Traceability of Animal by Product Meals in Broilers Fed Sugarcane Yeast Using Stable Isotopes

The objective of this study was to verify if the dietary inclusion of sugarcane yeast at levels commonly used in broiler diets influences the traceability of cattle meat meal and poultry offal meal, using the technique of stable carbon and nitrogen isotopes in the breast muscle of chickens. A number of 325 one-d-old male broilers were randomly distributed into 13 treatments with 25 birds each. Treatments consisted of a control diet based on corn and soybean meal, and the inclusion of 1, 2, 4, or 6% meat and bone meal, poultry offal meal or sugarcane yeast. At 42 days of age, six birds per treatment were randomly selected, sacrificed, and their breast muscle was collected for isotopic ration analysis. The isotopic ratio of birds fed the diet with inclusion of 6% sugarcane yeast was different from those fed the control treatment, but not from those fed diets with the inclusion of 2, 4 and 6% meat and bone meal or 4 and 6% poultry offal meal. The inclusion of 6% sugarcane yeast in broiler diets based on corn and soybean meal may affect the traceability of animal by product meals.


INTRODUCTION
According to the Brazilian Association of Broiler Producers and Exporters (ABEF, 2009), Brazil is the third largest broiler producer and the world largest chicken meat exporter.However, the markets importing Brazilian chicken meat has imposed several restriction relative to rearing, feeding, and trade practices.
The increasing consumer concerns with food safety have led the industry to propose methods to provide the certification of origin and of the quality of animal and plant products around the world (Denadai, 2009).One of them is traceability, a tool used in the meat production system that has been critical for the survival and success of meat companies in an increasingly competitive and demanding market (Iba et al., 2003), and which still requires much research and development, particularly in terms of methods.
Many importers of Brazilian chicken meat, such as countries of the European Union and Middle East, require that broilers are not fed any animal by of product feedstuff (Bellaver et al., 2005).One way to detect the presence of animal feedstuffs in broilers feeds is to use stable isotopes, because the dietary isotopic signature can be detected in the bird's body in up to ± 2.0‰ for δ 13 C and up to ± 3.0‰ for δ 15 N, according to DeNiro & Epstein (1976, 1978).
The isotopic ratio of carbon to nitrogen allows identifying the geographic origin and feeding method in sheep (Piasentier et al., 2003), characterizing and differentiating dietary regimes in Iberian pigs (González-Martin et al., 1999) and tracing animal feedstuffs in broiler diets (Oliveira, 2005;Carrijo et al., 2006).However, some feedstuffs,

Traceability of Animal by Product Meals in Broilers
Fed Sugarcane Yeast Using Stable Isotopes numbered leg band.These birds were slaughtered at the experimental processing plant of FMVZ/UNESP -Botucatu.Birds were electrically stunned, bled, plucked and eviscerated, and the carcasses were cut up for the removal of the breast muscle (Pectoralis major).
Breast muscle samples were collected by cutting a slice measuring approximately 5 mm transversal to the longitudinal middle third of the left Pectoralis major (Gottmann, 2007).Samples were duly identified, placed in plastic bags and frozen at -20 ºC until analysis.Isotopic analyses were carried out at the Center Stable Isotopes of the Biosciences Institute, UNESP, Botucatu, SP, Brazil.
Samples for isotopic analyses were thawed, rinsed in distilled water, and dried in a forced-ventilation oven (Marconi -Ma 035) at 56 °C for 48 hours.Then, samples were placed in a tube and ground in a liquidnitrogen cryogenic mill (Spex freezer/mill -model 6750), at -190 ºC for 3 minutes (tissue) and at maximal frequency to obtain a homogenous material with very fine particle size and microscopic aspect (Rosa et al., 2002).
Samples of approximately 50-60 µg and 500-600 µg were weighed in tin capsules to determine carbon and nitrogen isotopic ratios, respectively.The capsules were introduced by means of an automatic sampler in the element analyzer (EA 1108 -CHN -Fisons Instruments, Rodano, Italy), where, in the presence of oxygen (O 2 ) and copper oxide (CuO), samples were quantitatively burnt to obtain CO 2 and NOx, which was then reduced to N 2 in the presence of copper.The obtained gases were separated in gas chromatography column, and analyzed in isotopic ratio mass spectrometer (Delta S -Finnigan MAT, Bremen, Germany).
Isotopic ratio values were expressed as delta per thousand ( ‰) relative to the international standard PeeDee Belemnite (PDB) for δ 13 C and atmospheric air nitrogen for δ 15 N (Ducatti, 2004), according to the following general equation: where δX represents the enrichment of the heaviest isotope of the chemical element X (C or N) of the sample relative to the international standard and R represents the ratio between the least and the most abundant isotope, specifically 13 C/ 12 C and 15 N/ 14 N.
The obtained isotopic results were submitted to multivariate analysis of variance (MANOVA) using the GLM procedure of SAS statistical program (2004).Based on the data generated by error matrices, the such as sugarcane yeast and wheat bran, may interfere in the traceability of animal by product meals when the technique of stable isotopes due to its high protein content and consequent Nitrogen-15 and Carbon-13 enrichment (Gottmann, 2007).
Poultry production in Brazil uses a wide range of sugarcane yeast inclusion levels in feeds because it is usually applied as a feed additive.However, according to Grangeiro et al. (2001), levels of up to 7.5% yeast can be used as protein source in broiler diets without any negative effect on broiler performance.
The objective of the present study was to determine if the inclusion of sugarcane yeast (Saccharomyces cerevisiae) at the levels commonly used by the broiler industry interferes in the traceability of animal by product meals when the carbon and nitrogen stable isotopes method is applied.

MATERIALS AND METHODS
The experiment was carried out at the Poultry Nutrition Laboratory of the School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP, Brazil.
A total number of 325 one-day-old male Cobb ® broiler chicks, vaccinated at the hatchery against Infectious Bursal Disease and Marek's Disease, was used.Birds were housed in an experimental broiler house, where birds were randomly distributed in 13 pens measuring 2.5 m 2 , corresponding to 13 treatments, with 25 birds/pen at a density of 10 birds /m 2 .There were six replicates per treatment, and each slaughtered bird was considered one replicate.
A three-phase feeding program was adopted: starter (1 to 21 days), grower (22 to 35 days) and finisher (36 to 42 days), as shown in Table 1.

RESULTS AND DISCUSSION
Because sugarcane has the isotopic signals δ 13 C = -10.49‰ and δ 15 N = 4.36‰, its inclusion in the diet may lead to the dietary enrichment of carbon-13 and nitrogen-15, which also happens with the inclusion of poultry offal meal ( δ 13 C= -16.28‰ and δ 15 N = 4.29‰) and cattle meat meal ( δ 13 C = -12.82‰and δ 15 N = 7.72‰) in broiler diets (Gottmann et al., 2008).The carbon-13 and nitrogen-15 enrichment when animal by product meals are included in broiler diets was also observed by Oliveira (2005) and Carrijo et al. (2006).
It is possible to determine the presence of animal by products in broiler feeds by detecting their isotopic signature in the body of broilers.This can be applied to other animal species, as the animal is what it isotopically eats, or up to ± 2.0‰ for δ 13 C and up to ± 3.0‰ for δ 15 N, according to DeNiro & Epstein (1976, 1978).
The mean isotopic signals ( δ 13 C and δ 15 N) in the breast muscle of broilers showed that there was carbon-13 and nitrogen-15 enrichment with increasing dietary inclusion levels of meat meal, poultry offal meal and yeast (Table 2).Mean isotopic signals ( δ 13 C and δ 15 N) in the breast meat samples of each treatment were statistically compared with the means of the control treatment (Figure1) and of the treatments with different yeast levels (Figure2), generating regions of 95% confidence level.

Figure 1 -
Figure 1 -Confidence regions established by the difference among δ 13 C and δ 15 N isotopic values in the muscle Pectoralis major of 42-d-old broilers of each treatment compared with the control treatment (Vegetable).

Figure 2 -
Figure 2 -Confidence regions established by the difference among δ 13 C and δ 15 N isotopic values in the muscle Pectoralis major of 42-d-old broilers of each treatment compared with the treatment containing 6% sugarcane yeast.
Traceability of Animal by Product Meals in Broilers Fed Sugarcane Yeast Using Stable Isotopes

Traceability of Animal by Product Meals in Broilers Fed Sugarcane Yeast Using Stable IsotopesTable 1 -
Ingredient and calculated nutritional compostition of the starter (1 to 21 days) and finisher (22 to 42 dayss) experimental diets.

Table 2 -
Mean and standard deviation value of δ 13 C and δ 15 N in the breast muscle of 42-d-old broilers.