Effects of flaxseed supplementation on omega-6 to omega-3 fatty acid ratio, lipid mediator profile, proinflammatory cytokines and stress indices in laying hens

Figures & data

Table 1. Nutrient composition of experimental diet.

Items (g/kg)	Diet
	Commercial basal diet^a	Lintex170^c (Flaxseed)
Dry matter	910.1	936.0
Crude protein	165.0	205.9
Fat (ether extract)	25.0	344.1
Crude fiber	80.0	352.7
Crude ash	200.0	53.8
Calcium^b	40.6	-
Phosphorous^b	2.0	-
Methionine + Cysteine	6.0	-
Metabolizable energy, Mcal/kg	2.7	5.3

^a The commercial basal diet was a commercial laying hen feed product termed ‘Laying hen mid laying period No.4’, which was manufactured by a domestic feed manufacturing company.

^b The contents of calcium and phosphorus were included in the crude ash content.

^c The content of ALA in Lintex170 is 17% (w/w) of the total product.

Table 2. Fatty acid compositions of experimental diets.

Items	Group
Fatty acid composition (%)	C	T1	T2	T3
Feed composition	Commercial basal diet (C)	C + 0.9% (w/w) Lintex170	C + 1.8% (w/w) Lintex170	C + 3.6% (w/w) Lintex170
Myristic acid (C14:0)	0.42	0.37	0.35	0.26
Myristoleic acid (C14:1)	0.09	0.05	0.05	0.06
Pentadecanoic acid (C15:0)	0.11	0.04	0.04	0.05
Palmitic acid (C16:0, PA)	15.58	14.90	14.20	12.71
Palmitoleic acid (C16:1)	0.63	0.68	0.54	0.41
Margaric acid (C17:0)	0.17	0.17	0.15	0.13
Margaroleic acid (C17:1)	0.09	0.09	0.08	0.07
Stearic acid (C18:0, SA)	3.73	3.67	3.60	3.29
Elaidic acid (C18:1n9t)	0.28	0.31	0.25	0.18
Oleic acid (C18:1n9c, OA)	27.89	26.91	27.43	26.21
Linolelaidic acid (C18:2n6t)	1.23	0.67	1.13	1.03
Linoleic acid (C18:2n-6c, LA)	46.38	44.69	42.76	40.40
Arachidic acid (C20:0)	0.34	0.35	0.34	0.31
Gamma-linolenic acid(C18:3n6, GLA)	0.11	0.00	0.06	0.10
Gondoic acid (C20:1)	0.33	0.35	0.30	0.28
α-linolenic acid (C18:3n-3, ALA)	2.19	5.64	8.32	14.14
Eicosadienoic acid (C20:2)	0.09	0.07	0.06	0.07
Behenic acid (C22:0)	0.15	0.19	0.15	0.15
Erucic acid (C22:1n9)	0.00	0.13	0.00	0.00
Lignoceric acid(C24:0)	0.17	0.18	0.18	0.15
Total SFA	20.68	19.87	19.01	17.05
Total MUFA	29.32	28.51	28.65	27.21
Total n-6 PUFA	47.72	45.36	43.95	41.53
Total n-3 PUFA	2.19	5.64	8.32	14.14
n-6 / n-3	21.82	8.04	5.28	2.94

SFA, Saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-6, omega-6 fatty acid; n-3, omega-3 fatty acid.

Table 3. Fatty acid compositions of egg yolk samples at the end of the experiment¹.

Items	Group				SEM	p-value^2
Fatty acid composition (%)	C	T1	T2	T3		Trt	Lin	Quad
Feed composition	Commercial basal diet (C)	C + 0.9% (w/w) Lintex170	C + 1.8% (w/w) Lintex170	C + 3.6% (w/w) Lintex170
Myristic acid (C14:0)	0.43	0.44	0.41	0.36	0.016*	0.010**	0.002**	0.2578
Myristoleic acid (C14:1)	0.10	0.12	0.10	0.09	0.008**	0.116	0.060	0.546
Pentadecanoic acid (C15:0)	0.07	0.07	0.07	0.07	0.003**	0.9415	0.5684	0.911
Palmitic acid(C16:0, PA)	27.47	27.04	26.36	25.08	0.338	<.001***	<.001***	0.748
Palmitoleic acid (C16:1)	3.68	4.09	3.64	3.49	0.220	0.290	0.265	0.415
Margaric acid (C17:0)	0.17	0.16	0.17	0.18	0.009**	0.698	0.361	0.630
Margaroleic acid(C17:1)	0.13	0.14	0.14	0.15	0.006**	0.184	0.034*	0.815
Stearic acid (C18:0, SA)	7.83	7.50	7.61	7.51	0.1840	0.574	0.346	0.527
Elaidic acid (C18:1n9t)	0.21	0.20	0.14	0.18	0.019*	0.134	0.222	0.090
Oleic acid (C18:1n9c, OA)	38.98	38.76	39.00	38.60	0.853	0.984	0.786	0.914
Linolelaidic acid (C18:2n6t)	1.39	1.45	1.38	1.31	0.048*	0.304	0.146	0.380
Linoleic acid (C18:2n-6c, LA)	15.56	15.21	15.53	15.91	0.684	0.913	0.617	0.695
Arachidic acid (C20:0)	0.02	0.02	0.02	0.02	0.001**	0.176	0.060	0.434
Gamma-linolenic acid (C18:3n6, GLA)	0.15	0.13	0.12	0.12	0.008**	0.050*	0.011*	0.235
Gondoic acid (C20:1)	0.2	0.19	0.20	0.21	0.009**	0.743	0.407	0.515
α-linolenic acid (C18:3n-3, ALA)	0.26	0.85	1.35	2.55	0.1208	<.001***	<.001***	0.807
Heneicosanoic acid (C21:0)	0.06	0.07	0.07	0.07	0.003**	0.677	0.370	0.447
Eicosadienoic acid (C20:2n6)	0.15	0.16	0.16	0.16	0.009**	0.809	0.593	0.444
Behenic acid (C22:0)	0.06	0.00	0.00	0.00	0.003**	<.001***	<.001***	<.001***
Dihomogamma-linolenic acid (C20:3n-6)	0.18	0.18	0.17	0.16	0.008**	0.357	0.088	0.821
Erucic acid (C22:1n9)	0.02	0.02	0.02	0.01	0.001**	0.002**	<.001***	0.603
Tricosanoic acid (C23:0)	0.00	0.03	0.03	0.06	0.003**	<.001***	<.001***	0.008**
Arachidonic acid (C20:4n6)	2.37	2.06	1.86	1.61	0.044*	<.001***	<.001***	0.013*
Docosadienoic acid (C22:2n6)	0.00	0.01	0.01	0.02	0.001**	<.001***	<.001***	<.001***
Eicosapentaenoic acid (C20:5n-3, EPA)	0.00	0.02	0.03	0.06	0.003**	<.001***	<.001***	0.227
Docosahexaenoic acid (C22:6n-3, DHA)	0.50	1.09	1.41	2.04	0.081	<.001***	<.001***	0.073
Total SFA	36.11	35.33	34.74	33.35	0.369	<.001***	<.001***	0.958
Total MUFA	43.32	43.52	43.24	42.73	0.713	0.879	0.481	0.741
Total n-6 PUFA	19.80	19.20	19.23	19.29	0.712	0.925	0.696	0.630
Total n-3 PUFA	0.76	1.96	2.79	4.65	0.116	<.001***	<.001***	0.281
n-6 / n-3	26.05	9.80	6.89	4.15	0.291	<.001***	<.001***	<.001***

SEM, standard error of means; Trt, a treatment effect; Lin, a linear effect; Quad, a quadratic effect; SFA, Saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-6, omega-6 fatty acid; n-3, omega-3 fatty acid

¹ Each value represents the least squares means of five replicate eggs.

² Linear, and quadratic contrasts were tested (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 1. (A) Partial least squares discriminant analysis (PLS-DA) plot of lipid mediators obtained from the serum of laying hens fed 0, 0.9, 1.8 and 3.6% (w/w) flaxseed for 4 weeks. (B) Variable importance in projection (VIP) plot of 15 lipid mediators (VIP scores Top 15) that were differentially regulated among groups. C, 0% (w/w) flaxseed; T1, C + 0.9% (w/w) flaxseed; T2, C + 1.8% (w/w) flaxseed; T3, C + 3.6% (w/w) flaxseed. The colored boxes indicate the relative concentration of each group per lipid mediator. Lipid mediators whose relative concentrations were significantly differed among group are marked in bold italic (one-way ANOVA, P < 0.05).

Figure 2. Bar plots of relative concentrations of polyunsaturated fatty acids from the serum of laying hens fed 0, 0.9, 1.8 and 3.6% (w/w) flaxseed for 4 weeks. AA (A), EPA (B), and DHA (C) were determined in the sera of laying hens fed diets containing flaxseed for 4 weeks and measured by LC-MS/MS with 5 replicates per group. AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; C, 0% (w/w) flaxseed; T1, C + 0.9% (w/w) flaxseed; T2, C + 1.8% (w/w) flaxseed; T3, C + 3.6% (w/w) flaxseed.

Figure 3. Bar plots of relative concentrations of lipid mediators from serum of laying hens fed 0, 0.9, 1.8 and 3.6% (w/w) flaxseed for 4 weeks. 9-HOTrE, ALA-derived lipid mediator (A), PGF3α, EPA-derived lipid mediator (B), and 4-HDoHE, DHA-derived lipid mediator (C) were determined in the sera of laying hens fed diets containing flaxseed for 4 weeks and measured by LC-MS/MS with 5 replicates per group. HOTrE, hydroxyoctadecatrienoic acid; PGF, prostaglandin F; HDoHE, hydroxydocosahexaenoic acid; C, 0% (w/w) flaxseed; T1, C + 0.9% (w/w) flaxseed; T2, C + 1.8% (w/w) flaxseed; T3, C + 3.6% (w/w) flaxseed.

Table 4. The levels of serum proinflammatory cytokine (TNF-alpha, IL-1beta and IL-6), serum corticosterone, and heterophil to lymphocyte ratio in experimental hens at week 0 and week 4¹

Items	Group				SEM	p-value^2
	C	T1	T2	T3
Feed composition	Commercial basal diet (C)	C + 0.9% (w/w) Lintex170	C + 1.8% (w/w) Lintex170	C + 3.6% (w/w) Lintex170		Trt	Lin	Quad
Serum pro-inflammatory cytokines (pg/mL)
TNF-alpha
Week 0	27.13	23.54	39.35	23.93	25.902	0.485	0.558	0.654
Week 4	31.58^b	23.34^ab	10.55^a	12.06^a	24.341	0.380	0.119	0.545
IL-1beta
Week 0	137.67	84.07	123.47	101.21	10.800	0.725	0.970	0.540
Week 4	137.36	80.53	92.84	63.30	8.233	0.380	0.147	0.378
IL-6
Week 0	12.68	13.07	12.55	17.68	1.490	0.060	0.109	0.195
Week 4	15.05	12.79	12.68	13.66	1.272	0.533	0.593	0.191
Serum corticosterone (ng/mL)
Week 0	16.81	13.05	11.45	22.28	4.340	0.347	0.331	0.160
Week 4	12.18	11.15	13.39	9.15	2.071	0.549	0.394	0.459
Heterophil:Lymphyocyte ratio
Week 0	1.85	1.89	1.95	1.95	0.142	0.945	0.602	0.789
Week 4	1.68^b	1.56^ab	1.42^ab	1.21^a	0.097	0.021*	0.002**	0.846

SEM, standard error of means; Trt, a treatment effect; Lin, a linear effect; Quad, a quadratic effect; TNF, Tumor necrosis factor; IL, Interleukin.

¹ Each value represents the least squares means of 5 replicates per group.

² Linear, and quadratic contrasts were tested (*p < 0.05, **p < 0.01, ***p < 0.001).

^a-b Means in a row without a common superscript letter differ significantly as analyzed by one-way ANOVA with post hoc TUKEY HSD test.

Table 5. Effects of flaxseed in the diet on the performance and egg quality of laying hens during 33–37 weeks of age¹

Items	Group				SEM	p-value^2
Feed composition	Commercial basal diet (C)	C+0.9%(w/w) Lintex170	C+1.8%(w/w) Lintex170	C+3.6%(w/w) Lintex170		Trt	Lin	Quad
Laying performance
Week 0–4
Hen-day egg production (%)	82.36	87.93	87.71	87.57	2.681	0.391	0.265	0.243
Egg mass production (g day^−1 hen^−1)	47.64	52.05	52.94	51.65	1.731	0.143	0.171	0.065
Average egg weight (g/egg)	57.52^a	59.10^bc	60.20^b	58.83^c	0.352	<0.001***	0.022*	<0.001***
Feed intake (g day^−1 hen^−1)	118.74	119.56	119.25	119.22	0.472	0.673	0.669	0.402
Feed conversion ratio (g feed/g egg mass)	2.07^a	2.02^bc	1.98^b	2.03^ac	0.012	<0.001***	0.018*	<0.001***
Egg quality
Haugh unit
Week 4	101.85	99.261	99.52	102.12	1.678	0.499	0.694	0.154
Eggshell thickness (mm)
Week 4	0.395	0.397	0.404	0.408	0.007	0.596	0.193	0.830

SEM, standard error of means; Trt, a treatment effect; Lin, a linear effect; Quad, a quadratic effect.

¹ Each value represents the least squares means of 10 replicate cages (5 hens per cage).

² Linear, and quadratic contrasts were tested (*p < 0.05, **p < 0.01, ***p < 0.001).

^a–c Means in a row without a common superscript letter differ significantly as analyzed by one-way ANOVA with post hoc TUKEY HSD test.