45 0.56 0.90 1.26 −16.22 ± 0.10 2.67 ± 0.11
3,100 19.30 3.00 0.88 0.41 0.51 0.83 1.16 −17.09 ± 0.16 1.85 ± 0.24
3,100 19.30 2.57 0.88 0.41 0.51 0.83 1.16 −15.76 ± 0.07 3.26 ± 0.10
1Starter vitamin-mineral supplement Vaccinar (levels per kilogram of diet, Vaccinar Nutrição and Saúde Animal, Belo Horizonte, Minas Gerais, Brazil): vitamin A, 14,000 IU; vitamin D3, 2,500 IU; vitamin E, 25 mg; vitamin K3, 3 mg; thiamine, 2 mg; riboflavin, 5 mg; pyridoxine, 4 mg; vitamin B12, 25 µg; niacin, 35 mg; pantothenic acid, 12 mg; biotin, 0.10 mg; folic acid, 1 mg; choline, 800 mg; butylated hydroxytoluene antioxidant, 2 mg; selenium, 0.18 mg; iron, 50.10 mg; manganese, 78 mg; iodine, 0.70 mg; copper, 10 mg; and zinc, 55 mg. Grower vitamin-mineral supplement Vaccinar (levels per kilogram of diet): vitamin A, 10,000 IU; vitamin D3, 2,000 IU; vitamin K3, 2 mg; thiamine, 2 mg; riboflavin, 4 mg; pyridoxine, 4 mg; vitamin B12, 20 µg; niacin, 30 mg; pantothenic acid, 10 mg; biotin, 0.06 mg; folic acid, 1 mg; choline, 600 mg; antioxidant, 2 mg; selenium, 0.18 mg; iron, 50.10 mg; manganese, 78 mg; iodine, 0.70 mg; copper, 10 mg; and zinc, 55 mg. 2Average isotopic values of δ13C and δ15N relative to the international standard Peedee Belemnite and atmospheric nitrogen (N ), respectively. 2
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offal meal (8% OM) diet after a certain age. The opposite was done to G5 and G6 birds that started the rearing phase with an OM diet and then were changed to a vegetable diet. The G2 birds received the OM diet throughout the experimental period (OM up to 42 d). Tissues from all broiler groups were compared with the respective standard tissues of green chicken (G1, the control group), without animal ingredients in the diet throughout the rearing period (vegetable diet up to 42 d old). The diets were formulated to meet the nutritional requirements by Rostagno et al. (2000) of a feeding program with 2 nutritional levels from 1 to 21 d old (starter diet) and from 22 to 42 d old (grower diet). The nutritional contents of both diets were equal for energy, protein, calcium, phosphorus, methionine, methionine + cystine, and lysine. Table 1 shows the percentage of composition, calculated nutritional contents, and isotopic values of the experimental diets. Each ingredient used in the diet came from the same batch. The poultry OM used in this experiment was from a poultry slaughterhouse located in the city of Tietê, State of São Paulo, Brazil, and had the following average values: δ13C = −16.85 ± 0.13‰ and δ15N = 4.07 ± 0.10‰. All procedures involving animals were approved by the São Paulo State University Committee for Animal Care and Use.
OFFAL MEAL TRACEABILITY WITH ISOTOPES
(13C/12C)
(15N/14N)
to determine carbon and nitrogen isotopic ratios according to the method described by Ducatti et al. (1979). The results of the analyses were expressed in parts per thousand (‰) in relation to Peedee Belemnite international standard and atmospheric nitrogen (N2) for carbon and nitrogen elements, respectively, according to the expression: δ‰ (sample, standard) = [(Rsample – Rstandard)/Rstandard] × 1,000,
Statistical Analyses The data obtained in the carbon and nitrogen isotopic analysis were submitted to multivariate ANOVA with the help of GLM of the SAS statistical program (SAS Institute, 2000). From the data generated by the error matrices for each tissue, regions (ellipses) were defined within 95% confidence to verify differences among the averages of the experimental treatments and the average of the control group (a strictly vegetable diet).
RESULTS AND DISCUSSION The results of the isotopic analyses (δ13C and δ15N) of the diets used in this study are in Table 1. It was observed that the values of δ15N enriched both the vegetable diet (0%) as well as the OM diet (8%) like the starter diet (1–21 d old) and the grower diet (22–42 d old) within the same diet. This probably occurred due to the variation in the percentage of composition of the ingredients in the diet. The feed of the OM diet had less soybean meal and more corn and OM when compared with that of the vegetable diet feed. A similar enrichment observed in the starter diet is also verified in the grower diet and this may be due to the same fact, there was a smaller percentage of soybean meal in the grower diet. Corn (δ13C = −12.15 ± 0.04‰ and δ15n = 6.81 ± 0.04‰), soybean meal (δ13C = −26.15 ± 0.10‰ and δ15n = 0.55 ± 0.04‰), and poultry OM (δ13C = −16.85 ± 0.13‰ and δ15n = 4.07 ± 0.10‰) used to make the feed were also isotopically analyzed for nitrogen and carbon. It is known that soybean meal has a value of δ15N that is close to the standard value of atmospheric N2 (δ15N ≅ 0.0 ± 1.0‰) because the soy plants fix the air nitrogen and present a fractioning factor of δ15N around one unity, generally lower than 1.003‰ (Kohl and Shearer, 1980; Handley and Raven, 1992; Werner and Schmidt, 2002). The value of δ15N presented by plants that cannot fix atmospheric nitrogen depends
greatly on the abundance of this isotope in the managed soil and fertilizers, like in corn (Choi et al., 2002). Sleiman et al. (2004), assessing corn samples from some Brazilian states, found average values of 4.77 ± 1.16‰ for δ15N and −11.74 ± 0.40‰ for δ13C, respectively. Similar to the enrichment that occurred for the values of δ15N described above, the same was observed for the values of δ13C in the experimental diets because when OM is included in the diet formulation, a smaller quantity of soybean meal and oil was required to balance it in relation to the corresponding isocaloric and isonitrogenous vegetable diet. Besides that, the inclusion of an energetic ingredient (i.e., corn) was increased. This happens because plants from the C3 photosynthetic cycle (e.g., soybean meal, rice, and wheat) have a modal value of δ13C = −27.6‰ in relation to the Peedee Belemnite international standard, and in plants from the C4 photosynthetic cycle (e.g., corn, sorghum, and sugarcane) this modal value is δ13C = −12.6‰ (Vogel, 1993). Grower diets would be richer in 13C when compared with that of starter diets because they are nutritionally more energetic and less proteic, making the percentage of composition of the ingredients change within the same diet, although these values may not present significant differences. Therefore, grower diets would have more corn and less soybean meal. These figures related to the values of δ13C and δ15N in broiler diets, as well as in other species’ diets, are fundamental to identifying the presence of animal byproducts in food once the isotopic signature of the diet is reflected in the animals’ organism. According to DeNiro and Epstein (1976, 1978), an animal is what it consumes isotopically, up to 2.0‰ for δ13C and up to 3.0‰ for δ15N, although each tissue of the same animal may present a particular isotopic signature, fractioning factor (Hobson and Clark, 1992b), and isotopic turnover (Hobson and Clark, 1992a). The average values of δ13C and δ15N for broiler breast, drumstick, thigh, and wing meat in the end of the experimental period are presented in Table 2, and the results of the statistical analysis for isotopic pairs (δ13C and δ15N) for the tissues of the birds from groups G2, G3, G4, G5, and G6 when compared with that of the control group (G1) are illustrated in Figure 1. In the G3 and G4 treatments, the birds were fed with a vegetable diet at first, and then after a determined age (21 and 35 d, respectively) 8% OM was included in the diet until slaughtering age (42 d). In all analyzed tissues, it was observed that these treatments differed from the control group (G1) because their confidence regions did not overlap any axis of the graph (Figure 1). Likewise, when analyzing the data from the breast (Figure 1A), drumstick (Figure 1B), thigh (Figure 1C), and wing (Figure 1D), besides treatments G5 and G6 where birds had an 8% OM diet in the first breeding phase followed by a vegetable diet after a specific age (21 and 35 d, respectively), treatment G2 also differed from the control group showing the biggest distance
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where R represents the ratio between the heaviest and the lightest isotope, specifically 13C/12C and 15N/14N. Each sample was analyzed twice to obtain average values. The measurements were replicated when the analytical SD was greater than 0.2‰ for δ‰13C.
481
482 1G1 = vegetable diet with corn and soybean meal without animal-origin by-product (basal diet) from 1 to 42 d old; G2 = 8% poultry offal meal (OM) diet from 1 to 42 d old; G3 = basal diet from 1 to 21 d old and 8% OM diet from 22 to 42 d old; G4 = basal diet from 1 to 35 d old and 8% OM diet from 36 to 42 d old; G5 = 8% OM diet from 1 to 21 d old and basal diet from 22 to 42 d old; and G6 = 8% OM diet from 1 to 35 d old and basal diet from 36 to 42 d old.
0.09 0.09 0.12 0.08 0.03 0.14 ± ± ± ± ± ± 2.75 3.57 3.47 3.01 3.01 3.47 ± ± ± ± ± ± −18.99 −17.00 −17.42 −18.43 −18.46 −17.49 0.09 0.13 0.19 0.13 0.21 0.18 ± ± ± ± ± ± 3.25 4.30 4.13 3.62 3.58 4.15 ± ± ± ± ± ± −19.18 −17.19 −17.50 −18.52 −18.66 −17.62 0.12 0.32 0.27 0.05 0.17 0.26 ± ± ± ± ± ± ± ± ± ± ± ± −19.06 −16.94 −17.32 −18.38 −18.56 −17.56 0.11 0.05 0.13 0.06 0.16 0.08 ± ± ± ± ± ± ± ± ± ± ± ± −19.28 −17.18 −17.57 −18.56 −18.69 −17.69 G1 G2 G3 G4 G5 G6
Diet1
δ13C
0.15 0.14 0.08 0.19 0.17 0.09
2.36 3.26 3.15 2.74 2.55 3.05
δ15N
δ13C
0.13 0.09 0.06 0.20 0.13 0.12
3.30 4.36 4.25 3.78 3.72 4.26
δ15N
δ13C
0.14 0.11 0.11 0.15 0.07 0.09
Thigh (‰) Drumstick (‰) Breast (‰)
Table 2. Average δ13C and δ15N values and their respective SD in different tissues of 42-d-old broilers
between the x- and y-axes among the analyzed groups. This is possibly because G2 consisted of birds having an OM diet throughout the experimental period. Overall, the differences among the treatment δ15N averages in relation to the control group are smaller than the differences among the δ13C averages when comparing all tissues, making δ15N important for the detection of OM utilization in the broiler diet. Thus, the confidence regions in the presented graphics become more distant along the carbon axis than along the nitrogen one. This probably resulted from the carbon and nitrogen isotopic differences between the vegetable diet and the OM diet where the latter is, on average, 1.41‰ richer for δ13C and 1.16‰ richer for δ15N. Carrijo et al. (2006) verified that the 8% meat and bone meal diet was 1.17‰ richer for δ13C and 1.20‰ richer for δ15N, on average, when compared with the respective vegetable diet. Oliveira et al. (2010), when feeding broilers an 8% poultry offal meal diet, observed that it was 2.27‰ richer for δ13C and 1.05‰ richer for δ15N. The differences of nitrogen isotopic ratios observed among the analyzed tissues must be greatly related to the composition of these tissues in muscle fibers. Broiler chickens’ muscle, when transformed into meat during the postmortem period, is an important source of highquality protein for human nutrition. Regardless of not performing the muscle functions anymore, it is important to consider that the meat is still a live substance whose metabolic processes are partly dependent on the originating muscle. The skeletal muscle presents striated fibers and an important physiological function in providing movement to the living bird. Its varied fiber composition allows the adaptation to different environments or selection realities and may be reflected in the also varied quality of the meat (Scheuermann, 2008). When the ellipsis formed by the data from the drumstick (Figure 1B) and thigh (Figure 1C) were observed, a very similar behavior among them was noticed. This is probably due to the composition of their muscles that are red, richly vascularized, and formed by oxidative and oxidative-glycolytic fibers. Simultaneously, when the ellipsis formed by data from the breast (Figure 1A) are observed, they behave differently from the previous ones. This is probably because this tissue is formed predominantly by glycolytic fibers and oxidative-glycolytic fibers, which is different from the muscle constitution of drumsticks and thighs. On the other hand, the breast ellipses were similar to the ellipses from the wing data (Figure 1D); it was possibly because the function of this muscle in broilers does not have the same function as in flying poultry once they have lost their flying capacity along the species evolution, showing a similar behavior to the breast. Dubowitz and Brooke (1984) reported that the difference in metabolic and contractile behaviors among muscle fibers can be shown by histochemical techniques, classifying them into 3 muscle fibers: white, red, and intermediate ones. The white fibers, rich in glycolytic enzymes, are adapted to the anaerobic metabolism and
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δ15N
δ13C
0.15 0.04 0.07 0.06 0.11 0.06
Wing (‰)
δ15N
Cruz et al.
OFFAL MEAL TRACEABILITY WITH ISOTOPES
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Figure 1. A) Confidence regions formed by the difference among the 42-d-old broiler breast isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). B) Confidence regions formed by the difference among the 42-d-old broiler drumstick isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). C) Confidence regions formed by the difference among the 42-d-old broiler thigh isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing). D) Confidence regions formed by the difference among the 42-d-old broiler wing isotopic values of δ13C and δ15N when compared with that of the control group (G1 = axis crossing).
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in the percentage of composition of their biochemical fractions, such as lipids, carbohydrates, and proteins, comparatively in which the lipid fraction can present depletion of 13C (DeNiro and Epstein, 1978). Analyzing the composition of CP, ether extract, and mineral matter of the breast, legs (drumstick and thigh), and wings of 49-d-old broilers, Faria Filho et al. (2006) found higher values of protein in the breast (72.6%), followed by the legs (50.5%) and wings (45.3%); higher values of ether extract were found for the wing (42.9%), followed by the legs (37.8%) and breast (21.2%). The latter had significantly lower values of mineral matter (6.2%) compared with that of the legs (11.7%) and wing (11.8%), which did not differ among them. In addition to what has been reported until now, it is probable that the management of diets richer in 15N and 13C to animals that have a larger contrast in the isotopic signatures in relation to the vegetable diet allows for an easier determination of significant differences between the treatments and the basal diet. This is because, at first, the isotopic enrichment of nitrogen and carbon in tissues is related to the isotopic abundance of the diet and the analyzed tissue type (DeNiro and Epstein, 1978; Tieszen et al., 1983; Shoeninger and DeNiro, 1984). The results found in this study seem to be important for the technological improvement involved in the identification process of broiler carcasses of birds fed without poultry OM in their diet to meet the demands of specific markets. However, complementary studies have to be done to evaluate the practical potential of this technique in the poultry segment, helping the traceability process that is an important tool for the certification of poultry products. This is because the stable isotopes serve as natural tracers in the investigation of food programs (Jones et al., 1979; Tyrrell et al., 1984; DeNiro, 1987; Peterson and Fry, 1987; Carrijo et al., 2006; Móri et al., 2007, 2008; Gottmann et al., 2008; Denadai et al., 2009; Oliveira et al., 2010), using only the natural isotopic variation of ingredients without the need for using specifically marked or radioactive compounds. Besides, other tissues deserve to be studied for this purpose. Still, it is worthwhile to note that the analyses were carried out in 42-d-old broilers. The application of this technique can be viable for the same kind of study in younger birds because they would have the isotopic signal of a stabilized diet in their tissues when they are around 2 wk old (Oliveira et al., 2010). Therefore, if this technique were validated in practice in the poultry industry, it would be suggested that even before the slaughtering age, the birds would be sampled for isotopic analysis still in the poultry farms, rather than in the slaughter house or in the exportation container. Thus, the use of the technique of carbon- and nitrogen-stable isotopes is promising for the detection of animal ingredient use in a broiler diet through the isotopic analyses of their tissues.
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use glycogen as an energetic substrate. The red fibers, rich in oxidative enzymes, have aerobic metabolism and use fat as the main energetic substrate. The white fibers, also called fast glycolytic (FG), presented a larger area and fast contraction, whereas the red fibers are called slow oxidative (SO) and have a small area and slow contraction. The intermediate fibers are called fast oxidative glycolytic (FOG) and present an area size that is intermediate to the white and red ones, aerobic and glycolytic metabolism and fast contraction. They also have red color (Peter et al., 1972). According to the metabolic characteristics, demonstrated by histochemical techniques, the fibers are classified as type I with low contraction and oxidative metabolism; type IIA with fast contraction and oxidative-glycolytic metabolism; and type IIB with fast contraction and gycolytic metabolism. These types correspond to SO, FOG, and FG fibers, respectively. Type I or SO fibers are small, have numerous mitochondria and abundant myoglobin that gives them a red color. Mitochondria are large and have several crysts; therefore, the oxygen storage by myoglobin in these fibers avoids fatigue. Type II fibers are large muscle cells that have a small amount of myoglobin and mitochondria. Type IIA (FOG) fibers are resistant to fatigue, whereas IIB (FG) fibers are easily fatigued and latic acid accumulates in them easily. Type IIA fibers are considered intermediate between I and IIB and adaptable, becoming more glycolytic or more oxidative according to the demand of the muscle activity. Type I and IIA fibers are aerobic and depend on a high rate of oxygen and metabolic exchanges. However, anaerobic fibers (IIB) have a lower rate of metabolic exchanges and oxygen and present a larger area, and therefore, a greater hypertrophic process. In the presence of one or more types of fibers, their distribution and subtype frequency is what determines the metabolic and contractile characteristics of the skeletal muscle tissue, showing their biochemical and physiological properties (Macari et al., 2002). In laying hens and broilers, the breast muscle has predominantly FG and FOG fibers and, histologically, presents a small density of blood capillaries; their cells have a small number of mitochondria. The red muscle of the drumstick and thigh, on the other hand, is richly vascularized with many mitochondria in their fibers, mainly SO and FOG types (Macari et al., 2002). The muscle fiber distribution in a determined muscle in different animal species and individuals is a functional adaptation to their multiple types of activity. Thus, the rate of different muscle fiber types is directly correlated to its function in the animal organism. The definitive phenotype of adult skeletal muscle fibers is the result of events that start in the embryo and are continuously modulated and refined throughout the organism’s life. The observed variations among the tissues for the δ13C values can be partly related to the differences
OFFAL MEAL TRACEABILITY WITH ISOTOPES
Under experimental conditions, it can be concluded that the application of the technique of carbon- and nitrogen-stable isotopes can trace poultry OM use in a broilers’ diet during breeding or when OM substitutes a strictly vegetable diet, even up to 35 d old. When a poultry OM diet is substituted by a strictly vegetable diet, the animal-origin ingredient must be part of the diet for 21 d or longer so that its detection can be possible using this method.
ACKNOWLEDGMENTS
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The authors thank the National Council for Scientific and Technological Development (CNPq, Brasília, Distrito Federal, Brazil) for their financial support. We also thank the postdoc fellowship [Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, Brazil] for the publication financial support, the company Frangoeste (Tietê, São Paulo, Brazil) for the donation of the poultry offal meal, the Laboratory of Poultry Nutrition (Botucatu, São Paulo, Brazil) and The Center of Stable Isotopes of UNESP—Campus of Botucatu (Botucatu, São Paulo, Brazil) for allowing the development of this study and the samples and data processing.
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