Effects of dietary ω-3 fatty acid-enriched microalgae supplementation on growth performance, blood profiles, meat quality, and fatty acid composition of meat in broilers

Abstract

A total of 300 2-d-old ROSS 308 (BW of 42.6±0.1 g) broiler chicks were randomly allotted to one of the three dietary treatments as follows: (1) CON (basal diet), (2) MA0.1 [basal diet +0.1% microalgae (Schizochytrium JB5) powder], and (3) MA0.2 [basal diet+0.2% microalgae (Schizochytrium JB5) powder]. There were five replicate pens per treatment with 20 birds per pen. Dietary microalgae did not affect the growth performance, red blood cells, white blood cells, and relative organ weight of liver, spleen, gizzard, abdominal fat, bursa of Fabricius, and breast meat (P>0.05). However, the inclusion of microalgae increased lymphocyte concentration compared to those that were fed a basal diet (P<0.05). The stearic acid composition of breast muscle was lower in MA0.1 treatment than CON treatment (P<0.05). Dietary microalgae powder increased the oleic acid, DHA, ω-3 fatty acid, and USFA compositions compared to the CON group (P<0.05). Microalgae supplementation reduced ω-6/ω-3 fatty acid ratio, saturated fatty acid contents and SFA/USFA ratio than those fed a basal diet (P<0.05). In conclusion, dietary ω-3-fatty-acid-enriched microalgae supplementation can improve the fatty acid composition of breast meat without affecting the growth performance in broilers.

Keywords:

• broilers
• fatty acid contents
• growth performance

1. Introduction

With the increasing awareness of the nutritional quality and health benefits of food, people are growing focused on the ways to improve the fatty acid composition of their foods. It is well accepted that broiler meat contains high protein and low fat content and has been considered as one of the main sources of polyunsaturated fatty acid for human diet (Swain et al. 2012). Mori et al. (2000) had previously suggested that long-chain n-3 fatty acid is very important in human nutrition because of its improvement role in the maintenance of human health. Hulan et al. (1988) also suggested that the n-3 fatty acid in poultry meat could be improved by increasing the levels of n-3 polyunsaturated fatty acid in poultry diets through the inclusion of oily fish by-products. Therefore, providing n-3 PUFA to the broilers could be considered as a good method to modify fatty acid composition and improve the nutritional value of broiler meat.

Our study was concerned about a kind of n-3-fatty-acid-enriched marine microalgae (Schizochytrium type), which was reported as a cheaper and interesting source n-3 fatty acid than the other sources such as fish oil and plant oils (Rymer et al. 2010). To the best of our knowledge, the utilisation of this n-3 fatty acid source is not well investigated in the poultry industry. Therefore, the objective of the current study was to try and evaluate the effects of microalgae (MA) as an ω-3 fatty acid source supplementation on growth performance, blood profiles, meat quality, and fatty acid composition of breast meat in broilers.

2. Materials and methods

2.1. Animals and sampling

A total of 300 2-d-old ROSS 308 (BW of 42.6±0.1g) broiler chicks were obtained from a commercial hatchery (Yang Ji Company, Cheonan, Choongnam, South Korea). All birds were randomly raised in-house with stainless steel pens of identical size (1.75×1.55 m), with concrete floors covered with clean rice bran, in an area that was provided with continuous light. The temperature of the room was maintained at 33±1°C for the first 3 d, and decreased to 24°C until the end of the experiment. The diets were fed during the experiment in two phases consisting of a starter phase from d 0 to 21 and a finisher phase from d 22 to 35. The chicks were given free access to water and mash feed. All birds used in this trial were handled in accordance with the guidelines set forth by the Animal Care and Use Committee of Dankook University.

The broilers were randomly allotted to any one of the three dietary treatments with five replicate pens per treatment and 20 broilers per pen. The composition of the basal diet is shown in Table 1. The dietary treatments were as follows: (1) CON (basal diet), (2) MA0.1 [basal diet+0.1% marine microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source (Table 2)], and (3) MA0.2 [basal diet+0.2% marine microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source]. All diets were formulated to meet or exceed the dietary nutritional requirements of the broilers reported by the NRC (1994). The marine microalgae (Schizochytrium JB5) were obtained in the form of powder (JINIS Co., LTD., Wanju, Jeonbuk, Korea). Fatty acid content of microalgae powder is shown in Table 2.

Table 1. Basal diet composition for broilers (as-fed basis).

Ingredient, g/kg
Ground maize	674.5
Soybean meal	181.4
Rice bran	50.0
Molasses	50.0
Animal fat	20.0
Defluorinated phosphate	11.2
Calcium carbonate	6.8
L-Lysine·HCL	2.0
Salt	1.5
Vitamin premix^a	0.5
Mineral premix^b	1.5
Choline chloride	0.4
L-Threonine	0.2
Chemical composition^c
DE, MJ/kg	14.1
CP, g/kg	148.0
Lys, g/kg	8.9
Ca, g/kg	7.4
P, g/kg	5.4

^a Provided per kg of complete diet: 4000 IU of vitamin A; 800 IU of vitamin D3; 17 IU of vitamin E; 2mg of vitamin K; 4mg of vitamin B2; 1 mg of vitamin B6; 16 µg of vitamin B12; 11mg of pantothenic acid; 20 mg of niacin and 0.02 mg of biotin.

^b Provided per kg of complete diet: 220 mg of Cu; 175 mg of Fe; 191 mg of Zn; 89 mg of Mn; 0.3 mg of I; 0.5 mg of Co and 0.15 mg of Se.

^c All calculated values are based on NRC (1998) tabular values.

Table 2. Fatty acid composition of microalgae (Schizochytrium JB5) powder.

Fatty acid (% total fatty acids)
12:0	0.20
14:0	8.95
15:0	2.19
16:0	38.01
17:0	0.85
18:0	1.47
18:2 n-6	0.23
18:3 n-3	0.59
18:3 n-6	0.23
20:0	0.52
21:0	0.33
20:3 n-6	0.33
20:4 n-6	0.49
22:0	0.39
20:5 n-3	0.72
22:5 n-6	6.74
22:6 n-3	37.63
Saturated fatty acid	52.91
Unsaturated fatty acid	46.96
ω-3/ω-6 fatty acid	5.58

2.2. Procedures, sampling, and analysis

2.2.1. Growth performance and blood profiles

Broilers were weighed and the feed intake (FI) was recorded on d 0, 7, 21, and 35. This information was then used to calculate Body Weight gain (BWG) and feed conversion rate (FCR). At the end of the experiment, 15 broilers were randomly selected from each treatment (three birds per pen) and blood samples were selected from the wing vein into a sterile syringe and stored at 4°C. The white blood cells (WBC), red blood cells (RBC), and lymphocyte counts in the whole blood were then determined using an automatic blood analyzer (ADVIA 120, Bayer, NY).

2.2.2. Relative organ weight

Three broilers per pen were weighed individually and slaughtered by cervical dislocation. The gizzard, bursa of Fabricius, liver, spleen, abdominal fat, and breast and thigh meat were then removed by trained personnel and weighed (Table 3). The breast muscle was stored at 20°C for the following analysis. Organ weight was expressed as a percentage of BW.

2.2.3. Fatty acid analysis

Lipid from the breast meat was extracted with hexane/isopropanol (3:2 v/v). Fatty acids were converted into methyl esters. Briefly, 0.5 mL of toluene and 2 mL of 5% KOH-MeOH were added to the lipid and the samples were vortex-mixed and heated at 70°C for 8 min and then cooled in cold water; 2 mL of 14% BF3-MeOH was added to the sample and heated at 70°C for another 8 min. The sample was cooled, and then 3 mL of 5% NaCl was added to the sample and mixed; 5 mL of distilled water and 0.5 mL of hexane were added to extract the fatty acid methyl esters (FAME). The mixture was vortexed and centrifuged at 3000×g for 5 min, and then the upper phase was collected and dried with sodium sulphate. Samples were analysed for total fatty acids using an HP5890 gas chromatograph with a flame ionisation detector (Hewlett Packard 5890 Series II, USA). The FAME were separated using a Supelcowax-10 fused silica capillary column (100 m, 0.32 mm i.d., 0.25 µm film thickness; Supelco, Inc., Bellefonte, PA, USA) with a 1.2 mL/min of helium flow. Oven temperature was increased from 220 to 240°C at the rate of 2°C/min. Temperatures of the injector and detector were 240 and 250°C, respectively; 1 µL of sample was injected into the column in the split mode (50:1). The peak of fatty acids was identified and quantified by comparing the retention time and peak area of each fatty acid standard (Sigma, USA). Fatty acid content was expressed as the percentage of the total fatty acids. The recovery of methylated fatty acids calculated in a comparison to the internal standard was higher than 80%.

2.3. Statistical analysis

Data were statistically analysed by ANOVA using GLM procedure SAS (SAS Institute 1996) for a randomised complete block design. Differences among all treatments were separated by Duncan's (1998) multiple range tests. Mean values and standard error of means (S.E.M.) were reported. In addition, orthogonal comparisons were conducted to measure the linear and quadratic effects for increasing dietary concentrations of supplemental microalgae powder. Statements of statistical significance were based on P<0.05, while P<0.10 was considered indicative of a tendency.

3. Results and discussion

3.1. Growth performance

The effect of marine microalgae on the growth performance is summarised in Table 4. The inclusion of MA did not affect the BWG, feed intake (FI), and feed conversion ratio (FCR) throughout the experiment (P>0.05), which is in agreement with Rymer et al. (2010), who suggested that algal (Schizochytrium) supplementation did not affect the growth performance in broiler chickens. Similarly, Inborr and Waldenstedt (2000) also suggested that the supplementation of natural astaxanthin, in the form of aglgal meal, did not affect the live weight gain in broilers, although the FCR were slightly reduced. Therefore our study suggested that the growth performance will not be affected by the algal sources used in different experiments.

Table 3. Effects of dietary microalgae (Schyzochytrium JB5) as ω-3 fatty acid source supplementation on relative organ weight in broilers.

					P-value
Items,%	CON^a	MA0.1^a	MA0.2^a	S.E.M.^b	Linear	Quadratic
Liver	2.62	2.64	2.61	0.10	0.887	0.458
Spleen	0.14	0.13	0.13	0.03	0.798	0.909
Gizzard	1.75	1.82	1.89	0.23	0.736	0.859
Abdominal fat	1.35	1.62	1.63	0.16	0.413	0.443
Bursa of Fabricius	0.21	0.21	0.23	0.02	0.940	0.410
Breast meat	7.56	8.22	7.53	0.26	0.927	0.150
Thigh meat	3.31	3.15	3.32	0.15	0.974	0.967

^a Abbreviation:CON, basal diet; MA0.1, basal diet+0.1% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source; MA0.2, basal diet+0.2% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source.

^b Standard error mean.

3.2. Blood profiles and relative organ weight

The effect of marine microalgae supplementation on the blood profile is presented in Table 5. Dietary microalgae powder linearly increased the lymphocyte concentration (P<0.05). However, no difference was observed on the red blood cells (RBC) and white blood cells (WBC) count among treatments (P>0.05).

Table 4. Effects of dietary microalgae (Schyzochytrium JB5) as ω-3 fatty acid source supplementation on growth performance in broilers.

					P-value
Items	CON^a	MA0.1^a	MA0.2^a	S.E.M.^b	Linear	Quadratic
BWG, g
1 weeks	92	85	100	7.3	0.377	0.228
3 weeks	592	594	574	13.4	0.685	0.734
5 weeks	876	872	935	38.5	0.361	0.535
Overall	1560	1,551	1,609	38.3	0.350	0.460
FI, g
1 weeks	125	122	133	5.2	0.189	0.220
3 weeks	959	920	963	24.6	0.982	0.226
5 weeks	2075	2,167	1,951	125.2	0.780	0.676
Overall	3159	3,209	3,047	108.0	0.696	0.894
FCR
1 weeks	1.36	1.44	1.33	0.07	0.675	0.253
3 weeks	1.62	1.55	1.68	0.07	0.852	0.369
5 weeks	2.37	2.49	2.08	0.23	0.794	0.500
Overall	2.03	2.07	1.89	0.11	0.861	0.597

^a Abbreviation:CON, basal diet; MA0.1, basal diet+0.1% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source; MA0.2, basal diet+0.2% microalgae (Schiz ochytrium JB5) powder as ω-3 fatty acid source.

^b Standard error mean

Previously, it is well suggested that the blood characteristics could be used as a good way to evaluate the feed quality for the broilers (Alzawqari et al. 2011; Masoudi et al. 2011). Kotrbácek et al. (1994) have reported that dietary algae Cholrella vularis supplementation improved the immune response of broilers by increasing phagocytic activity of leucocyte in blood. Hossein et al. (2010) also suggested that algae extracts might stimulate the immune system by activating macrophages and lymphocytes, resulting in the augmentation of the defence mechanism of the host. In the current study, lymphocyte concentration in blood was increased with microalgae supplementation, which again confirmed the lymphocyte-enhanced effect of n-3 fatty acid supplementation.

In terms of the relative organ weight, the inclusion of the microalgae powder did not affect the relative weight of liver, spleen, gizzard, abdominal fat, bursa of Fabricius, and breast and thigh meat (P>0.05). It is well accepted that the measurement of immune organ weight is a common method for evaluation of the immune status in broiler chickens (Heckert et al. 2002; Ghobadi & Karimi 2012). Therefore, we hypothesised that the relative organ weight of the broilers could be affected by the microalgae supplementation. The results were out of anticipation, which is in agreement with Venkataraman et al. (1994), who reported that the sun-dried Spirulina platensis algae supplementation did not affect the organ weight in broilers.

Table 5. Effects of dietary microalgae (Schyzochytrium JB5) as ω-3 fatty acid source supplementation on blood profile in broilers.

					P-value
Items	CON^1	MA0.1^1	MA0.2^1	S.E.M.^2	Linear	Quadratic
Red blood cell,×10^6/µl	2.32	2.41	2.57	0.12	0.737	0.981
White blood cell,×10^3/µl	349.36	405.38	355.96	31.61	0.218	0.127
Lymphocyte,%	71.2^b	76.0^a	78.8^a	3.65	<0.001	<0.001

¹ Abbreviation: CON, basal diet; MA0.1, basal diet+0.1% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source; MA0.2, basal diet+0.2% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source.

² Standard error mean.

^a,b Means in the same row with different superscripts differ (P<0.05).

3.3. Fatty acid composition of breast meat

The effect of microalgae powder on the fatty acid composition of breast meat is presented in Table 6. Broilers fed MA0.5 treatment reduced (quadratic effect; P=0.044) stearic acid (C18:0) compared to those fed CON treatment (P<0.05). Dietary MA treatment increased oleic acid (C18:1 n-9), DHA (C22:6 n-3), ω-3 fatty acid (P=0.008), and USFA (P=0.024) contents of breast meat compared to those in CON treatment (P<0.05). Moreover, the inclusion of MA treatment linearly decreased the ω-6/ω-3 fatty acid ratio (P=0.022), saturated fatty acid content (P=0.038), and SFA/USFA ratio (P=0.026) compared to basal diet.

Table 6. Effects of dietary microalgae (Schyzochytrium JB5) as ω-3 fatty acid source supplementation on fatty acid composition of breast meat in broilers.

					P-value
Items	CON^1	MA0.1^1	MA0.2^1	S.E.M.^2	Linear	Quadratic
C14:0 (Myristic acid),%	0.53	0.38	0.36	0.05	0.091	0.367
C16:0 (Palmitic acid),%	28.17	27.82	26.30	1.32	0.374	0.736
C16:1 (Palmitoleic acid),%	2.76	3.18	2.97	0.21	0.499	0.285
C18:0 (Stearic acid),%	14.99^a	11.44^b	12.86^ab	0.70	0.097	0.044
C18:1n9 (Oleic acid),%	25.87^b	27.92^a	28.81^a	0.29	0.002	0.174
C18:2n6 (Linoleic acid),%	20.90	22.13	21.53	0.67	0.546	0.329
C20:5n3 (Eicosapataenoic acid),%	0.60	0.84	0.76	0.08	0.234	0.174
C22:6n3 (Docosahexaenoic acid),%	0.17^c	0.48^b	0.71^a	0.05	0.002	0.608
ω-6 fatty acid,%	21.44	22.61	21.84	0.74	0.722	0.346
ω-3 fatty acid,%	1.35^b	2.16^a	2.27^a	0.13	0.008	0.092
ω-6/ω-3 ratio	15.84^a	10.45^b	9.63^b	1.25	0.022	0.202
SFA,%	46.83^a	42.09^b	42.14^b	1.09	0.038	0.146
USFA,%	52.68^b	57.33^a	57.25^a	0.91	0.024	0.101
SFA/USFA	0.89^a	0.73^b	0.74^b	0.03	0.026	0.105

¹ Abbreviation:CON, basal diet; MA0.1, basal diet+0.1% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source; MA0.2, basal diet+0.2% microalgae (Schizochytrium JB5) powder as ω-3 fatty acid source.

² Standard error mean.

^a,b,c Means in the same row with different superscripts differ (P<0.05).

In this study, the inclusion of MA significantly increased the n-3 fatty acid and USFA compared to the CON group; this decrease was primarily due to the oleic acid and DHA. It is well accepted that the long-chain n-3 fatty acid plays an important role in the maintenance of human health (Mori et al. 2000). Katan (2000) also demonstrated a strong relationship between the total fat intake and composition and a number of diseases such as coronary heart disease (CHD), cancer, diabetes, and depression. Moreover, Hulan et al. (1988) had suggested that the n-3 fatty acid in poultry meat could be improved by increasing the levels of n-3 polyunsaturated fatty acid in poultry diets. Choi et al. (2004) also reported that DHA-enriched 2% Euglena treatment yielded a higher DHA concentration of breast muscle, which was 3.9 times of that of the control. Therefore, we hypothesised that the new n-3 fatty acid source could produce meat with good nutrition quality. Indeed, our study indicated that birds fed the diet supplemented with MA had a lower concentration of SFA but higher PUFA and n-3 fatty acid concentration, indicating the consumption of this meat could reduce the risk of diseases such as CHD. Similar results were also observed by López-Ferrer et al. (1999, 2001), who also reported an increased USFA concentration with ω-3 fatty acid-enriched fish oil.

4. Conclusion

In conclusion, dietary ω-3-fatty-acid-enriched microalgae supplementation can improve the fatty acid composition of breast meat without affecting the growth performance in broilers, indicating that the microalgae could be used as an ω-3 fatty acid source in the broiler's diet.