The omega-3 fatty acid (n-3 FA) content of broiler tissues can be increased through dietary supplementation of hens with n-3 FA-rich microalgae. The aim of this study was to evaluate the effect of three different dietary inclusion levels of a docosahexaenoic acid (DHA)-rich microalgae (AURA) on broiler performance and the enrichment of tissues with n-3 FA. The randomized study was conducted using 352 birds, housed in 32 pens with 11 birds per pen. Pens were randomly assigned to one of four treatments, with each treatment replicated 8 times. The treatments included one unsupplemented control (0%) and three wheat-soya based experimental diets supplemented with AURA at a level of 0.5%, 1.5% and 2.5% for the starter, grower and finisher periods. Birds were weighed on days 0, 10, 24, 35 and 41, and feed intake was recorded per pen. On day 41, five birds per treatment were euthanized and individually weighed. Thigh muscle, breast muscle, liver, kidney and skin samples were taken post-mortem, freeze dried and DHA content quantified, following fat extraction and methylation, by GC-FID (AOAC 996.06 method). Performance and tissue data were analyzed by ANOVA with Dunnett’s (2-sided) post-hoc test to determine the differences between the mean values for each treatment. Dietary supplementation with AURA had no effect on body weight or feed intake during any period of the study. For thigh muscle, kidney and skin the DHA increased linearly (P < 0.05) with increasing level of dietary AURA, whilst there was a quadratic response in uptake of DHA in breast muscle and liver. The study demonstrated the potential of efficiently enriching broiler meat and organs with DHA by feeding AURA.
Consuming adequate amounts of omega-3 fatty acids (n-3 FA) can reduce the risk of various diseases including cardiovascular disease and depressive disorders [
Due to the simplicity of modifying FA composition of animal meat, milk or eggs through the diet, early attempts focused on two strategies, either through supplementation with fish meal or fish oils to provide EPA and DHA directly or alternatively to supplement flaxseed as a source of alpha linoleic acid (ALA n-3) [
Flaxseed can be a good source of omega-3, however it is required in higher doses which cause issues for formulators due to dilution of other nutrients in the feed and changes in processing quality when pelleting and crumbling, for example. Moreover, the addition of a high percentage of flaxseed in the diet has been associated with a laxative effect and poor uptake of nutrients in laying hens [
The aim of the study was to evaluate the efficacy of the dietary inclusion of an unextracted Aurantiochytrium limacinum biomass, on the enrichment of select edible broiler chicken tissues, including breast meat, thigh meat, liver, kidney and skin with adhering fat.
The research protocol and animal care were in accordance with European Union Directive 2010/63/EU covering the protection of animals used for experimental or other scientific purposes. The 41-day trial was conducted in the broiler house of Alimetrics Ltd. in Southern Finland. 352-day-old broiler chicks were housed in 88 open pens (1.125 m2 each) with wood shavings litter, in an environmentally controlled room. Feed and water were supplied ad libitum for the 41-day study. Pens were randomly assigned to one of four dietary treatments, with each treatment replicated 8 times (11 birds per pen). Each chick was marked with permanent color to feathers to identify the treatment but not the individual animal. A veterinarian checked the health of the chicks at the beginning of the trial. The birds were observed twice a day. Chicks with compromised health were excluded from the trial.
The temperature of the barn was raised to 32˚C two days before chicks arrived. Luminosity was adjusted to 20 lux and air humidity was adjusted according to recommendations. Brooder lamps were adjusted to provide extra heating to the chicks during the first week. The temperature was gradually decreased to 22˚C over the rearing period. Temperature, ventilation and humidity was monitored and recorded throughout the experiment on a daily basis. The dark hours were gradually increased within a week, so that light-dark cycle was 18 hours light and 6 hours dark daily. The standard temperature and lighting program were followed.
The diet was wheat-soy based feed for broiler chicks as specified in the
The analytical composition of AURA was determined using standardized and validated procedures (Association of Analytical Chemists, AOAC, USA) prior to the start of the study at Eurofins Nutrition Analysis Centre (Des Moines, USA): crude protein (AOAC 990.03), crude fat (AOAC 954.02), moisture (AOAC 930.15) and ash (AOAC 942.05). The fatty acid composition (AOAC 996.06) of AURA was determined at Mérieux NutriSciences (Burnaby, BC, Canada) in compliance with ISO 17025. In brief, fat and fatty acids were extracted from the algae biomass powder by acidic hydrolytic methods and subsequently into ether followed by methylation resulting in Fatty Acid Methyl Esters (FAMEs) using boron trifluoride (BF3) in methanol. The FAME reference solution (GLC-85) and triundecanoin (C11:0) used as an internal standard for sample extraction were purchased from Nu-Chek-Prep Inc. (Minnesota, USA). The FAMEs were quantitively measured by gas chromatography equipped with a hydrogen flame ionization detector as described in Section 2.4.
Performance observations included live weight (d0, 10, 24, 35 and 41), feed intake (pen) and daily mortality. The feed conversion ratio was calculated and corrected for mortality. On day 41, five birds per treatment (20 birds total) were euthanized by cervical dislocation, coded, weighed individually, abdominal cavity opened and representative samples of the thigh and breast muscles (skinless), liver, kidney and skin (with adhering fat) were removed and stored in polyethylene bags frozen at −20˚C immediately.
Starter % 0 - 10 days | Grower % 10 - 24 days | Finisher % 24 - 41 days | |
---|---|---|---|
Wheat | 59.84 | 64.17 | 65.79 |
Soybean meal 48 | 31.80 | 27.90 | 25.50 |
Sunflower oil | 3.60 | 3.46 | 4.50 |
Monocalcium phosphate | 1.60 | 1.50 | 1.40 |
Limestone | 1.65 | 1.55 | 1.48 |
NaCl | 0.40 | 0.40 | 0.40 |
Mineral premix* | 0.20 | 0.20 | 0.20 |
Vitamin premix** | 0.20 | 0.20 | 0.20 |
Methionine | 0.26 | 0.23 | 0.21 |
L-Lysine HCl | 0.36 | 0.31 | 0.25 |
Threonine | 0.09 | 0.08 | 0.07 |
Total | 100 | 100 | 100 |
MJ/kg | 12.42 | 12.55 | 12.92 |
CP g/kg | 220.97 | 206.13 | 195.54 |
aContents of the mineral premix: calcium 296.8 g/kg, iron 12.5 g/kg, copper 4 g/kg, manganese 25 g/kg, zinc 32.5 g/kg, iodine 0.225 g/kg, selenium 0.1 g/kg. bContents of the vitamin premix: calcium 331.3 g/kg, vitamin A 6,000,000 IU, vitamin D3 2,250,000 IU, vitamin E 30000, tocopherol 27,270 mg/kg, vitamin K3 1505 mg/kg, vitamin B1 1257.3 mg/kg, vitamin B2 3000 mg/kg, vitamin B6 2009.7 mg/kg, vitamin B12 12.5 mg/kg, biotin 75 mg/kg, folic acid 504 mg/kg, niacin 20,072 mg/kg, pantothenic acid 7506.8 mg/kg
Fatty acid profiles of tissue and organ samples were determined at Mérieux NutriSciences (Burnaby, BC, Canada) in compliance with ISO 17025. Recently, a GC-FID method (AOAC 996.06) was validated and verified to demonstrate the fitness for purpose in analyzing seven fatty acids in five different chicken tissues; i.e. breast, thigh, skin, kidney and liver [
The following fatty acid methyl ester standards were sourced from Nu-Chek-Prep Inc. (Minnesota, USA); methyl 4,7,10,13,16,19-docosahexaenoate; methyl hexadecanoate; methyl 9-octadecenoate; methyl trans 9-octadecenoate; 9,12-methyl octadecadienoate; methyl trans 9,12-octadecadienoate, methyl 5,8,11,14,17 eicosapentaenoate. The internal standard for sample extraction, 1,2,3-triundecanoylglycerol (common name: triundecanoin), the internal standard for calibration curve and QCs, methyl undecanoate and docosahexaenoic acid (DHA) were also all purchased from Nu-Chek-Prep Inc. (Minnesota, USA). The Omega-3 Concentrate (Standard Reference Material 3275) was purchased from NIST (Gaithersburg, USA). FAMEs were quantitatively measured on an Agilent 6890 N gas chromatograph (Agilent, Ontario, Canada) equipped with a SP2560 100 cm long capillary column, a hydrogen flame ionization detector (FID) set at 250˚C temperature and an Agilent 7683 autosampler (Agilent, Ontario, Canada). Total fat was calculated as the sum of the individual fatty acids expressed as triglyceride equivalents and calculated back to wet tissue weight.
Performance and tissue data were analyzed by the general linear model procedure of Minitab® (Minitab® version 18, State College, USA) with Dunnett’s (2-sided) or Tukey’s post-hoc tests used to determine the differences between the mean values for each treatment. For the ANOVA model, p < 0.05 indicated a significant difference. Regression analysis was used to determine the relationship between DHA intake and the DHA content of the broiler tissues or organs.
The test article, AURA, used in this study primarily consisted of 70.2 g crude fat/100 g DM biomass and was composed of a significant level of palmitic acid and DHA, 35.97% and 17.04% respectively (
The mean DHA content detected in the breast, thigh, liver and kidney and skin with adhering fat for each treatment group is presented in
Fatty Acid | g/100 g | Fatty Acid | g/100 g |
---|---|---|---|
C4:0 (Butyric acid) | <0.01 | C18:4 (Octadecatetraenoic acid) | 0.05 |
C6:0 (Caproic acid) | <0.01 | C20:0 (Arachidic acid) | 0.15 |
C8:0 (Caprylic acid) | <0.01 | C20:1 (Gadoleic acid + isomers) | <0.01 |
Total Short Chain Fatty Acids | <0.01 | C20:2 (Eicosadienoic acid) | <0.01 |
C20:3 (Eicosatrienoic acid − Omega 3) | <0.01 | ||
C10:0 (Capric acid) | <0.01 | C20:3 (di-homo-gamma-linolenic acid − Omega 6) | 0.10 |
C11:0 (Undecanoic acid) | <0.01 | C20:4 (Eicosatetraenoic acid − Omega 3) | 0.31 |
C12:0 (Lauric acid) | 0.10 | C20:4 (Arachidonic acid − Omega 6) | 0.05 |
C14:0 (Myristic acid) | 3.31 | C20:5 (Eicosapentaenoic acid − Omega 3) | 0.21 |
C14:1 (Myristoleic acid) | <0.01 | C21:5 (Heneicosapentaenoic acid − Omega 3) | <0.01 |
C15:0 (Pentadecanoic acid) | 0.24 | C22:0 (Behenic acid) | 0.08 |
C15:1 (Pentadecenoic acid) | <0.01 | C22:1 (Erucic + isomers) | <0.01 |
Total Medium Chain Fatty Acids | 3.65 | C22:2 (Docosadienoic acid − Omega 6) | <0.01 |
C22:3 (Docosatrienoic acid − Omega 3) | <0.01 | ||
C16:0 (Palmitic acid) | 35.97 | C22:4 (Docosatetraenoic acid − Omega 6) | <0.01 |
C16:1 Total (Palmitoleic acid + isomers) | 0.18 | C22:5 (Docosapentaenoic acid − Omega 3) | 0.04 |
C17:0 (Margaric acid) | 0.08 | C22:5 (Docosapentaenoic acid − Omega 6) | 4.22 |
C17:1 (Heptadecenoic acid) | <0.01 | C22:6 (Docosahexaenoic acid − Omega 3) | 17.04 |
C18:0 (Stearic acid) | 0.99 | Total Long chain fatty acids | 59.63 |
C18:2 (Linoleic acid + isomers) | 0.11 | ||
C18:3 (alpha linolenic acid − Omega 3) | 0.02 | ||
C18:3 (gamma linolenic acid − Omega6) | 0.03 | Total Fatty Acids | 63.68 |
0.0% | 0.50% | 1.00% | 2.50% | SEM | P-value | |
---|---|---|---|---|---|---|
Breast | 15.2d | 188.6c | 323.8b | 445.4a | 20.3 | <0.001 |
Thigh | 16.2d | 201.6c | 469.2b | 835.8a | 26.7 | <0.001 |
Liver | 62.1d | 514.0c | 835.0b | 1100.0a | 37.1 | <0.001 |
Kidney | 42.7d | 196.5c | 290.0b | 423.8a | 10.4 | <0.001 |
Skin with fat | 15.5c | 204.8c | 560.2b | 1208.6a | 49.0 | <0.001 |
diet [
The Control thigh meat (dark) averaged 16.2 mg/100 g fresh edible tissue and in general agreement with previous findings, 9 - 24 mg/100 g fresh edible tissue [
A strong relationship was found between the dietary concentration of DHA and the DHA concentration in all tissues (
The incorporation of dietary DHA from fish or marine algae sources into poultry meat tissue has a long history, going back to the 1960’s [
contrast to our study findings, in a review of the literature, Rymer and Givens [
This study shows the possibility of efficiently feeding broilers a source of sustainable n-3 FA (DHA) thereby producing a healthier DHA-enriched meat. An enriched poultry meat product that redresses nutritional imbalances in today’s diet [
In conclusion, the dietary supplementation of an unextracted DHA-rich Aurantiochytrium limacinum biomass to a broiler diet for the entire production cycle (41 days) resulted in the significant and efficient transfer of DHA to the fat of the muscle, skin and organs, thereby improving the nutritional quality of the tissues for human consumption.
Funding for this work was provided by Alltech SARL (France). The authors would like to express their gratitude to Ms. Rebecca Timmons (Alltech USA) for her technical input on microalgae and Dr. Tuoying Ao for his knowledge of the application of microalgae in broiler nutrition
The authors Colm A. Moran and Jason D. Keegan work for Alltech who manufacture and market the ingredient All-G-Rich.
Moran, C.A., Keegan, J.D., Vienola, K. and Apajalahti, J. (2018) Broiler Tissue Enrichment with Docosahexaenoic Acid (DHA) through Dietary Supplementation with Aurantiochytrium limacinum Algae. Food and Nutrition Sciences, 9, 1160-1173. https://doi.org/10.4236/fns.2018.910084