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Effect of reduced dietary protein and supplementation with a docosahexaenoic acid product on broiler performance and meat quality a
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T. Ribeiro , M. M. Lordelo , P. Costa , S. P. Alves , W. S. Benevides , R. J. B. Bessa , J. P. a
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C. Lemos , R. M. A. Pinto , L. M. A. Ferreira , C. M. G. A. Fontes & J. A. M. Prates a
CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal b
Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal c
Faculdade de Veterinária da Universidade Estadual do Ceará, Av. Paranjana, Fortaleza, Brasil d
iMed UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisboa, Portugal Accepted author version posted online: 03 Oct 2014.
To cite this article: T. Ribeiro, M. M. Lordelo, P. Costa, S. P. Alves, W. S. Benevides, R. J. B. Bessa, J. P. C. Lemos, R. M. A. Pinto, L. M. A. Ferreira, C. M. G. A. Fontes & J. A. M. Prates (2014): Effect of reduced dietary protein and supplementation with a docosahexaenoic acid product on broiler performance and meat quality, British Poultry Science, DOI: 10.1080/00071668.2014.971222 To link to this article: http://dx.doi.org/10.1080/00071668.2014.971222
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Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2014.971222 CBPS-2014-042
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Ed. Kjaer, August 2014; MacLeod, September 2014
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product on broiler performance and meat quality
T. RIBEIRO, M. M. LORDELO1, P. COSTA, S. P. ALVES, W.S. BENEVIDES2, R. J. B.
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BESSA, J. P. C. LEMOS, R. M. A. PINTO 3, L. M. A. FERREIRA, C. M. G. A. FONTES J. A. M. PRATES
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CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do
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Alto da Ajuda, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal, 1Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal, 2
Faculdade de Veterinária da Universidade Estadual do Ceará, Av. Paranjana, Fortaleza,
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Brasil and 3iMed UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisboa, Portugal
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Effect of reduced dietary protein and supplementation with a docosahexaenoic acid
Running title: Dietary protein and DHA supplementation
Correspondence to: Teresa Ribeiro, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal. Tel. +351 213652800. Fax +351 213652889. E-mail: [email protected] Accepted for publication 16th July 2014
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Abstract. 1. Chicken breast meat is a lean meat due to its low content of intramuscular fat (IMF) resulting in an overall lower acceptability by consumers due to a decrease in juiciness, flavour and increased chewiness. Recently, studies performed in pigs suggested the possibility of increasing IMF by decreasing dietary crude protein (CP) content, an effect possibly mediated through an increased lipogenesis.
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2. Dietary supplementation with lipids rich in omega 3 long-chain polyunsaturated fatty acids
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monogastric animals and, thus, promote the daily intake of n-3 LC-PUFA by humans.
quality and consumers acceptability.
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3. LC-PUFA are very susceptible to oxidation, resulting in off-flavours that affect meat
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4. This trial was conducted to assess the effect of reducing dietary CP, from 21% to 17%, on chicken’s meat IMF content and, simultaneously, to evaluate if a complementary supplementation with a proprietary n-3 LC-PUFA source (DHA Gold™) could improve meat
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quality. These effects were assessed by measuring productive performance and meat quality,
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oxidative stability, sensory traits and fatty acid profile. 5. A reduction in CP content of broiler diets, from 21% to 17%, balanced for lysine, improved performance while it was not sufficient to increase IMF content in chicken meat. In contrast,
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DHA Gold™ supplementation had a positive impact both in broiler productive parameters and meat fatty acid profile.
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(n-3 LC-PUFA) may modulate an increase in the content of these fatty acids in meat from
6. In addition, incorporation of 7.4% of DHA Gold™ in the diet promoted carcass yield but negatively affected chicken meat acceptability by consumers, due to a decrease of meat
oxidative stability. 7. Overall the data suggest that neither a dietary supplementation with DHA Gold™ nor a reduction in CP have a direct positive effect in the levels of IMF present in broiler meat.
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INTRODUCTION In western countries poultry meat is highly appreciated with annual per capita consumptions of approximately 22 kg and 38 kg in Europe and the United States of America (USA), respectively (Eurostat, 2008; USDA, 2010). In addition, chicken meat, mainly breast, is a lean meat, with low contents of intramuscular fat (IMF) (Palmquist, 2009). IMF is one of the key
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meat quality traits affecting directly meat sensory properties (Chizzolini et al., 1999; Ruiz et
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acceptability (Oddy et al., 2001). It is well known that meat sensory traits are of extreme
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importance for meat acceptability by consumers (Guo-Bin et al., 2010). Therefore, production of poultry meat with high sensory quality, by increasing levels of IMF, without affecting
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carcass yields, is desirable for poultry industry and consumers.
In pigs, the use of reduced protein diets (RPD) (Doran et al., 2006) or low lysine incorporations (D’Souza et al., 2008) have been proved to be a successful nutritional strategy
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to enhance fat accumulation in muscle, without affecting other fat depots such as the
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abdominal or the subcutaneous (improved fat partitioning). In addition, it was showed that the increased IMF induced by RPD may enhance pork sensory acceptability (Teye et al., 2006; Madeira et al., 2013). Although the principle of these nutritional strategies is to restrict
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muscle development, the mechanism involved in the increasing of IMF remains unknown (Hocquette et al., 2010). One of the possible explanations might be the tissue-specific
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al., 2001; Chen et al., 2005), in addition to tenderness, juiciness, flavour and overall
expression of lipogenic enzymes under RPD which, in turn, could lead to the increase of de novo fatty acid synthesis (Hocquette et al., 2010). However, it remains unknown whether this nutritional strategy is also effective to ameliorate fat partitioning in broilers. Omega 3 (n-3) long chain-polyunsaturated fatty acids (LC-PUFA), especially eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are recognised as beneficial compounds for animal growth and development (Zhang et al., 2010). These important PUFA have been shown to reduce the risk of several chronic disorders,
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including cardiovascular diseases (Lopez-Garcia et al., 2004). Due to the high consumption of poultry meat in developed countries, this type of meat has been considered one of the main potential sources of n-3 LC-PUFA for humans (Rymer and Givens, 2005; Givens and Gibbs, 2008). Nevertheless, there are some disadvantages related to meat oxidative stability. LCPUFAs are very susceptible to oxidation, producing off-flavours and odours in meat that are
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often associated with fish flavour (Wood et al., 2008). This oxidative instability can influence
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known that marine algae are excellent sources of n-3 LC-PUFA (Schmitz and Ecker, 2008),
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the impact of novel algae-derived products on broiler performance and meat quality remains to be established. DHA GoldTM is a product derived from Schizochytrium marine algae with
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the golden hue due to the high content of naturally occurring carotenoids, thus providing high PUFA stabilisation (Barclay et al., 1994).
There is no information in the literature concerning the use of RDP to produce poultry
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meat with higher levels of IMF and a more favourable fatty acid composition, without
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affecting broiler growth performance and increasing the amount of other fat depots. In addition, the interaction between dietary protein concentration and DHA Gold™ supplementation has not been explored in different broiler feeding systems. Therefore, we
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hypothesised that RPD might increase meat IMF content in broilers, thus increasing meat sensory traits, without major undesirable effects on growth rate. In addition, complementary
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negatively meat quality and, consequently, consumers’ acceptability. Although it is well
dietary supplementation with DHA Gold™ may potentiate the incorporation of higher concentrations of n-3 LC-PUFA in broiler meat. The objective of the present study was to
assess the combined effect of a RPD with a DHA Gold™ supplementation using a maizebased diet on broiler performance and meat quality, oxidative stability, sensory attributes and fatty acid composition.
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MATERIALS AND METHODS Animals, diets and management Animal experiments were conducted in accordance with the principles and guidelines of the European Union (1986), reviewed by the Ethics Committee of CIISA (Faculdade de Medicina Veterinária) and approved by the Animal Care Committee of the National Veterinary
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Authority (Direção-Geral de Veterinária, Lisboa, Portugal).
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with 3 birds per cage, in a controlled environmental room under standard brooding practices,
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and were fed ad libitum with a maize-based diet during the first 21 d of the trial (Table 1). The experimental period was taken from d 21 to 35 and animals were fed on 4 different
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experimental treatments (grower/finisher diet) summarised in Table 1. All diets were formulated to achieve the National Research Council (1994) requirements. The 4 treatments consisted on a maize-based control diet with 210 g/kg (NPD21) or 170 g/kg (RPD17) of crude
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protein. NPD21 and RPD17 diets were also supplemented with 74 g/kg of DHA Gold™,
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leading to diets DHA21 and DHA17, respectively. The determined composition of DHA Gold™, a product extracted from the marine Schizochytrium algae (Novus, Brussels, Belgium) was 110 g/kg of crude protein, 5 g/kg of crude fibre, 210 g/kg of crude fat and 28
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MJ of ME/kg. Pelleted feed and water were offered ad libitum. The 4 dietary treatments were formulated to obtain, at least, the minimum content of amino acids recommendations: 11 g/kg
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A total of 120 1-d old male Ross 308 birds were distributed in 40 battery brooders
of lysine, 4.1 g/kg of methionine, 7.4 g/kg of threonine and 8 g/kg of methionine plus cysteine. The two supplemented treatments were formulated to achieve 20 g/kg of total n-3 PUFA. The fatty acid composition and vitamin E profile of the diets is also given in Table 1. DHA Gold™ and the final feed were analysed for crude protein, crude fibre, crude fat and gross energy. Feeds were analysed for dry matter (DM) by drying a sample at 100 °C to a constant weight. Nitrogen content was determined by Kjeldahl (AOAC, 1990) and crude protein was calculated as 6.25 × N. The samples were extracted with petroleum ether, using
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an automatic Soxhlet extractor (Gerhardt Analytical Systems, Königswinter, Germany), to determine crude fat. Feed gross energy was determined by adiabatic bomb calorimetry (Parr 1261, Parr Instrument Company, Moline, IL, USA).
Table 1 near here
Body weight and feed consumption were recorded weekly for performance evaluation (body weight gain, feed conversion ratio and feed intake). Environment and cage
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temperatures were controlled by an air conditioning unit and forced ventilation to guarantee
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in a commercial slaughterhouse, after a fast of 24 h and other bird per pen was slaughtered
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with an intravenous injection of an aqueous isotonic solution of 125 mg thiopental (Braun, Barcelona, Spain). Blood samples were collected in a Sarstedt™ tube (Numbrecht, Germany)
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for serum separation to assess birds’ health. Birds slaughtered with the intravenous injection were dissected to collect the gastro-intestinal (GI) contents. Size and weight of the GI tract (crop, gizzard, liver, duodenum, ileum and caecum) was measured and weighed emptied,
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respectively.
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Carcasses from the slaughterhouse were maintained at 4 ºC after 4 h in the air-chilled circuit, where carcass final temperature of 4 ºC was achieved. The final temperature of the carcass was monitored with a probe thermometer. The carcasses dissection was started after
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12 h after slaughter. The pectoralis major muscle and whole thighs were obtained without skin. In addition, thighs were also deboned. Left breast (pectoralis major) and thigh of each
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the requirements of Ross 308 management manual. One bird per pen was slaughtered at d 35
animal were collected and approximately 50 g were minced and stored at -20 ºC in a vacuum sealed bag to determine fatty acid composition, total cholesterol and vitamin E homologues. Approximately 20 g of breast and thigh meat were minced for determination of meat oxidation. The entire right breast (pectoralis major) of each animal was collected and frozen in individual vacuum sealed bags at -20 ºC for sensory evaluation. Samples were stored at -20 ºC for approximately 1 week, when the samples began to be used for the several
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determinations and analysis to 2 months, when all the analysis have been completed. Freezing rate was approximately -2 ºC/min accordingly with the manufacturers’ information. Determination of carcass yield and pH Carcass yield was evaluated by measuring the ratio of carcass weight (after removal of viscera, neck, head and feet) in relation to bird live weight. The carcass was weighed after
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approximately 4 h in the air-chilled circuit (until final carcass temperature of 4 ºC). Final meat
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breast muscle. Meat pH was determined in triplicate in the right breast muscle, before the
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dissection of the whole carcass and using a HI9025 potentiometer (Hanna instruments, Woonsocket, RI). The final values were the average of the replicates. All the procedures
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referred above were performed in a 4 ºC controlled room. Carcasses were maintained in a dark 4 ºC chamber in covered up boxes between the different determinations. Determination of meat oxidation
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Approximately 15 g of meat from the left breast and thigh of each animal were minced,
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divided in 4 portions and kept for 0, 2, 4 or 6 d at 4 ºC, exposed to air, in plastic bags. Meat oxidation was determined at d 0, 2, 4 and 6 by the technique of thiobarbituric acid-reactive substances (TBARS) based on the Botsoglou et al. (1994) and Grau et al. (2000) method.
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Duplicate measurements were obtained and the results were expressed as mg of malondialdehyde (MDA) per kg of meat.
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temperature was determined with a manual probe thermometer in the thickest part of the
Sensory analysis Meat sensory analysis was performed only on breast muscle due to the difficulty in separating muscles from thighs. Right skinless breast muscle (only pectoralis major) were removed from whole carcass and frozen in a vacuum sealed bag at -20 ºC, as well as a portion of the left breast muscles for sensory analysis. For sensory analysis, left and right breast meat were thawed at 4 ºC during 24 h and were grilled in a plate grill (67/70 FTES electric griddle, Modelar Catering Equipment, Italy) at 250 ºC until the final internal temperature of 80 ºC was
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reached. Meat temperature was monitored by a thermocouple (Lufft C120, Munchen, Germany). Meat was turned the first time when the internal temperature was 55 ºC and from then on was turned frequently until the final temperature was reached. Before and after grilling (when breast meat temperature reached 60 ºC), breast muscle was weighed to determine cooking loss.
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For sensory analysis, muscles were trimmed of external connective tissue, cut into
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identified, from sample preparation to the samples been provided to panellists. The trained
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sensory panel (Faculty of Veterinary Medicine, Lisbon, Portugal), composed by 9 members, tasted the meat samples to assess tenderness (defined as the opposite of the force required to
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bite through the sample with the molars), juiciness (the amount of liquid drained from the sample during the initial chews), flavour (the intensity with which the meat sample is recognised as chicken), presence of off-flavours and overall appreciation (the perception of
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how the meat is palatable taking into account the aforementioned attributes) in 4 panel
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sessions, with 10 random samples per session where all the 4 animal treatments were included. All the attributes were scored in a numeric scale from 1 to 8, where 1 is the low score and 8 is the high score. For off-flavour evaluation, the scale was from 0 (absence of off-
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flavour) to 8 (maximum of off-flavour). Determination of meat dry matter and total lipids
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cubes of approximately 1 cm3 and maintained at 60 ºC, in heated plaques previously
Dry matter (DM) content of breast and thigh meats was determined, in duplicate, by microwave desiccation using a SMART System5 apparatus, model sp1141 (CEM Corporation™, Matthews, NC). Control analyses of DM were performed, in duplicate, by lyophilisation. The difference between the two methods for DM determination did not exceed 1%. Samples were lyophilised to constant weight using a lyophilisator Edwards Modulyo (Edwards High Vacuum International, Crawley, UK) at -60 ºC and 2.0 hPa. After lyophilisation, samples were maintained in desiccators at room temperature and analysed for
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fatty acids within two weeks. For total lipid determination, IMF was extracted from feed and from lyophilised breast and thigh muscles using the method of Folch et al. (1957) upon mincing and homogenisation. Total lipids were measured gravimetrically, in duplicate, by weighing the fatty residue obtained after solvent evaporation. Determination of fatty acid composition of meat and diets
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Intramuscular fat of lyophilised meat (0.25 g) and feed (0.10 g) samples were analysed as
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transesterification by a basic/acid sequential reaction as described by Raes et al. (2001). The
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fatty acid composition was determined by gas chromatography of fatty acids methyl esters, performed by a chromatograph HP6890A (Hewlett-Packard, Avondale, PA, USA), equipped
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with a flame ionisation detector and a CP-Sil 88 capillary column (100 m; 0.25 mm i.d. × 0.20 µm film thickness; Chrompack, Varian Inc., Walnut Creek, CA, USA). The chromatographic conditions were as follows: injector temperature: 250 ºC; detector temperature: 280 ºC;
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helium was used as carrier gas and the split ratio was 1:30. The gas chromatograph oven
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temperature was programmed to start at 50 ºC (maintained for 4 min) followed by a 13 ºC/min ramp to 175 ºC (maintained for 20 min), followed by a 4 ºC/min ramp to 275 ºC (maintained for 40 min). Identification was made by comparing the retention times of peaks from samples
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with those of FAME standard mixtures (Sigma® Aldrich Co, Buchs, Switzerland). Quantification of FAME was based on the internal standard technique, using nonadecanoic
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described by Ponte et al. (2008). The fatty acids were converted to methyl esters by
acid (19:0) as internal standard and on the conversion to relative peak areas to weight percentage. Fatty acids were expressed as a percentage of the sum of identified fatty acids (%
of fatty acids). Determination of cholesterol, tocopherols and tocotrienols of meat and diets The simultaneous quantification of total cholesterol, tocopherols and tocotrienols was performed as described by Prates et al. (2006). The method involves a direct saponification of the fresh meat (0.75 g) or feed (0.10 g of DM), a single n-hexane extraction, and analysis of
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the extracted compounds by normal-phase HPLC, using fluorescence (tocopherols and tocotrienols) and UV-visible photodiode array (cholesterol) detections in tandem. The contents of total cholesterol, tocopherols and tocotrienols were calculated in duplicate for each sample based on the external standard technique from a standard curve of peak area vs. compound concentration.
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Statistical analysis
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experimental unit (4 treatments with 10 replicates of three birds each). Data were analysed
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using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA), considering a treatment factorial 2x2 (two crude protein concentrations and absence or presence of DHA
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Gold™ supplementation). The assumption of variance homogeneity between treatments was not observed for most variables and thus the GROUP option was included in the model to accommodate the heterogeneity of variances between treatments. As a consequence, the
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results are presented as mean ± standard error of mean. The model used for analysing data
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included the effect of protein reduction (RDP), the effect of supplementation with DHA Gold™ (DHA) and the interaction between RPD and DHA (RPD*DHA). Differences were
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considered significant when P < 0.05.
RESULTS AND DISCUSSION
Diet analysis
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The experimental design was completely randomised and the cage with three birds was the
The chemical composition, fatty acid profile, cholesterol and diterpene contents of diets used in this study are described in Table 1. RPD diets had lower CP contents but similar concentrations of essential amino acids, including L-lysine, when compared to supplemented diets. Most of the differences detected in the 4 diets were related with supplementation. Supplemented diets with DHA Gold™, were characterised by a high content of n-3 LC-PUFA (data not shown), especially DHA (22:6n-3), with a consequent increase of total n-3 PUFA and a decrease of total n-6 PUFA, when compared to the non-supplemented diets. DHA
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Gold™ supplemented diets were also characterised by high concentrations of myristic and palmitic acids (14:0 and 16:0, respectively), which increased the content of total saturated fatty acids (SFA), when compared to the non-supplemented diets. In addition, the content of cholesterol was similar among diets. Regarding diterpenes, while the most important lipidsoluble antioxidant in animal tissues (α-tocopherol) was present in similar concentrations in
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all diets, supplemented diets were characterised by lower contents of β-tocopherol, γ-
Birds’ health and performance
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other carotenoids were not detected in the supplemented diets.
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Table 2 near here
The profile of different serum parameters is presented in Table 2. Animals supplemented with
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DHA Gold™ had lower concentrations (P < 0.05) of total cholesterol, triacylglycerols, VLDL-cholesterol, total lipids and HDL-cholesterol. Harris (1989) and Sanz et al. (2000) suggested that n-3 fatty acids may reduce hepatic fatty acid and triacylglycerol synthesis.
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Moreover, diets rich in PUFA, such as DHA, can increase the β-oxidation rate of unsaturated
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fats leading to higher uptakes of triacylglycerols from blood by tissues (Sanz et al., 2000). Data from our trial suggest that this mechanism has been potentiated by the presence of DHA in supplemented diets, leading to lower concentrations of the parameters mentioned above.
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Low-protein diets led to a decrease (P < 0.05) in serum concentrations of urea and creatinine. The lower concentrations of urea in RPD treatments, relative to NPD, are in agreement with
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tocopherol and δ-tocopherol, when compared to the non-supplemented diets. β-carotene or
previous studies, in which protein content in the diets were reduced (Aletor et al., 2000;
Corzo et al., 2005; Ruusunen et al., 2007). These studies indicated that the lowest contents in urea are positively correlated with the highest nitrogen retention coefficient, which demonstrate that there is a more efficient utilisation of amino acids in these RPDs than in control diets. This efficient utilisation of the dietary amino acids can also lead to less nitrogen excretion, which could represent a major advantage in the environmental impact of this sector (Aletor et al., 2000). RPDs also led to a decrease in creatinine and a tendency to decrease
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LDL cholesterol and to an increase in both AST and ALP, which in accordance with the Tableare 3 near here results obtained by Rymer and Givens (2005). However, the values obtained for these hepatic markers indicated that the treatments applied to the broilers did not promote liver toxicity. Broiler performance parameters are presented in Table 3. Productive performance was affected by diet protein content and by DHA Gold™ supplementation. In general, DHA
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Gold™ supplemented diets led to better performance, as also did diets with protein reduction.
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gain and body weight. Supplemented groups and control group had higher body weights,
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weight gains and feed intake, as well as lower feed conversion ratios, relative to the control group (P < 0.05). The differences appeared as soon as the 4 diets were introduced. Pellets
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from supplemented diets were more cohesive during the entire trial than the control diets, which could have a positive effect on feed consumption. Moreover, in a recent study, animals supplemented with DHA GoldTM were also shown to display better performance parameters
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(Ribeiro et al., 2013), which suggest that DHA Gold™ can improve body weight. Our results
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also indicate that it is possible to decrease dietary CP from 21% to 17% without affecting broilers productive parameters. Several authors revealed improvements in growth rate and feed efficiency (Bregendahl et al., 2002; Rahman et al., 2002; Si et al., 2004; Sterling et al.,
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2003) in birds fed diets with low CP, but supplemented with essential amino acids (AA) (Jennsen and Colnago, 1991). Waguespack et al. (2009) revealed that supplementation of a
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Birds of the NPD21 group presented lower feed intakes (P < 0.05), which led to lower weight
low-CP diet with non-limiting essential AA supported broiler growth performance when compared to broilers fed on a control diet with 22% of CP. Nevertheless, other authors have reported no effects on performance when broilers are fed low-protein diets (Parr and
Summers, 1991 ;Kamran et al., 2010), while several studies report a reduction in animal performance in RPDs (Bregendahl et al., 2002; Kamran et al., 2008; Namroud et al., 2008) even when essential AA were provided. Here, a reduction of dietary protein lead to an improvement in broiler productive parameters, and a similar observation was reported when
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DHA Gold™ was added to the diets. However, the interaction observed between these two factors suggest that DHA Gold™ supplementation only improves broilers performance when dietary crude protein is 21% but not 17%. The relative weight of crop, gizzard and liver of broilers, as well as the relative lengths of duodenum, jejunum, ileum and caecum, are presented on Table 3. Birds fed the NPD21
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diet had the heaviest gizzard and liver and longer intestinal portions (P < 0.05). Interactions
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and jejunum. Szabó et al. (2001) stated that liver weight increases with higher dietary protein
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content in pigs. Kamran et al. (2008) showed that higher liver weights are not found when the EM:CP ratio is maintained. However, some authors suggested that birds fed low-CP diets
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displayed increased liver weights as a consequence of a possible enhanced de novo lipogenesis (Rosebrough and McMurtry, 1993). This was also observed in the present study. Overall, the data suggest that when animals are supplemented with DHA Gold™ in low
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protein diets, there is a compensation on the effects on weights/lengths of gastrointestinal
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compartments of animals, resulting in shorter/smaller compartments. Meat quality
Meat quality characteristics are displayed in Table 4. Carcass yield, pH and cooking loss were
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not affected by dietary treatments.
Table 4 near here
Acceptability of breast meat was evaluated by a sensory panel with data presented in
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between protein concentration and supplementation were found in gizzard, liver, duodenum
Table 4. Meat from supplemented birds had differences in flavour, off-flavour and overall appreciation. These three parameters were strongly correlated (r = 0.87, P < 0.001) with each other. The sensory panel detected off-flavours, mainly in DHA Gold™ supplemented groups. The overall breast meat score was lower in animals fed on DHA Gold™ supplemented diets (P < 0.05). A reduction in dietary protein had no effect in meat sensory traits. As it is mentioned above, the IMF breast meat content was not improved in birds fed RPDs
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(Fernandez et al., 1999; Ruiz et al., 2001). However, there was a tendency (P = 0.083) to lower juiciness in meat from birds of the supplemented groups. The off-flavours detected in meat from DHA Gold™ supplemented groups are likely due to DHA oxidation. The presence of off-flavours affected meat overall score that was lower for breast meat originated in supplemented groups. Off-flavour showed to be
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determinant (r = 0.91, P < 0.001) for the overall score. The meat off-flavour increase as a
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previously by Mooney et al. (1998). Nevertheless, it seems that off-flavour score is less
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affected by marine algae supplementation than by fish oil probably because the oil droplets in algae are encapsulated within the algal cell, thus protecting them from oxidative deterioration
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(Mooney et al., 1998). In a recent study (Rymer et al., 2010) a higher content of aldehydes in meat supplemented with marine algae was described. Rymer and Givens (2006) showed that a supplementation with 4% of fish oil had no impact on meat sensory characteristics. However,
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other studies revealed that even a lower inclusion rate of fish oil generates an unacceptable
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meat due to lipid oxidation (Mirghelenj et al., 2009). Nevertheless, data described here suggest that at the incorporation concentrations tested in this study DHA Gold™ supplementation leads to a decrease in meat sensory properties.
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Meat oxidative stability
Levels of oxidative stability of breast and thigh meats are presented in Table 5. The oxidative
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consequence of dietary supplementation with fish oil and marine algae was reported
stability was not affected by CP, although some interactions were found for breast meat at d 2 and for thigh meat at d 0. The oxidative stability of supplemented groups was affected by the addition of LC-PUFA to the diet via DHA. After slaughter, oxidative susceptibility of thigh meat from birds of the DHA treatment was significantly higher when compared to the stability of meat originated in animals of other treatments. The reduced oxidative stability of meat from lipid supplemented birds was due to the enrichment of meat with n-3 LC-PUFA, which are molecules very susceptible to oxidation. In addition, the higher susceptibility of
15
thigh meat to oxidation, relative to breast meat, this might simply result from the higher lipid content of this meat (Leskanich and Noble, 1997; Betti et al., 2009). Vitamin E profile of both tissues is also present in Table 5. The supplemented groups had lower values of α-tocopherol comparatively with control groups. Despite α-tocopherol, the other tocopherols were presented at low concentrations. β-carotene was not detected in any of the meat samples
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analysed. Carotenoids from DHA Gold™ were not detected in the diet (Table 1), which
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does not provide oxidant stability to the meat, as we initially hypothesised. The diterpene
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profile was in agreement with that described by Prates et al. (2006), Ponte et al. (2008) and Ribeiro et al. (2013). The lowest values of α-tocopherol in meat from animals of the
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supplemented groups, when compared to control groups, were in agreement with a recent work of Ribeiro et al. (2013). Several other authors observed that diets rich in PUFA lead to lower concentrations of α-tocopherol in tissues, both in rats (Tijburg et al., 1997) and in
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poultry (Ruiz et al., 1999; Surai and Sparks, 2000; Sijben et al., 2002) due to a lower
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absorption of α-tocopherol as a result of a higher PUFA content. The low/residual concentrations of the other tocopherols are in agreement with the general properties displayed by the diets.
Table 5 near here
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Meat lipids and fatty acid composition Intramuscular fat content, total cholesterol and fatty acid profile of breast and thigh meats are
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suggest that this product do not maintain antioxidant properties after feed manufacture and
displayed in Table 6. IMF content in both cuts was affected neither by DHA Gold™ supplementation nor by CP concentration. Nevertheless, breast meat from supplemented animals presented lower levels of IMF (P < 0.05). In addition to IMF, total fatty acids of both
meats were also determined by gas chromatography – flame ionisation detector (GC-FID) (data not shown). Although IMF did not change in breast meat, total fatty acids were significantly higher in the low-protein treatment (data not shown), which is in accordance with several reports in pigs (e.g. Doran et al, 2006). The IMF content of breast and thigh
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meats is in accordance with the range of values described by other authors (Rabot, 1998; Zerehdaran et al., 2004; Zhao et al., 2007). In addition, a tendency (P = 0.11) for a higher level of IMF in thigh meat was found when a RPD was provided. These results suggest that an increment of IMF would most probably require concentrations of dietary crude protein lower than 17%.
Table 6 near here
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Comparatively, there was a marked difference between breast and thigh meats
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to 56 mg/g meat in thighs. Generally, accordingly to the criteria of the Food Advisory
thigh meat is considered a fat meat.
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Committee (1990), breast meat is considered to be a lean meat (fat content
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effect of reduced dietary protein and supplementation with a docosahexaenoic acid product on broiler performance and meat quality a
b
a
d
a
a
c
a
T. Ribeiro , M. M. Lordelo , P. Costa , S. P. Alves , W. S. Benevides , R. J. B. Bessa , J. P. a
a
a
C. Lemos , R. M. A. Pinto , L. M. A. Ferreira , C. M. G. A. Fontes & J. A. M. Prates a
CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal b
Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal c
Faculdade de Veterinária da Universidade Estadual do Ceará, Av. Paranjana, Fortaleza, Brasil d
iMed UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisboa, Portugal Accepted author version posted online: 03 Oct 2014.
To cite this article: T. Ribeiro, M. M. Lordelo, P. Costa, S. P. Alves, W. S. Benevides, R. J. B. Bessa, J. P. C. Lemos, R. M. A. Pinto, L. M. A. Ferreira, C. M. G. A. Fontes & J. A. M. Prates (2014): Effect of reduced dietary protein and supplementation with a docosahexaenoic acid product on broiler performance and meat quality, British Poultry Science, DOI: 10.1080/00071668.2014.971222 To link to this article: http://dx.doi.org/10.1080/00071668.2014.971222
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Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2014.971222 CBPS-2014-042
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Ed. Kjaer, August 2014; MacLeod, September 2014
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product on broiler performance and meat quality
T. RIBEIRO, M. M. LORDELO1, P. COSTA, S. P. ALVES, W.S. BENEVIDES2, R. J. B.
AND
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BESSA, J. P. C. LEMOS, R. M. A. PINTO 3, L. M. A. FERREIRA, C. M. G. A. FONTES J. A. M. PRATES
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CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do
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Alto da Ajuda, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal, 1Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal, 2
Faculdade de Veterinária da Universidade Estadual do Ceará, Av. Paranjana, Fortaleza,
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Brasil and 3iMed UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisboa, Portugal
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Effect of reduced dietary protein and supplementation with a docosahexaenoic acid
Running title: Dietary protein and DHA supplementation
Correspondence to: Teresa Ribeiro, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal. Tel. +351 213652800. Fax +351 213652889. E-mail: [email protected] Accepted for publication 16th July 2014
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Abstract. 1. Chicken breast meat is a lean meat due to its low content of intramuscular fat (IMF) resulting in an overall lower acceptability by consumers due to a decrease in juiciness, flavour and increased chewiness. Recently, studies performed in pigs suggested the possibility of increasing IMF by decreasing dietary crude protein (CP) content, an effect possibly mediated through an increased lipogenesis.
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2. Dietary supplementation with lipids rich in omega 3 long-chain polyunsaturated fatty acids
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monogastric animals and, thus, promote the daily intake of n-3 LC-PUFA by humans.
quality and consumers acceptability.
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3. LC-PUFA are very susceptible to oxidation, resulting in off-flavours that affect meat
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4. This trial was conducted to assess the effect of reducing dietary CP, from 21% to 17%, on chicken’s meat IMF content and, simultaneously, to evaluate if a complementary supplementation with a proprietary n-3 LC-PUFA source (DHA Gold™) could improve meat
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quality. These effects were assessed by measuring productive performance and meat quality,
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oxidative stability, sensory traits and fatty acid profile. 5. A reduction in CP content of broiler diets, from 21% to 17%, balanced for lysine, improved performance while it was not sufficient to increase IMF content in chicken meat. In contrast,
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DHA Gold™ supplementation had a positive impact both in broiler productive parameters and meat fatty acid profile.
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(n-3 LC-PUFA) may modulate an increase in the content of these fatty acids in meat from
6. In addition, incorporation of 7.4% of DHA Gold™ in the diet promoted carcass yield but negatively affected chicken meat acceptability by consumers, due to a decrease of meat
oxidative stability. 7. Overall the data suggest that neither a dietary supplementation with DHA Gold™ nor a reduction in CP have a direct positive effect in the levels of IMF present in broiler meat.
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INTRODUCTION In western countries poultry meat is highly appreciated with annual per capita consumptions of approximately 22 kg and 38 kg in Europe and the United States of America (USA), respectively (Eurostat, 2008; USDA, 2010). In addition, chicken meat, mainly breast, is a lean meat, with low contents of intramuscular fat (IMF) (Palmquist, 2009). IMF is one of the key
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meat quality traits affecting directly meat sensory properties (Chizzolini et al., 1999; Ruiz et
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acceptability (Oddy et al., 2001). It is well known that meat sensory traits are of extreme
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importance for meat acceptability by consumers (Guo-Bin et al., 2010). Therefore, production of poultry meat with high sensory quality, by increasing levels of IMF, without affecting
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carcass yields, is desirable for poultry industry and consumers.
In pigs, the use of reduced protein diets (RPD) (Doran et al., 2006) or low lysine incorporations (D’Souza et al., 2008) have been proved to be a successful nutritional strategy
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to enhance fat accumulation in muscle, without affecting other fat depots such as the
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abdominal or the subcutaneous (improved fat partitioning). In addition, it was showed that the increased IMF induced by RPD may enhance pork sensory acceptability (Teye et al., 2006; Madeira et al., 2013). Although the principle of these nutritional strategies is to restrict
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muscle development, the mechanism involved in the increasing of IMF remains unknown (Hocquette et al., 2010). One of the possible explanations might be the tissue-specific
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al., 2001; Chen et al., 2005), in addition to tenderness, juiciness, flavour and overall
expression of lipogenic enzymes under RPD which, in turn, could lead to the increase of de novo fatty acid synthesis (Hocquette et al., 2010). However, it remains unknown whether this nutritional strategy is also effective to ameliorate fat partitioning in broilers. Omega 3 (n-3) long chain-polyunsaturated fatty acids (LC-PUFA), especially eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are recognised as beneficial compounds for animal growth and development (Zhang et al., 2010). These important PUFA have been shown to reduce the risk of several chronic disorders,
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including cardiovascular diseases (Lopez-Garcia et al., 2004). Due to the high consumption of poultry meat in developed countries, this type of meat has been considered one of the main potential sources of n-3 LC-PUFA for humans (Rymer and Givens, 2005; Givens and Gibbs, 2008). Nevertheless, there are some disadvantages related to meat oxidative stability. LCPUFAs are very susceptible to oxidation, producing off-flavours and odours in meat that are
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often associated with fish flavour (Wood et al., 2008). This oxidative instability can influence
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known that marine algae are excellent sources of n-3 LC-PUFA (Schmitz and Ecker, 2008),
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the impact of novel algae-derived products on broiler performance and meat quality remains to be established. DHA GoldTM is a product derived from Schizochytrium marine algae with
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the golden hue due to the high content of naturally occurring carotenoids, thus providing high PUFA stabilisation (Barclay et al., 1994).
There is no information in the literature concerning the use of RDP to produce poultry
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meat with higher levels of IMF and a more favourable fatty acid composition, without
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affecting broiler growth performance and increasing the amount of other fat depots. In addition, the interaction between dietary protein concentration and DHA Gold™ supplementation has not been explored in different broiler feeding systems. Therefore, we
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hypothesised that RPD might increase meat IMF content in broilers, thus increasing meat sensory traits, without major undesirable effects on growth rate. In addition, complementary
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negatively meat quality and, consequently, consumers’ acceptability. Although it is well
dietary supplementation with DHA Gold™ may potentiate the incorporation of higher concentrations of n-3 LC-PUFA in broiler meat. The objective of the present study was to
assess the combined effect of a RPD with a DHA Gold™ supplementation using a maizebased diet on broiler performance and meat quality, oxidative stability, sensory attributes and fatty acid composition.
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MATERIALS AND METHODS Animals, diets and management Animal experiments were conducted in accordance with the principles and guidelines of the European Union (1986), reviewed by the Ethics Committee of CIISA (Faculdade de Medicina Veterinária) and approved by the Animal Care Committee of the National Veterinary
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Authority (Direção-Geral de Veterinária, Lisboa, Portugal).
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with 3 birds per cage, in a controlled environmental room under standard brooding practices,
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and were fed ad libitum with a maize-based diet during the first 21 d of the trial (Table 1). The experimental period was taken from d 21 to 35 and animals were fed on 4 different
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experimental treatments (grower/finisher diet) summarised in Table 1. All diets were formulated to achieve the National Research Council (1994) requirements. The 4 treatments consisted on a maize-based control diet with 210 g/kg (NPD21) or 170 g/kg (RPD17) of crude
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protein. NPD21 and RPD17 diets were also supplemented with 74 g/kg of DHA Gold™,
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leading to diets DHA21 and DHA17, respectively. The determined composition of DHA Gold™, a product extracted from the marine Schizochytrium algae (Novus, Brussels, Belgium) was 110 g/kg of crude protein, 5 g/kg of crude fibre, 210 g/kg of crude fat and 28
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MJ of ME/kg. Pelleted feed and water were offered ad libitum. The 4 dietary treatments were formulated to obtain, at least, the minimum content of amino acids recommendations: 11 g/kg
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A total of 120 1-d old male Ross 308 birds were distributed in 40 battery brooders
of lysine, 4.1 g/kg of methionine, 7.4 g/kg of threonine and 8 g/kg of methionine plus cysteine. The two supplemented treatments were formulated to achieve 20 g/kg of total n-3 PUFA. The fatty acid composition and vitamin E profile of the diets is also given in Table 1. DHA Gold™ and the final feed were analysed for crude protein, crude fibre, crude fat and gross energy. Feeds were analysed for dry matter (DM) by drying a sample at 100 °C to a constant weight. Nitrogen content was determined by Kjeldahl (AOAC, 1990) and crude protein was calculated as 6.25 × N. The samples were extracted with petroleum ether, using
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an automatic Soxhlet extractor (Gerhardt Analytical Systems, Königswinter, Germany), to determine crude fat. Feed gross energy was determined by adiabatic bomb calorimetry (Parr 1261, Parr Instrument Company, Moline, IL, USA).
Table 1 near here
Body weight and feed consumption were recorded weekly for performance evaluation (body weight gain, feed conversion ratio and feed intake). Environment and cage
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temperatures were controlled by an air conditioning unit and forced ventilation to guarantee
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in a commercial slaughterhouse, after a fast of 24 h and other bird per pen was slaughtered
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with an intravenous injection of an aqueous isotonic solution of 125 mg thiopental (Braun, Barcelona, Spain). Blood samples were collected in a Sarstedt™ tube (Numbrecht, Germany)
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for serum separation to assess birds’ health. Birds slaughtered with the intravenous injection were dissected to collect the gastro-intestinal (GI) contents. Size and weight of the GI tract (crop, gizzard, liver, duodenum, ileum and caecum) was measured and weighed emptied,
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respectively.
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Carcasses from the slaughterhouse were maintained at 4 ºC after 4 h in the air-chilled circuit, where carcass final temperature of 4 ºC was achieved. The final temperature of the carcass was monitored with a probe thermometer. The carcasses dissection was started after
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12 h after slaughter. The pectoralis major muscle and whole thighs were obtained without skin. In addition, thighs were also deboned. Left breast (pectoralis major) and thigh of each
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the requirements of Ross 308 management manual. One bird per pen was slaughtered at d 35
animal were collected and approximately 50 g were minced and stored at -20 ºC in a vacuum sealed bag to determine fatty acid composition, total cholesterol and vitamin E homologues. Approximately 20 g of breast and thigh meat were minced for determination of meat oxidation. The entire right breast (pectoralis major) of each animal was collected and frozen in individual vacuum sealed bags at -20 ºC for sensory evaluation. Samples were stored at -20 ºC for approximately 1 week, when the samples began to be used for the several
7
determinations and analysis to 2 months, when all the analysis have been completed. Freezing rate was approximately -2 ºC/min accordingly with the manufacturers’ information. Determination of carcass yield and pH Carcass yield was evaluated by measuring the ratio of carcass weight (after removal of viscera, neck, head and feet) in relation to bird live weight. The carcass was weighed after
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approximately 4 h in the air-chilled circuit (until final carcass temperature of 4 ºC). Final meat
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breast muscle. Meat pH was determined in triplicate in the right breast muscle, before the
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dissection of the whole carcass and using a HI9025 potentiometer (Hanna instruments, Woonsocket, RI). The final values were the average of the replicates. All the procedures
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referred above were performed in a 4 ºC controlled room. Carcasses were maintained in a dark 4 ºC chamber in covered up boxes between the different determinations. Determination of meat oxidation
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Approximately 15 g of meat from the left breast and thigh of each animal were minced,
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divided in 4 portions and kept for 0, 2, 4 or 6 d at 4 ºC, exposed to air, in plastic bags. Meat oxidation was determined at d 0, 2, 4 and 6 by the technique of thiobarbituric acid-reactive substances (TBARS) based on the Botsoglou et al. (1994) and Grau et al. (2000) method.
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Duplicate measurements were obtained and the results were expressed as mg of malondialdehyde (MDA) per kg of meat.
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temperature was determined with a manual probe thermometer in the thickest part of the
Sensory analysis Meat sensory analysis was performed only on breast muscle due to the difficulty in separating muscles from thighs. Right skinless breast muscle (only pectoralis major) were removed from whole carcass and frozen in a vacuum sealed bag at -20 ºC, as well as a portion of the left breast muscles for sensory analysis. For sensory analysis, left and right breast meat were thawed at 4 ºC during 24 h and were grilled in a plate grill (67/70 FTES electric griddle, Modelar Catering Equipment, Italy) at 250 ºC until the final internal temperature of 80 ºC was
8
reached. Meat temperature was monitored by a thermocouple (Lufft C120, Munchen, Germany). Meat was turned the first time when the internal temperature was 55 ºC and from then on was turned frequently until the final temperature was reached. Before and after grilling (when breast meat temperature reached 60 ºC), breast muscle was weighed to determine cooking loss.
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For sensory analysis, muscles were trimmed of external connective tissue, cut into
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identified, from sample preparation to the samples been provided to panellists. The trained
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sensory panel (Faculty of Veterinary Medicine, Lisbon, Portugal), composed by 9 members, tasted the meat samples to assess tenderness (defined as the opposite of the force required to
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bite through the sample with the molars), juiciness (the amount of liquid drained from the sample during the initial chews), flavour (the intensity with which the meat sample is recognised as chicken), presence of off-flavours and overall appreciation (the perception of
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how the meat is palatable taking into account the aforementioned attributes) in 4 panel
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sessions, with 10 random samples per session where all the 4 animal treatments were included. All the attributes were scored in a numeric scale from 1 to 8, where 1 is the low score and 8 is the high score. For off-flavour evaluation, the scale was from 0 (absence of off-
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flavour) to 8 (maximum of off-flavour). Determination of meat dry matter and total lipids
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cubes of approximately 1 cm3 and maintained at 60 ºC, in heated plaques previously
Dry matter (DM) content of breast and thigh meats was determined, in duplicate, by microwave desiccation using a SMART System5 apparatus, model sp1141 (CEM Corporation™, Matthews, NC). Control analyses of DM were performed, in duplicate, by lyophilisation. The difference between the two methods for DM determination did not exceed 1%. Samples were lyophilised to constant weight using a lyophilisator Edwards Modulyo (Edwards High Vacuum International, Crawley, UK) at -60 ºC and 2.0 hPa. After lyophilisation, samples were maintained in desiccators at room temperature and analysed for
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fatty acids within two weeks. For total lipid determination, IMF was extracted from feed and from lyophilised breast and thigh muscles using the method of Folch et al. (1957) upon mincing and homogenisation. Total lipids were measured gravimetrically, in duplicate, by weighing the fatty residue obtained after solvent evaporation. Determination of fatty acid composition of meat and diets
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Intramuscular fat of lyophilised meat (0.25 g) and feed (0.10 g) samples were analysed as
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transesterification by a basic/acid sequential reaction as described by Raes et al. (2001). The
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fatty acid composition was determined by gas chromatography of fatty acids methyl esters, performed by a chromatograph HP6890A (Hewlett-Packard, Avondale, PA, USA), equipped
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with a flame ionisation detector and a CP-Sil 88 capillary column (100 m; 0.25 mm i.d. × 0.20 µm film thickness; Chrompack, Varian Inc., Walnut Creek, CA, USA). The chromatographic conditions were as follows: injector temperature: 250 ºC; detector temperature: 280 ºC;
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helium was used as carrier gas and the split ratio was 1:30. The gas chromatograph oven
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temperature was programmed to start at 50 ºC (maintained for 4 min) followed by a 13 ºC/min ramp to 175 ºC (maintained for 20 min), followed by a 4 ºC/min ramp to 275 ºC (maintained for 40 min). Identification was made by comparing the retention times of peaks from samples
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with those of FAME standard mixtures (Sigma® Aldrich Co, Buchs, Switzerland). Quantification of FAME was based on the internal standard technique, using nonadecanoic
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described by Ponte et al. (2008). The fatty acids were converted to methyl esters by
acid (19:0) as internal standard and on the conversion to relative peak areas to weight percentage. Fatty acids were expressed as a percentage of the sum of identified fatty acids (%
of fatty acids). Determination of cholesterol, tocopherols and tocotrienols of meat and diets The simultaneous quantification of total cholesterol, tocopherols and tocotrienols was performed as described by Prates et al. (2006). The method involves a direct saponification of the fresh meat (0.75 g) or feed (0.10 g of DM), a single n-hexane extraction, and analysis of
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the extracted compounds by normal-phase HPLC, using fluorescence (tocopherols and tocotrienols) and UV-visible photodiode array (cholesterol) detections in tandem. The contents of total cholesterol, tocopherols and tocotrienols were calculated in duplicate for each sample based on the external standard technique from a standard curve of peak area vs. compound concentration.
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Statistical analysis
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experimental unit (4 treatments with 10 replicates of three birds each). Data were analysed
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using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA), considering a treatment factorial 2x2 (two crude protein concentrations and absence or presence of DHA
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Gold™ supplementation). The assumption of variance homogeneity between treatments was not observed for most variables and thus the GROUP option was included in the model to accommodate the heterogeneity of variances between treatments. As a consequence, the
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results are presented as mean ± standard error of mean. The model used for analysing data
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included the effect of protein reduction (RDP), the effect of supplementation with DHA Gold™ (DHA) and the interaction between RPD and DHA (RPD*DHA). Differences were
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considered significant when P < 0.05.
RESULTS AND DISCUSSION
Diet analysis
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The experimental design was completely randomised and the cage with three birds was the
The chemical composition, fatty acid profile, cholesterol and diterpene contents of diets used in this study are described in Table 1. RPD diets had lower CP contents but similar concentrations of essential amino acids, including L-lysine, when compared to supplemented diets. Most of the differences detected in the 4 diets were related with supplementation. Supplemented diets with DHA Gold™, were characterised by a high content of n-3 LC-PUFA (data not shown), especially DHA (22:6n-3), with a consequent increase of total n-3 PUFA and a decrease of total n-6 PUFA, when compared to the non-supplemented diets. DHA
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Gold™ supplemented diets were also characterised by high concentrations of myristic and palmitic acids (14:0 and 16:0, respectively), which increased the content of total saturated fatty acids (SFA), when compared to the non-supplemented diets. In addition, the content of cholesterol was similar among diets. Regarding diterpenes, while the most important lipidsoluble antioxidant in animal tissues (α-tocopherol) was present in similar concentrations in
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all diets, supplemented diets were characterised by lower contents of β-tocopherol, γ-
Birds’ health and performance
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other carotenoids were not detected in the supplemented diets.
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Table 2 near here
The profile of different serum parameters is presented in Table 2. Animals supplemented with
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DHA Gold™ had lower concentrations (P < 0.05) of total cholesterol, triacylglycerols, VLDL-cholesterol, total lipids and HDL-cholesterol. Harris (1989) and Sanz et al. (2000) suggested that n-3 fatty acids may reduce hepatic fatty acid and triacylglycerol synthesis.
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Moreover, diets rich in PUFA, such as DHA, can increase the β-oxidation rate of unsaturated
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fats leading to higher uptakes of triacylglycerols from blood by tissues (Sanz et al., 2000). Data from our trial suggest that this mechanism has been potentiated by the presence of DHA in supplemented diets, leading to lower concentrations of the parameters mentioned above.
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Low-protein diets led to a decrease (P < 0.05) in serum concentrations of urea and creatinine. The lower concentrations of urea in RPD treatments, relative to NPD, are in agreement with
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tocopherol and δ-tocopherol, when compared to the non-supplemented diets. β-carotene or
previous studies, in which protein content in the diets were reduced (Aletor et al., 2000;
Corzo et al., 2005; Ruusunen et al., 2007). These studies indicated that the lowest contents in urea are positively correlated with the highest nitrogen retention coefficient, which demonstrate that there is a more efficient utilisation of amino acids in these RPDs than in control diets. This efficient utilisation of the dietary amino acids can also lead to less nitrogen excretion, which could represent a major advantage in the environmental impact of this sector (Aletor et al., 2000). RPDs also led to a decrease in creatinine and a tendency to decrease
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LDL cholesterol and to an increase in both AST and ALP, which in accordance with the Tableare 3 near here results obtained by Rymer and Givens (2005). However, the values obtained for these hepatic markers indicated that the treatments applied to the broilers did not promote liver toxicity. Broiler performance parameters are presented in Table 3. Productive performance was affected by diet protein content and by DHA Gold™ supplementation. In general, DHA
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Gold™ supplemented diets led to better performance, as also did diets with protein reduction.
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gain and body weight. Supplemented groups and control group had higher body weights,
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weight gains and feed intake, as well as lower feed conversion ratios, relative to the control group (P < 0.05). The differences appeared as soon as the 4 diets were introduced. Pellets
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from supplemented diets were more cohesive during the entire trial than the control diets, which could have a positive effect on feed consumption. Moreover, in a recent study, animals supplemented with DHA GoldTM were also shown to display better performance parameters
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(Ribeiro et al., 2013), which suggest that DHA Gold™ can improve body weight. Our results
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also indicate that it is possible to decrease dietary CP from 21% to 17% without affecting broilers productive parameters. Several authors revealed improvements in growth rate and feed efficiency (Bregendahl et al., 2002; Rahman et al., 2002; Si et al., 2004; Sterling et al.,
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2003) in birds fed diets with low CP, but supplemented with essential amino acids (AA) (Jennsen and Colnago, 1991). Waguespack et al. (2009) revealed that supplementation of a
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Birds of the NPD21 group presented lower feed intakes (P < 0.05), which led to lower weight
low-CP diet with non-limiting essential AA supported broiler growth performance when compared to broilers fed on a control diet with 22% of CP. Nevertheless, other authors have reported no effects on performance when broilers are fed low-protein diets (Parr and
Summers, 1991 ;Kamran et al., 2010), while several studies report a reduction in animal performance in RPDs (Bregendahl et al., 2002; Kamran et al., 2008; Namroud et al., 2008) even when essential AA were provided. Here, a reduction of dietary protein lead to an improvement in broiler productive parameters, and a similar observation was reported when
13
DHA Gold™ was added to the diets. However, the interaction observed between these two factors suggest that DHA Gold™ supplementation only improves broilers performance when dietary crude protein is 21% but not 17%. The relative weight of crop, gizzard and liver of broilers, as well as the relative lengths of duodenum, jejunum, ileum and caecum, are presented on Table 3. Birds fed the NPD21
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diet had the heaviest gizzard and liver and longer intestinal portions (P < 0.05). Interactions
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and jejunum. Szabó et al. (2001) stated that liver weight increases with higher dietary protein
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content in pigs. Kamran et al. (2008) showed that higher liver weights are not found when the EM:CP ratio is maintained. However, some authors suggested that birds fed low-CP diets
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displayed increased liver weights as a consequence of a possible enhanced de novo lipogenesis (Rosebrough and McMurtry, 1993). This was also observed in the present study. Overall, the data suggest that when animals are supplemented with DHA Gold™ in low
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protein diets, there is a compensation on the effects on weights/lengths of gastrointestinal
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compartments of animals, resulting in shorter/smaller compartments. Meat quality
Meat quality characteristics are displayed in Table 4. Carcass yield, pH and cooking loss were
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not affected by dietary treatments.
Table 4 near here
Acceptability of breast meat was evaluated by a sensory panel with data presented in
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between protein concentration and supplementation were found in gizzard, liver, duodenum
Table 4. Meat from supplemented birds had differences in flavour, off-flavour and overall appreciation. These three parameters were strongly correlated (r = 0.87, P < 0.001) with each other. The sensory panel detected off-flavours, mainly in DHA Gold™ supplemented groups. The overall breast meat score was lower in animals fed on DHA Gold™ supplemented diets (P < 0.05). A reduction in dietary protein had no effect in meat sensory traits. As it is mentioned above, the IMF breast meat content was not improved in birds fed RPDs
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(Fernandez et al., 1999; Ruiz et al., 2001). However, there was a tendency (P = 0.083) to lower juiciness in meat from birds of the supplemented groups. The off-flavours detected in meat from DHA Gold™ supplemented groups are likely due to DHA oxidation. The presence of off-flavours affected meat overall score that was lower for breast meat originated in supplemented groups. Off-flavour showed to be
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determinant (r = 0.91, P < 0.001) for the overall score. The meat off-flavour increase as a
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previously by Mooney et al. (1998). Nevertheless, it seems that off-flavour score is less
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affected by marine algae supplementation than by fish oil probably because the oil droplets in algae are encapsulated within the algal cell, thus protecting them from oxidative deterioration
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(Mooney et al., 1998). In a recent study (Rymer et al., 2010) a higher content of aldehydes in meat supplemented with marine algae was described. Rymer and Givens (2006) showed that a supplementation with 4% of fish oil had no impact on meat sensory characteristics. However,
d
other studies revealed that even a lower inclusion rate of fish oil generates an unacceptable
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meat due to lipid oxidation (Mirghelenj et al., 2009). Nevertheless, data described here suggest that at the incorporation concentrations tested in this study DHA Gold™ supplementation leads to a decrease in meat sensory properties.
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Meat oxidative stability
Levels of oxidative stability of breast and thigh meats are presented in Table 5. The oxidative
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consequence of dietary supplementation with fish oil and marine algae was reported
stability was not affected by CP, although some interactions were found for breast meat at d 2 and for thigh meat at d 0. The oxidative stability of supplemented groups was affected by the addition of LC-PUFA to the diet via DHA. After slaughter, oxidative susceptibility of thigh meat from birds of the DHA treatment was significantly higher when compared to the stability of meat originated in animals of other treatments. The reduced oxidative stability of meat from lipid supplemented birds was due to the enrichment of meat with n-3 LC-PUFA, which are molecules very susceptible to oxidation. In addition, the higher susceptibility of
15
thigh meat to oxidation, relative to breast meat, this might simply result from the higher lipid content of this meat (Leskanich and Noble, 1997; Betti et al., 2009). Vitamin E profile of both tissues is also present in Table 5. The supplemented groups had lower values of α-tocopherol comparatively with control groups. Despite α-tocopherol, the other tocopherols were presented at low concentrations. β-carotene was not detected in any of the meat samples
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analysed. Carotenoids from DHA Gold™ were not detected in the diet (Table 1), which
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does not provide oxidant stability to the meat, as we initially hypothesised. The diterpene
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profile was in agreement with that described by Prates et al. (2006), Ponte et al. (2008) and Ribeiro et al. (2013). The lowest values of α-tocopherol in meat from animals of the
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supplemented groups, when compared to control groups, were in agreement with a recent work of Ribeiro et al. (2013). Several other authors observed that diets rich in PUFA lead to lower concentrations of α-tocopherol in tissues, both in rats (Tijburg et al., 1997) and in
d
poultry (Ruiz et al., 1999; Surai and Sparks, 2000; Sijben et al., 2002) due to a lower
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absorption of α-tocopherol as a result of a higher PUFA content. The low/residual concentrations of the other tocopherols are in agreement with the general properties displayed by the diets.
Table 5 near here
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Meat lipids and fatty acid composition Intramuscular fat content, total cholesterol and fatty acid profile of breast and thigh meats are
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suggest that this product do not maintain antioxidant properties after feed manufacture and
displayed in Table 6. IMF content in both cuts was affected neither by DHA Gold™ supplementation nor by CP concentration. Nevertheless, breast meat from supplemented animals presented lower levels of IMF (P < 0.05). In addition to IMF, total fatty acids of both
meats were also determined by gas chromatography – flame ionisation detector (GC-FID) (data not shown). Although IMF did not change in breast meat, total fatty acids were significantly higher in the low-protein treatment (data not shown), which is in accordance with several reports in pigs (e.g. Doran et al, 2006). The IMF content of breast and thigh
16
meats is in accordance with the range of values described by other authors (Rabot, 1998; Zerehdaran et al., 2004; Zhao et al., 2007). In addition, a tendency (P = 0.11) for a higher level of IMF in thigh meat was found when a RPD was provided. These results suggest that an increment of IMF would most probably require concentrations of dietary crude protein lower than 17%.
Table 6 near here
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Comparatively, there was a marked difference between breast and thigh meats
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to 56 mg/g meat in thighs. Generally, accordingly to the criteria of the Food Advisory
thigh meat is considered a fat meat.
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Committee (1990), breast meat is considered to be a lean meat (fat content
