This article was downloaded by: [UQ Library] On: 16 June 2014, At: 11:55 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20 Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3 long-chain polyunsaturated fatty acids T. Ribeiro a , M.M. Lordelo b , S.P. Alves a , R.J.B. Bessa a , P. Costa a , J.P.C. Lemos a , L.M.A. Ferreira a , C.M.G.A. Fontes a & J.A.M. Prates a a CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Pólo Universitário do Alto da Ajuda, Lisboa, Portugal b Instituto Superior de Agronomia, Lisboa, Portugal Published online: 08 Jan 2014. To cite this article: T. Ribeiro, M.M. Lordelo, S.P. Alves, R.J.B. Bessa, P. Costa, J.P.C. Lemos, L.M.A. Ferreira, C.M.G.A. Fontes & J.A.M. Prates (2013) Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3 long-chain polyunsaturated fatty acids, British Poultry Science, 54:6, 753-765, DOI: 10.1080/00071668.2013.841861 To link to this article: http://dx.doi.org/10.1080/00071668.2013.841861 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
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This article was downloaded by: [UQ Library]On: 16 June 2014, At: 11:55Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK
British Poultry SciencePublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/cbps20
Direct supplementation of diet is the most efficientway of enriching broiler meat with n-3 long-chainpolyunsaturated fatty acidsT. Ribeiroa, M.M. Lordelob, S.P. Alvesa, R.J.B. Bessaa, P. Costaa, J.P.C. Lemosa, L.M.A.Ferreiraa, C.M.G.A. Fontesa & J.A.M. Pratesa
a CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, PóloUniversitário do Alto da Ajuda, Lisboa, Portugalb Instituto Superior de Agronomia, Lisboa, PortugalPublished online: 08 Jan 2014.
To cite this article: T. Ribeiro, M.M. Lordelo, S.P. Alves, R.J.B. Bessa, P. Costa, J.P.C. Lemos, L.M.A. Ferreira, C.M.G.A.Fontes & J.A.M. Prates (2013) Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3long-chain polyunsaturated fatty acids, British Poultry Science, 54:6, 753-765, DOI: 10.1080/00071668.2013.841861
To link to this article: http://dx.doi.org/10.1080/00071668.2013.841861
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Direct supplementation of diet is the most efficient way of enriching broilermeat with n-3 long-chain polyunsaturated fatty acidsT. RIBEIRO, M.M. LORDELO1, S.P. ALVES, R.J.B. BESSA, P. COSTA, J.P.C. LEMOS,L.M.A. FERREIRA, C.M.G.A. FONTES AND J.A.M. PRATES
CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Pólo Universitário do Alto da Ajuda,Lisboa, Portugal, and 1Instituto Superior de Agronomia, Lisboa, Portugal
Abstract 1. Concentrations of beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs)in poultry meat can be improved by increasing the concentration of n-3 PUFA in poultry diets.2. A decrease in flavour quality is, however, usually associated with the dietary supplementation with n-3PUFA, which is due to the susceptibility of PUFA to oxidation.3. This experiment was conducted to study the effects of introducing two different n-3 fatty acid sources(extruded linseed and DHA Gold™, a proprietary algal product rich in docosahexaenoic acid), eitherseparately or together, on broiler productive performance, and meat quality, oxidative stability, sensorytraits and LC-PUFA profile.4. Birds given the algal product displayed better productive performances than animals from other groups.5. The data revealed an improvement in the fatty acid nutritional value of meat from birds receiving thealgal product and an inefficient conversion of α-linolenic acid (LNA) into LC-PUFA.6. Metabolisation of LNA in vivo is not sufficient to improve meat quality in n-3 LC-PUFA and directsupplementation of the diet with n-3 LC-PUFA is a better alternative to modulate an increase in beneficialfatty acids of broiler meat.7. The overall acceptability of meat was negatively affected by the dietary supplementation with 7.4% ofDHA, in contrast to the supplementation with 3.7% of DHA, which showed to be efficient in improvingLC-PUFA meat content without affecting its sensory properties.
INTRODUCTION
The human intake of omega-3 polyunsaturatedfatty acids (n-3 PUFAs), particularly eicosapentae-noic acid (EPA, 20:5n-3) and docosahexaenoicacid (DHA, 22:6n-3), has been decreasing inWestern diets (Prates and Bessa, 2009; Adkinsand Darsham, 2010), in contrast to the increasedintake of n-6 PUFA, especially linoleic acid (LA,18:2n-6), as a result of an increased consumptionof vegetable oils (Schmitz and Ecker, 2008).
The essential α-linolenic acid (LNA) and LAare the precursors of n-3 and n-6 LC-PUFAs,respectively. LA is converted to arachidonic acid(AA, 20:4n-6) and, then, to docosapentaenoic acid(DPA, 22:5n-6) (Schmitz and Ecker, 2008). LNA is
converted to stearidonic acid and eicosatetraenoicacid (20:4n-3) to form EPA. EPA is further meta-bolised to DHA (Schmitz and Ecker, 2008). Theconversion of these essential fatty acids, mainlyLNA, along the entire pathway, is a limited processas a result of its low concentration in diets relativeto LA (Lopez-Ferrer et al., 2001a) and the competi-tion for desaturase enzymes, which are the samefor both n-6 and n-3 pathways (Emken et al., 1994).Thus, it is important to assure an adequate dietaryLA/LNA ratio to obtain an efficient conversion ofLNA into EPA and DHA (Griffin, 2008).
The n-3 LC-PUFAs are recognised as impor-tant biomolecules for animal growth and develop-ment and are known to prevent humancardiovascular diseases (Zhang et al., 2010).
Correspondence to: Teresa Ribeiro, Faculdade de Medicina Veterinária, DPASA, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal. E-mail: [email protected]
Accepted for publication 28 June 2013.
British Poultry Science, 2013Vol. 54, No. 6, 753–765, http://dx.doi.org/10.1080/00071668.2013.841861
Reports have consistently demonstrated that n-3LC-PUFAs, especially EPA and DHA, are recog-nised as important fatty acids for animal growthand development, particularly neural tissue(Zhang et al., 2010), as well as to reduce the riskof human cardiovascular diseases (Lopez-Garciaet al., 2004). The primary source of n-3 LC-PUFAis marine fish, seafood and some plants (Schmitzand Ecker, 2008), such as algae (Alasalvar et al.,2002; Arterburn et al., 2006).
Poultry meat is one of the most consumedmeats in both Europe and the United States, withan annual average per capita consumption of 22 kg(Eurostat, 2008) and 39 kg (USDA, 2010), respec-tively. Thus, poultry meat has been considered anattractive route to increase n-3 LC-PUFA in humandiets (Lopez-Ferrer et al., 2001a; Cortinas et al.,2004; Rymer and Givens, 2005; Givens and Gibbs,2008; Poureslami et al., 2010). Dietary supplemen-tation with lipids and oils rich in n-3 PUFA is anefficient method to increase the content of thesefatty acids in animal muscle (Lopez-Ferrer et al.,2001b). However, there are some disadvantages ofusing this route, which are related with meat oxi-dative stability. In fact, LNA, EPA and DHA are verysusceptible to oxidation, producing off-flavours andodours in meat that are often associated with fishoil (Bou et al., 2001; Wood et al., 2008).
Although it is well known that marine algaeare excellent sources of n-3 LC-PUFAs, the impactof novel algae-derived products, alone or in com-bination with other n-3 PUFA sources, on broilerperformance, and meat quality and lipid profileremains to be established. DHA GoldTM is a pro-duct derived from Schizochytrium marine algae withthe golden hue due to naturally occurring carote-noids, which may provide n-3 PUFA stabilisation(Barclay et al., 1994).
Here we aim to compare two different dietarysources of n-3 PUFA, extruded linseed (LS, rich inthe precursor of n-3 LC-PUFA) and an algalproduct (DHA; direct supplementation of n-3LC-PUFA, specifically DHA), to improve broilermeat quality. This comparison meant to elucidatethe conversion efficiency of LNA (provided by LS)into LC-PUFA and evaluate the use of an algaeproduct to improve the functional, oxidativestability and sensory characteristics of meat.
MATERIALS AND METHODS
Animals, diets and management
One hundred and twenty 1-d-old male Ross 308birds were housed using 40-battery brooders withthree birds per cage in a controlled environmen-tal room under standard brooding practices. Theanimals were fed ad libitum with a maize-based dietduring the first 21 d. The experimental period wasfrom d 21 to 35, where animals were given 4
different treatments (see finisher diet describedin Table 1). All diets were formulated to achievethe National Research Council (1994) require-ments. The 4 treatments consisted of a maize-based control diet (CN), and the CN diet supple-mented with 154 g/kg of LS (Reagro, Lisboa,Portugal), 74 g/kg of DHA Gold™ (DHA;Novus, Brussels, Belgium) or 37 g/kg of DHAGold™ plus 77 g/kg of extruded linseed(DHALS). Feed was offered in pelleted form.The 4 dietary treatments were formulated toobtain 80 g/kg of fat, from which 40 g/kg wasfrom the different lipid supplementation sourcesto achieve 20 g/kg of total n-3 PUFA in the threesupplemented diets.
DHALS as well as the experimental diets wereanalysed for crude protein, crude fibre, crude fatand gross energy. Samples were analysed for drymatter (DM) by drying a sample at 100°C to aconstant weight. Nitrogen content was deter-mined by Kjeldahl (AOAC, 1990) and crude pro-tein was calculated as 6.25 × N. The samples wereextracted with petroleum ether, using an auto-matic Soxhlet extractor (Gerhardt AnalyticalSystems, Königswinter, Germany), to determinecrude fat. Feed gross energy in the feed was deter-mined by adiabatic bomb calorimetry (Parr 1261,Parr Instrument Company, Moline, IL, USA). LS(with concentration of hydrocyanic (HCN)acid <10 mg/kg – manufacturer’s specifications)and DHA Gold™ determined content in crudeprotein, crude fibre, crude fat and gross energywere of 170 g/kg, 120 g/kg, 210 g/kg and 22MJ/kg for linseed and 110 g/kg, 5 g/kg, 210 g/kg and 28 MJ/kg for DHA Gold™, respectively.The fatty acid composition and vitamin E profileof the diets are also given in Table 1. Body weight(BW) and feed consumption were recordedweekly for performance evaluation. At d 35, onebird per cage was slaughtered in a commercialslaughterhouse after a fast of 24 h.Gastrointestinal (GI) contents were collected dur-ing evisceration and the size and weight of the GItract were measured and weighed, respectively,after its emptying. Carcasses were maintained inthe air-chilled circuit until the final carcass tem-perature of 4°C was obtained.
Animal experiments were conducted inaccordance with the principles and specific guide-lines of the European Union (1986), reviewed bythe Ethics Committee of CIISA (Faculdade deMedicina Veterinária) and approved by theAnimal Care Committee of the NationalVeterinary Authority (Direcção-Geral deVeterinária, Lisboa, Portugal).
Determination of carcass yield, pH and colour
Carcass yield was determined by the percentageof weight of the commercial carcass (after the
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removal of viscera, neck, head and feet) in pro-portion to the bird live weight. Carcasses wereweighed after being air-chilled. Meat pH wasdetermined in the right breast muscle (in tripli-cate) using a HI9025 potentiometer (HannaInstruments, Woonsocket, RI, USA). Meat andskin colour measurements were determinedwith a Minolta™ Chroma Meter CR-300 Series(Osaka, Japan), attached with a data processorDP-301, in homologous locations of the skin of allcarcasses. After skin colour measurements, theskin was removed and other determinationswere performed in homologue locations of the
right breast and thigh of all carcasses. All colourmeasurements were replicated three times andthe values were expressed in the InternationalCommission on Illumination (CIELAB) systemof lightness (L*), redness (a*) and yellowness(b*) (CIE, 1978).
Sensory evaluation
Meat sensory analysis was performed only onbreast muscle (Pectoralis major). Thigh meat hasan important impact on consumers’ preferences.Nevertheless, due to the difficulty to individualise
Table 1. Ingredients, calculated analysis, fatty acid composition (g/100 g fatty acid, FA) and diterpene profile (µg/100 g DM) of theexperimental diets
Control diet, CN, and control diet supplemented with DHA GoldTM, DHA, extruded linseed, LS, or DHA GoldTM plus extruded linseed, DHALS. 1Mineral–vitamin premix provided the following per kilogram of feed: retinol, 2.7 mg; cholecalciferol, 0.05 mg; α-tocopherol, 20 mg; nicotinic acid, 30 mg; cyanoco-balamin, 0.12 mg; calcium pantothenate, 10 mg; menadione, 2 mg; thiamin, 1 mg; riboflavin, 4.2 mg; pyridoxine hydrochloride, 1.7 mg; folic acid, 0.5 mg;biotin, 0.5 mg; Fe, 80 mg; Cu, 10 mg; Mn, 100 mg; Zn, 80 mg; Co, 0.2 mg; I, 1.0 mg; Se, 0.3 mg; monensin, 100 mg/kg; nd = not detected. 2Co-eluted with asmall proportion of α-tocotrienol; 3Co-eluted with a small proportion of β-tocotrienol.
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the muscles in thigh cuts, we chose not to evaluatethis part of the carcass. After the air-chilled circuit,carcasses were desiccated. Thighs were deboned.A portion of left-side breast meat and the com-plete right-side breast meat were stored at –20°Cin a vacuum-sealed bag until sensory analysis. Forsensory analysis, these portions were thawed at4°C during 24 h and grilled in a plate grill(65/70 FTES Electric Griddle, Modelar CateringEquipment, Italy) until an internal temperature of80°C was reached, which was monitored by aninternal thermocouple (Lufft C120, München,Germany). The meat was turned the first timewhen the internal temperature was 55°C andfrom then on was turned frequently until thefinal temperature was reached. Before and aftergrilling, breast muscles were weighed to deter-mine cooking loss. Breast meat from the left sidewas cooled at room temperature during 2 h aftergrilling to measure shear force. Shear force wasdetermined as described by Ponte et al. (2008) ina maximum of 2 cm wide strips of the muscles,which were subjected to a Warner–Bratzler blade,coupled to a texture analyser TA.XT.plus® fromStable Microsystems™ (Surrey, UK).Measurements (cuts) were replicated extensivelydepending on the size of available meat sample (aminimum of 5 replications). Data were collectedwith specific software (Texture Expert Exceed,Stable Micro Systems, Surrey, UK). Meat shearforce was presented as the mean of the peakvalue of replicates.
For sensory analysis, muscles were cut incubes of 1 cm3 and added to a box previouslyidentified. A trained sensory panel (Faculty ofVeterinary Medicine, Lisbon, Portugal), com-posed by 9 members, tasted the meat samples toanalyse tenderness, juiciness, chicken flavour, pre-sence of off-flavours and overall appreciation in4 panel sessions, with 10 random samples persession that included all the 4 animal treatments.The sensory tests were made by blind tasting. Allthe attributes were tested in a numeric scale from1 to 8, where 1 is the low/negative score and 8 thehigh/positive score. For off-flavour evaluation, thescale was from 0 (absence of off-flavour) to 8(maximum of off-flavour).
Determination of meat oxidation
Approximately 15 g of meat from the left breastand deboned thigh of each animal were minced,divided in 4 portions and kept for 0, 2, 4 or 6 d at4°C exposed to air in plastic bags. Meat oxidationwas determined at d 0, 2, 4 and 6, by measuringthe thiobarbituric acid-reactive substances(TBARSs) based on the procedures ofBotsoglou et al. (1994) and Grau et al. (2000),with the resulting colour measured at 532 nmwith a UV/VIS Spectrophotometer (Pharmacia
LKB Biochrom Ltd., UK). Duplicate measure-ments were obtained and the results wereexpressed as mg of malondialdehyde (MDA)per kg of meat.
Determination of tocopherols and tocotrienols
The quantification of tocopherols and tocotrienolsin meat was performed as described by Prates et al.(2006). The method involved a direct saponifica-tion of the fresh meat (0.75 g) or feed (0.10 g ofDM), a single n-hexane extraction, and analysis ofthe extracted compounds by normal-phase high-performance liquid chromatography (HPLC),using fluorescence detection. The contents of toco-pherols and tocotrienols were calculated in dupli-cate for each sample based on the externalstandard technique from a standard curve of peakarea versus compound concentration.
Determination of DM and total lipids
DM content of breast and thigh meats was deter-mined, in duplicate, by microwave desiccationusing a SMART System5 apparatus, modelsp1141 (CEM Corporation™, Matthews, NC,USA). Control analyses of DM were performed,in duplicate, by lyophilisation using a lyophilisatorEdwards Modulyo™ (Edwards High VacuumInternational, Crawley, UK), at –60°C and 2.0hPa. After lyophilisation, samples were maintainedin desiccators at room temperature and analysedfor fatty acids within 2 weeks. The differencebetween the two methods for DM determinationdid not exceed 1%. For total lipid determination,intramuscular fat was extracted from lyophilisedbreast and thigh muscles using the method ofFolch et al. (1957) upon mincing and homogeni-sation. Samples from feed were also taken. Totallipids were measured gravimetrically, in duplicate,by weighing the fat residue obtained after solventevaporation.
Determination of fatty acid composition
Intramuscular fat from lyophilised meat (0.25 g)and from feed (0.10 g of DM) samples were trea-ted and analysed as described by Ponte et al.(2008). Fatty acids were converted to methyl estersby transesterification by a basic/acid sequentialreaction as described by Raes et al. (2001). Thefatty acid composition was determined by gaschromatography of fatty acid methyl esters, per-formed by a chromatograph HP6890A (Hewlett-Packard, Avondale, PA, USA), equipped with aflame ionisation detector and a CP-Sil 88 capillarycolumn (100 m; 0.25 mm i.d.; 0.20 µm film thick-ness; Chrompack, Varian Inc., Walnut Creek, CA,USA). The chromatographic conditions were asfollows: injector temperature, 250°C and detector
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temperature, 280°C. Helium was used as carriergas and the split ratio was 1:30. The gas chroma-tograph oven temperature was programmed tostart at 50°C (maintained for 4 min) followed bya 13°C/min ramp to 175°C (maintained for 20min), followed by a 4°C/min ramp to 275°C(maintained for 40 min). Identification wasaccomplished by comparing the retention timesof peaks from samples with those of FAME (fattyacid methyl esters) standard mixtures (Sigma®Aldrich Co, Buchs, Switzerland). Quantificationof FAME was based on the internal standard tech-nique, using nonadecanoic acid (19:0) as theinternal standard and the conversion to relativepeak areas to weight percentage. Fatty acids wereexpressed as a percentage of the sum of identifiedfatty acids (percentage of fatty acids).
Statistical analysis
The experimental design was completely rando-mised and a cage of three birds was the experi-mental unit. Data were analysed using the generallinear model (GLM) procedure of SAS (SAS Inst.Inc., Cary, NC, USA). Differences were consideredsignificant when P < 0.05. Regression analysis wasundertaken to assess and quantify the indepen-dent effect of several fatty acids and antioxidantsdetected in breast meat on sensory analysis andoxidative stability of breast meat. The model usedincluded the sensory panel attributes and the oxi-dative stability as dependent variables, and some
of the n-3 LC-PUFA (20:3n-3, EPA, DPA, DHA andthe sum of all n-3 LC-PUFAs) and antioxidantsdetected in breast meat as independent variables.This analysis was performed using the Regression(REG) procedure of SAS.
RESULTS AND DISCUSSION
Production performance
Bird performance, expressed as BW, BW gain,feed intake and feed conversion ratio are sum-marised in Table 2. There were significant differ-ences in BW and BW gain among treatments(P < 0.05), namely in birds from the DHA andLS groups. Growth rates were significantly higherand lower for birds of the DHA and LS treat-ments, respectively, when compared to controlanimals. LS treatment was also different fromthe other treatments for feed intake and feedconversion ratio (P < 0.05). The final BW ofbirds from the DHA treatment was on averagemore than 10% greater than in animals of thecontrol treatment. The relative weight of crop,gizzard and liver, as well as the relative length ofduodenum, jejunum, ileum and caecum are pre-sented in Table 2. The relative weight of gizzardand liver as well as the relative length of duode-num, jejunum, ileum and caecum were affected(P < 0.05) by lipid supplementation of diets. Inaddition, birds from the LS treatment, whencompared to those from the DHA treatment,
Table 2. Performance of broilers fed ad libitum with the experimental diets during the finisher period
Treatment
CN DHA LS DHALS SEM Significance
Body weight (g)21 d 962 970 971 931 15.4 NS28 d 1399b 1494a 1221c 1377b 19.9 0.00135 d 1844b 2048a 1514c 1840b 45.1 0.001Body weight gain (g)21-28 d 437b 525a 237c 438b 16.3 0.00128-35 d 458b 552a 292c 452b 27.2 0.00121-35 d 882b 1077a 537c 906b 40.7 0.001Feed intake (g)21-28 d 875b 927a 805c 828bc 22.4 0.00228-35 d 966b 1041a 849c 944bc 32.7 0.00221-35 d 1841a 1968a 1654b 1838a 43.8 0.001Gain:feed ratio21-35 d 0.47b 0.54b 0.32a 0.48b 0.017 0.001
Relative weight of GI tract, g/100 g BWCrop 0.31 0.34 0.35 0.28 0.026 NSGizzard 1.02ab 0.85b 1.06a 0.85b 0.059 0.030Liver 2.52a 1.92b 2.05ab 1.65b 0.184 0.017
Control diet, CN, and control diet supplemented with the algal product, DHA, extruded linseed, LS, or the algal product plus extruded linseed, DHALS. NS = nosignificant difference. a,b,c Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. GI, gastrointestinal tract.
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displayed heavier GI compartments and a longerGI tract.
The lower BW of birds from the LS treatmentcan, in part, be explained by a lower feed intake.Pellets from this treatment were less cohesive andvery susceptible to disintegration into flour. Thisphenomenon was also observed by Haug et al.(2007) and attributed to the melting point of lin-seed. Also, significant decreases in performancewhen animals are fed on mash diets versus pel-leted diets are well demonstrated (Kilburn andEdwards, 2001; Cerrate et al., 2009; Jafarnejadet al., 2010). Appearance of LS is significantlyunctuous and viscous when compared to thealgal product. Nevertheless, and despite the extru-sion process, it is also possible that the presence ofspecific anti-nutritive factor in linseed, even atlower concentrations, might have affected feedintake and/or digestibility. LS and DHA dietshad slight differences in fat (variation of 2.7%)and crude fibre (variation of 1.12%). The highestcontent of fat in the LS diet may explain some ofthe differences found in productive parameters aswell, as fat may contain some non-nutritive factors,as suggested by Jiménez-Moreno et al. (2009).Some other studies suggested that an increase indiet fibre, from a basal diet of 3%, will decreaseanimals’ performance (Jansen and Carré, 1985;Sklan et al., 2003). Nevertheless, these results sug-gest that while DHA could improve weight, incor-poration of LS at the concentrations reported inthis experiment decreased feed intake and, con-sequently, growth rates.
The inclusion of linseed increased the relativeweight of the different GI compartments. Barley-,wheat- or rye-based diets rich in soluble non-starch polysaccharides (NSPs) are also known tolead to an increase in the size and length of GItract compartments (Choct, 1997) as a conse-quence of an increase in digesta viscosity. The
effect of LS on intestinal length reported herehas not been observed previously. However,given the difficulty in pelleting the LS diet, it ispossible that the dietary incorporation of LS con-tributes to a change in the physical properties ofthe digesta, decreasing feed passage rates, thuscontributing to impose a pressure towards theincrease in organ size and leading to a distensionof the GI organs.
Meat quality parameters and sensory evaluation
Data on carcass yield, breast meat pH, cookingloss and meat shear force are shown in Table 3.Lipid supplementation had no effect on breastmeat pH, cooking loss and shear force value(P > 0.05). In contrast, and reflecting the abovereported production parameters, carcass yield washigher in birds from the DHA treatment whencompared to the LS animals. This result is likelyexplained by the higher final BW of birds fromthe DHA group and the larger size of the GI tractin LS birds. Skin and meat colour were also eval-uated (data not shown) and were not affected(P > 0.05) by the dietary treatments. However,lightness (L*) of broiler skin tended to be nega-tively affected by supplementation with the algalDHA product (P < 0.10) and yellowness (b*) washigher in thighs of DHA treatment and lower in LStreatment. This may result from the higher β-carotenoid content of the algal DHA Gold™ product.
Sensory traits of breast meat are presented inTable 3. Breast meat from lipid-supplementedbirds displayed significant differences (P <0.05)in flavour, off-flavours and overall acceptabilitywhen compared to those from non-supplementedanimals. Meat tenderness and juiciness were notaffected by treatments. Thus, meat flavour fromDHA birds was less intense and had lower scoresin overall acceptability (P < 0.0001), with a higher
Table 3. Carcass yield (%) and breast meat pH, cooking loss (%), shear force (N) and sensory panel traits of breast meat of broilers fedad libitum with the experimental diets during the finisher period
Control diet, CN, and control diet supplemented with the algal product, DHA, extruded linseed, LS, or the algal product plus extruded linseed, DHALS. NS =not significant. The different attributes evaluated in sensorial traits were quantified on a rating scale from 1 (more negative) to 8 (more positive), with theexception of the flavour and off-flavour that were quantified from 0 (absent) to 8 (very intense). a,b Mean values within the same row sharing a commonsuperscript letter are not statistically different at P < 0.05.
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score for off-flavours, when compared to the othertreatments. Meat from the DHALS group had anintermediate score for off-flavour comparatively toLS and DHA treatments and scores for flavourand overall acceptability similar to LS and CNmeats. Overall appreciation showed that onlymeat from the DHA treatment had negativeacceptability (less than 4) and was different fromall the other treatments. Meat from non-supple-mented birds displayed a higher overall apprecia-tion (6–“moderately positive”) when compared tothe supplemented groups. LS and DHALS meatsshowed a favourable score of 5 (“slightly positive”)and were not different from the CN group.Overall, the data revealed that the type of dietarylipids strongly affects meat sensory traits with meatfrom DHA-fed birds displaying the lower scores inoverall appreciation and flavour and higher scoresin off-flavour. The deterioration of flavour qualityby dietary supplementation with fish oil and mar-ine algae is well known and has been previouslydescribed (Mooney et al., 1998). However, areduction in the flavour score is less marked withmarine algae supplementation than fish oil prob-ably because the oil droplets in algae are encapsu-lated within the algal cell, thus protecting themfrom oxidative deterioration (Mooney et al.,1998). In a more recent study (Rymer et al.,
2010), a higher content of aldehydes in meatsupplemented with marine algae was described.In addition, it is also possible that algae containmore natural antioxidants, mainly carotenoids,than fish oil. Rymer and Givens (2006) showedthat a supplementation with 4% of fish oil hadno impact on meat sensory characteristics, whichis a comparable supplementation rate to theDHALS treatment (3.7% of the algal product).However, other studies revealed that even lowerinclusion rates of fish oil generate an unaccepta-ble meat due to lipid oxidation (Mirghelenj et al.,2009). Nevertheless, data described here suggestthat meat from DHALS birds, in contrast to thatfrom the DHA treatment, had a minimal compro-mise of flavour quality when compared to themeat from non-supplemented animals.
Results of regression analysis are presented inTable 4. In this table, only independent variablesare shown for which the regression analysis wassignificant for the dependent variables that werestudied. Results suggested that there was a nega-tive effect between meat flavour and overallappreciation and the presence of some fattyacids such as 20:3n-3, EPA and DHA (P < 0.05).In addition, off-flavours in breast meat seem to beinfluenced directly by the same fatty acids.Overall, the results indicate that 20:3n-3, EPA,
Table 4. Regression analysis – sensory panel attributes and oxidative stability as dependent variables
1Co-eluted with a small proportion of α-tocotrienol; β, regression coefficient; SE, standard error. LC-PUFA, long-chain polyunsaturated fatty acid. TBARS,thiobarbituric acid-reactive substances. NS = not significant.
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DHA and the sum of n-3 LC-PUFA are responsiblefor the sensory attributes of breast meat detectedby the sensory panel.
The oxidative susceptibility of breast andthigh meats is presented in Table 5. Breast meatoxidative stability tended to be affected by diets(P < 0.10) after 2 and 6 d of storage, with meatfrom the DHA treatment revealing higher instabil-ity when compared to the other meats. Oxidativesusceptibility of thigh meat of the DHA treatmentwas also significantly higher (P < 0.05), immedi-ately after slaughter. After 2 and 4 d of storage,thigh meat from the DHA treatment tended to bedifferent from the other treatments (P < 0.10). Atd 6, the oxidative stability of thigh meat from theCN group was significantly higher when comparedto meat from supplemented birds. Regression ana-lysis also indicated that the oxidative stability wasdirectly influenced by the same fatty acids as thesensory attributes (20:3n-3, EPA, DPA and DHA).Meat vitamin E profiles are presented in Table 5.Unlike what was observed for thigh meat, concen-trations of δ-tocotrienol were not measurable inbreast meat. Lipid-supplemented diets had signifi-cantly lower contents of α-tocopherol in bothmeats, relative to the control meat. However, thetype (the algal product or extruded linseed) ofdietary lipid supplementation had no influenceon any of the vitamin E homologues analysed inthe various meats. In fact, DHA and linseed dietshad lower contents of vitamin E resulting in adecrease in meat α-tocopherol, the most impor-tant antioxidant compound in meat. Unlike theα-tocopherol, γ-tocopherol content seemed to
increase when dietary lipid supplementation wasincorporated, and linseed seemed to be particu-larly effective in that improvement. Regressionanalysis indicated a poor relationship betweenconcentration of antioxidants detected in breastmeat and the other attributes. However, α-tocopherol seemed to influence the oxidative stabilityof meat stored at 4°C for 2 d, as well as the overallappreciation of meat. Finally, β-tocopherolseemed to have a negative effect on juiciness.The reduced oxidative stability of meat fromlipid-supplemented birds most probably resultedfrom the enrichment of meat with n-3 LC-PUFAs,especially DHA, which are very susceptible to oxi-dation. Lipid oxidation increases linearly as theconcentration of PUFA increases and oxidativestability of unsaturated fatty acids decrease as thedegree of unsaturation increases (Cortinas et al.,2005). In addition, the higher susceptibility ofthigh meat to oxidation, relative to breast meat,might result from the higher lipid content of thismeat (Leskanich and Noble, 1997; Betti et al.,2009). Thus, broiler meat with higher concentra-tions of n-3 LC-PUFAs is more susceptible to oxi-dation than meat with PUFA with lower degrees ofunsaturation, such as LA (Rymer and Givens,2005). This is likely explained by the substitutionof the unsaturated membrane phospholipids withother PUFA (Jerónimo et al., 2009). Diterpeneprofile of meat, presented in Table 4, showedthat meat from DHA and LS treatments are lessprotected with natural antioxidants, especially α-tocopherol, when compared to the control group.In general, meat diterpene profile was in
Table 5. Oxidative susceptibility measured as thiobarbituric acid-reactive substances (mg malondialdehyde/kg meat), and diterpene profile(µg/100 g meat) of breast and thigh meats from broilers fed ad libitum with the experimental diets during the finisher period
Treatment
CN DHA LS DHALS SEM Significance
Breast0 d of storage 0.03 0.05 0.01 0.01 0.022 NS2 d of storage 0.04b 0.21a 0.09ab 0.15ab 0.051 0.0984 d of storage 0.09 0.49 0.20 0.17 0.125 NS6 d of storage 0.09b 0.97a 0.42ab 0.21ab 0.280 0.071α-Tocopherol1 939a 593b 574b 886a 113.5 0.052β-Tocopherol 12 10 12 11 1.3 NSγ-Tocopherol2 91a 107a 135b 84a 9.1 0.002γ-Tocotrienol 74 122 91 163 44.0 NSThigh0 d of storage 0.07b 0.34a 0.12b 0.09b 0.067 0.0262 d of storage 0.09b 0.32a 0.13ab 0.33a 0.075 0.0634 d of storage 0.13b 0.74a 0.36ab 0.34ab 0.170 0.0956 d of storage 0.19c 1.28a 0.66ab 0.39b 0.256 0.025α-Tocopherol1 1840b 1140a 1100a 1280a 148 0.004β-Tocopherol 17 14 12 16 1.6 NSγ-Tocopherol2 114a 154bc 172b 132c 13.0 0.018γ-Tocotrienol 164 195 141 182 49.7 NS
Control diet, CN, and control diet supplemented with the algal product, DHA, extruded linseed, LS, or the algal product plus extruded linseed, DHALS; NS =not significant. Meat was stored at 4°C; 1Co-eluted with a small proportion of α-tocotrienol. 2Co-eluted with a small proportion of β-tocotrienol. a,b,c Mean valueswithin the same row sharing a common superscript letter are not statistically different at P < 0.05.
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agreement with diet profiles despite the lowervalues of incorporation observed in meat. Thighshad higher diterpene concentrations than breastmeat, which is likely due to the higher fat content,although displaying a similar profile.α-Tocopherol was the major diterpene, while theothers vitamin E homologues were present atsmaller concentrations. This meat profile is inagreement with that usually found in animalmeats (Prates et al., 2006). Barclay et al. (1994)indicated that microalgae DHA GoldTM is rich inother antioxidant compounds, such as β-caroteneand canthaxanthin, which are responsible for thegolden hue. β-Carotene in meat was not detectedin this study. As the diets were stored undervacuum at –20°C, the algal product (which con-tains high concentrations of carotenoids,6.44 IU/100 g) and LS (which was subjected totreatment to reduce peroxide value to less than 10mEq O2/kg), are believed to remain stable duringstorage as claimed by the manufacturers (shelf lifeof 2 years and 5 m, respectively). For these rea-sons, it is unlikely that a major change in theoxidative stability of diets occurred upon storage.
Lipid and fatty acid composition
Diet
Themajor fatty acid present in all dietary treatmentswas LA (32–58% of total fatty acids) (Table 1).Nevertheless, the percentage of this fatty aciddecreased with the increase of the relative propor-tion of n-3 LC-PUFAs in the diet (DHA treatment).Total n-3 fatty acids in the supplemented diets var-ied from 21.2 g/100 g to 24.1 g/100 g of total fattyacids (1.9–2.6 g/100 g of feed DM), in contrast tothe 1.8 g/100 g of feed DM in the non-supplemen-ted diet. Compared to the CN, the three supplemen-ted diets had lower n-6/n-3 and LA/LNA ratiosbecause supplemented groups had increasedamounts of LNA and/or LC-PUFA. Hence, dietscontaining linseed (DHALS and LS) displayed alower LA/LNA ratio than the DHA diet, while thislatter diet had a lower LA/LNA ratio than the con-trol group. Linseed supplementation increasedLNA, especially in the LS treatment, where LNAcontent was 7.8-fold greater than the DHA treat-ment. Birds given DHA had higher DHA contentsin meat compared to the other diets.Concentrations of DHA were 12-fold higher in theDHA treatment than in meat from LS or controlbirds. The DHA diet had similar n-3 LC-PUFA con-centrations that described by Rymer and Givens(2010) in a treatment supplemented with 8% offish oil. The LA/LNA ratio can affect the efficiencyof the metabolic pathways since there is a competi-tion between n-6 and n-3 PUFAs for the desaturasesand elongases involved in the conversion of LC-PUFA. The affinity of Δ6 desaturase is greater for
LNA than for LA, although the typically greaterconcentration of LA in diets and consequently incells results in a greater conversion of n-6 LC-PUFA(Simopoulos, 2000). To achieve a better conversionof n-3 LC-PUFA, it is critical to increase LNA intakeand obtain a low LA/LNA ratio; LS treatment hadthe lowest ratio (1.7), while CN had the highestproportion (36.4). Also DHA treatment had 3-foldless LA/LNA than CN and DHALS had a ratio of2.8. These results suggest that there is a need tomodify lipid composition of the diets, to achievehealthier nutritional ratios.
Meat
Total lipids and fatty acid composition of breastand thigh meats are presented in Table 6. Breastmeat presented lower contents of total lipids(1.47–1.63 g/100 g) when compared to thighmeat (5.80–6.16 g/100g). Dietary lipid supple-mentation did not affect (P > 0.05) total lipidcontent in any of the meats (Komprda et al.,2005; Rebole et al., 2006). Also data from otherstudies suggest that lipid supplementation doesnot affect the total lipid content of breast andthigh meat of broilers (Scaife et al., 1994; Crespoand Esteve-Garcia, 2001).
The fatty acid composition of breast andthigh meats is also presented in Table 6. Theeffect of dietary lipid supplementation on meatFA composition was evident mostly for the indivi-dual FA, particularly on thigh meat, with theexception of some minor fatty acids (16:1c7,18:1c11). Although extensive modification ofmost FA was evident in breast meat, some majorfatty acids, such as 18:0 and 18:1c9, were notaffected (P > 0.05) by dietary lipid supplementa-tion. The general trend was increased n-3 PUFA,reflecting fatty acid composition of the diets.Nevertheless, differences between the types ofdietary lipid supplementation were observed. Inboth breast and thigh meats, LS supplementationresulted in a major increase in LNA (up to 10.4 g/100 g of total fatty acids), whereas DHA supple-mentation resulted in an improved content inDHA (up to 5.6 g/100 g in thigh meat and9.8 g/100 g in breast meat). In DHA supplemen-ted birds, there was a major depression of LA butnot of AA in both meat types, whereas in linseed-supplemented birds, the concentrations of LAremained elevated and close to the valuesobserved in meat of control animals. This mayresult from control of the degree of unsaturationof fatty acids in membrane phospholipids, tomaintain membrane fluidity and functionality(Jerónimo et al., 2009). In both meat types, DHAsupplementation resulted in increased 14:0, 15:0,16:0, 16:1c9, EPA and DHA in meat, compared toLS supplementation, reflecting the relative abun-dance of these FA in the experimental diets
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(Rymer and Givens, 2010). DHA content of meatfrom birds in the DHA treatment is in accordancewith the results of Mooney et al. (1998) and Rymerand Givens (2010). Sardi et al. (2006) evaluatedthe effects of dietary supplementation with a mar-ine algae product containing high concentrationsof DHA on heavy pig production performances.Pigs fed on marine algae diets showed an (P <0.01) improvement in DHA concentrations bothin loin and in subcutaneous fat. The deposition ofDHA and LNA, the major fatty acids in the lipidsupplements tested, showed an additive response,
presenting intermediate concentrations inDHALS when compared to LS and DHA animals.The 16:0, only in thigh meat, and LA, in bothmeats, presented higher proportions in DHALSbirds (P < 0.05) than the expected intermediatevalue obtained by additive interpolation.
The partial sums of fatty acids were calculatedto detect any effect of diets on PUFA elongase anddesaturase activities. Although the percentages ofshort-chain fatty acids (SFA), monounsaturatedfatty acids (MUFA), PUFA, total n-3 and total n-6seemed similar in breast and thighs, some other
Table 6. Total lipid content (g/100 g fresh meat), fatty acid composition and selected sums of fatty acids (g/100 g fatty acids) andnutritional ratios in breast meat of broilers fed ad libitum with the experimental diets during the finisher period
Treatment
SEM Significance
Treatment
SEM SignificanceCN DHA LS DHALS CN DHA LS DHALSBreast Thigh
Control diet, CN, and control diet supplemented with the algal product, DHA, extruded linseed, LS, or the algal product plus extruded linseed, DHALS; NS =not significant; short-chain fatty acids, SFAs = 12:0+14:0+15:0+16:0+17:0+18:0+20:0; Mono unsaturated fatty acids, MUFA = 14:1c9+16:1c7+16:1c9+18:1c9+18:1c11+20:1c11; Poly unsaturated fatty acids, PUFA = n-3 PUFA+n-6 PUFA; n-3 PUFA = 18:3n-3+18:4n-3+20:3n-3+20:5n-3+22:5n-3+22:6n-3; n-6PUFA = 18:2n-6+18:3n-6+20:2n-6+20:3n-6+20:4n-6+22:4n-6+22:5n-6; LC-PUFA = LC-PUFA n-3+LC-PUFA n-6; LC-PUFA n-3 = 20:3n-3+20:5n-3+22:5n-3+22:6n-3;LC-PUFA n-6 = 20:2n-6+20:3n-6+20:4n-6+22:4n-6+22:5n-6. a,b,c,d Mean values within the same row sharing a common superscript letter are not statisticallydifferent at P < 0.05.
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fatty acid sums such as LC-PUFA, n-3 LC-PUFAand n-6 LC-PUFA were higher in breast meat asa consequence of the lower concentrations ofintramuscular fat (Cortinas et al., 2004). Takentogether, the data suggest that lipid supplementa-tion did not affect SFA, MUFA, PUFA, n-6 PUFAand n-6 LC-PUFA but increased n-3 PUFA inbreast meat. Further, it did not change SFA andn-6 LC-PUFA but affected all other partial sums inthigh meat. Differential effects between lipid sup-plements were observed for SFA, total PUFA, n-6PUFA and n-3 LC-PUFA in breast meat, and for allpartial sums of fatty acids (excluding MUFA) forthighs. For both meat fractions, linseed supple-mentation resulted in a lower SFA and n-3LC-PUFA, and a higher total PUFA and n-6PUFA when compared to the DHA supplementa-tion. Birds given DHA accumulated more n-3LC-PUFA in breast and thigh meat when com-pared to the other treatments. Meat from supple-mented birds had similar PUFA/SFA ratiosrelative to the control group but much lower n-6/n-3 ratios for breast (15 vs. 2) and thigh (14 vs.3) meats. These differences reflect the pattern ofthe partial sums of fatty acids. For both meat frac-tions, PUFA/SFA ratio was affected by lipid sup-plementation and meat from LS animals had ahigher value than DHA birds. The n-6/n-3 ratiowas also affected by lipid supplementation in bothmuscles and higher in LS than in DHA breastmeat.
Lower levels of total fat in meat decreases thetriacylglycerol/phospholipid (PL) ratio sincePUFAs, mainly LC-PUFA, are mainly located in thePL fraction (Rymer et al., 2003) and PLs are inhigher proportion in breast than in thigh muscles(Ratnayake et al., 1989). The partial sums of fattyacids reflect the pattern followed by the major indi-vidual constituents. Birds given DHA accumulatedmore n-3 LC-PUFA in breast and thigh meat whencompared to the other treatments as a result of thehigher amount of DHA found in the diet (González-Esquerra and Leeson, 2000; Rymer and Givens,2010). Herber and van Elswyk (1996) revealed thatit is possible to enrich eggs with DHA and EPA bydiet supplementation with marine algae.Comparatively with the non-supplemented birdsand animals supplemented with fish oil, eggs fromthe algae supplemented group had also the highestcontent of total n-3 and significant differences inPUFA. Moreover, Farrell (1998) concluded thatconsumption of enriched eggs produced by birdssupplemented with fish oil contributes substantiallyto the intake of the recommended daily dose of n-3PUFAs. Current nutritional recommendations arethat the PUFA/SFA ratio in human diets should beabove 0.45 and, within the PUFA, the n-6/n-3 ratioshould not exceed 4.0 (Burghardt et al., 2010). Inview of the above guidelines, n-6/n-3 ratios in themeat of breast and thighs of the supplemented
birds, in contrast to those from control animals,are in accordance with the recommended guide-lines. Finally, as it can be calculated from the resultsshown in Table 6, that the beneficial DHA is higherin breast (0.12 g/100 g meat) and thigh(0.31 g/100 g meat) meats of DHA-treated animals,intermediate in meat of DHA plus linseed-fed birds(0.07 g/100 g breast meat–0.15 g/100 g thigh meat)and lower in linseed-fed animals (0.01 g/100 gbreast meat–0.03 g/100 g thigh meat).
Data presented here reveal that incorpora-tion of less than 8% of DHA in broiler dietsleads to an improvement in final BW and BWgain. In contrast, introduction of LS in dietsreduced feed intake and negatively affectedgrowth rates. Meat from birds supplemented with3.7% of the algal product and 7.7% of linseed(DHALS), in contrast to the supplementationwith 7.4% of DHA, had a chicken flavour and anoverall appreciation that was comparable withmeat from non-supplemented animals, suggestingthat it is possible to enrich meat with DHA withoutaffecting sensory attributes. Taken together, thedata showed that direct supplementation of thediet is a more appropriate strategy to enrich meatwith LC-PUFA when compared to the indirectsupplementation of the diet with an n-3 LC-PUFA precursor, such as linseed. Thus, datashowed that the conversion of precursor LNAinto n-3 LC-PUFA is very limited and inefficient.The results reported here indicate that dietarysupplementation with 3.7% of DHA GoldTM maybe a good strategy to improve broiler meat with n-3 LC-PUFA without affecting its quality.
ACKNOWLEDGEMENTS
We thank Sociedade Agrícola da Quinta da Freiria SA(Bombarral, Portugal) for supplying the 1-d-old broi-lers used in this experiments and Novus International(St. Charles, USA) for the generous gift of DHAGold™. We also thank all members of the sensorypanel from Faculdade de Medicina Veterinária(Lisbon, Portugal). Teresa Ribeiro, Susana Alves andPaulo Costa were supported by Fundação para aCiência e a Tecnologia (Lisbon, Portugal) throughthe individual fellowship SFRH/BD/32321/2006,SFRH/BPD/76836/2011 and SFRH/BPD/46135/2008, respectively. This work was supported byFundação para a Ciência e a Tecnologia, Lisbon,Portugal (grant PTDC/CVT/103942/2008).
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