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

ABSTRACT

Although polyunsaturated fatty acids (PUFAs) have gained attention because of their physiological effects on inflammation and stress, their derivatives, termed lipid mediators, have been rarely examined, especially in laying hens. In this study, we aimed to investigate PUFA and lipid mediator profiles of laying hens after feeding 0, 0.9, 1.8, or 3.6% (w/w) dietary flaxseed (Lintex170) for 4 weeks. We also monitored the indices of inflammation (serum proinflammatory cytokines) and stress (serum corticosterone, ratio of heterophils to lymphocytes). In this study, flaxseed reduced the omega-6 to omega-3 FA ratio and increased several omega-3 FA-derived lipid mediators. Furthermore, levels of the proinflammatory cytokine tumour necrosis factor alpha and two stress indices (corticosterone, heterophil to lymphocyte ratio) were decreased when fed the 3.6% flaxseed diet. Overall, laying performance indices were also significantly improved by flaxseed treatment. These findings suggest that flaxseed may alleviate the stress state of laying hens by enriching omega-3-derived lipid mediators, which could improve laying performance.

KEYWORDS:

• Flaxseed
• inflammation
• lipid mediator
• omega-6 to omega-3 fatty acid balance
• polyunsaturated fatty acid
• stress

1. Introduction

Polyunsaturated fatty acids (PUFAs) have been reported to broadly regulate homeostatic and inflammatory processes, either directly or through transformations to bioactive metabolic compounds such as lipid mediators (Zarate et al. 2017). PUFAs can be classified into two principal families, omega-3 and omega-6 fatty acids, depending on the position of the first double bond from the methyl group (Mariamenatu and Abdu 2021). Among them, linoleic acid (LA), an omega-6 fatty acid and alpha-linolenic acid (ALA), an omega-3 fatty acid, are essential fatty acids that must be obtained from the diet; omega-3 fatty acids cannot be converted to omega-6 fatty acids, and vice versa in mammals and poultry (Alagawany et al. 2021). When they enter the body, they undergo bioconversion by elongases and desaturases, which result in the conversion of LA to arachidonic acid (AA) and ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Shahidi and Ambigaipalan 2018). These PUFAs are incorporated into the cell membrane and used as resources to produce and release various derivatives termed lipid mediators, which are converted by cell membrane-incorporated enzymes such as lipoxygenases (LOXs), cyclooxygenases (COXs), and cytochrome P450 (CYP450) (Greene et al. 2011).

Lipid mediators are known for their roles in the onset and resolution of inflammation and the systemic profile of the lipid mediators can be nutritionally controllable depending on the quantity of ingested precursors such as omega-6 and omega-3 fatty acids (Serhan et al. 2015). Although the action mechanisms of the lipid mediators derived from both omega-3 and omega-6 fatty acids are too complex to be simplified in the physiological regulation of inflammation and its resolution (Shearer and Walker 2018), the various lipid mediators generally act as signal molecules that mainly mediate their anti-inflammatory and pro-resolution activity through G-coupled protein receptors (e.g. GPR32, GPR18, ChemR23, GPR37, and LGR6) (Hallisey et al. 2020). Omega-3-derived lipid mediators, including specialized pro-resolving lipid mediators, have been a focus because of their impact on the resolution of inflammation (Arnardottir et al. 2021). Cytokines are regulators of host responses to various immunological states, including inflammation. Some cytokines, such as tumour necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6), act as promoters of inflammation and are therefore called proinflammatory cytokines (Dinarello 2000). They play a key role in the acute phase response. However, when they persist in their actions so that inflammation is not resolved, chronic inflammation occurs, resulting in various problems in the body, such as tissue damage. Levels of circulating proinflammatory cytokines are elevated in several inflammatory diseases, such as Crohn’s disease (Gabay 2006; Nejm et al. 2017; Szczeklik et al. 2012) and obesity (Borges et al. 2018). Recently, the contribution of the network between lipid mediators and cytokines in immune homeostasis has been investigated. In general, the literature has reported that omega-6 derived lipid mediators increase proinflammatory cytokines, and omega-3-derived lipid mediators increase the level of anti-inflammatory cytokines and suppress that of proinflammatory cytokines (Schmitz and Ecker 2008). For these reasons, a balanced intake of omega-6 and omega-3 fatty acids is essential for homeostasis associated with inflammation control.

However, modern human diets and animal feeds commonly contain excessive levels of omega-6 fatty acids and low levels of omega-3 fatty acids. This is because of the high dependence on omega-6-rich resources, such as corn and soybean, to manufacture cooking oils and animal feeds. The ratio of omega-6 to omega-3 in modern diets was reported to have increased to approximately 20:1 from 1:1 in the past (Simopoulos 2016). Because fatty acid composition in the inner body has also been influenced by this omega-6 and omega-3 fatty acid imbalance in the diet, an increase in the incidence of chronic inflammatory diseases could occur (Khadge et al. 2018). Studies have found that omega-3 fatty acids counter the effects of chronic inflammatory diseases associated with cardiovascular diseases (Han et al. 2018; Nandish et al. 2018; Prasad 2019), central nervous system diseases (Czyż et al. 2016), diabetes (Zhu et al. 2020), obesity (Liu et al. 2021), and several cancers (D'Angelo et al. 2020; Liang et al. 2020; Tu et al. 2020). Similarly, the omega-6 to omega-3 fatty acid ratio in animal feed is also distorted. One of the rational inferences would be that livestock animals might experience problems such as chronic inflammation during feeding, which increases stress and adverse effects on their performance (Kang et al. 2020; Sevim et al. 2020). Therefore, restoration of the fatty acid composition through supplementation with omega-3 fatty acids in diets and feeds could be an applicable strategy to improve both human and animal health by recovering immune homeostasis.

Flaxseed has been widely used as a dietary supplement of omega-3 fatty acids in humans and animals because it contains abundant amounts of ALA, representing approximately 53% of total fatty acids (Moallem 2018). However, whole flaxseed has been known to contain anti-nutritional factors such as mucilage, cyanogenic glycosides, phytic acid and trypsin inhibitors, which could induce adverse effects such as malabsorption of primary bile acids and excretion of secondary bile acids (Mangi et al. 2021). One of the common treatments available to reduce the side effects of flaxseed is extrusion (Imran et al. 2015).

The literature has reported on omega-3 fatty acid-fortified livestock products through supplementation with omega-3 fatty acid-rich resources such as flaxseed (Antruejo et al. 2011; Gjerlaug-Enger et al. 2015; Zachut et al. 2010). However, according to a review of the literature, few reports have investigated the physiological effects of feeding omega-3 fatty acid-rich resources to livestock animals. In the present study, the effect of flaxseed supplementation was investigated in laying hens: specifically, the improvement in the omega-6 to omega-3 fatty acid ratio in eggs and animal physiology and health was assessed mainly by profiling lipid mediators after feeding. Based on the regulation of inflammation by lipid mediators and cytokines, this study’s hypothesis was that the supplementation of omega-3 fatty acids to laying hens could improve their performance by alleviating inflammation and stress during the laying period. To examine this hypothesis, the profile of lipid mediators and representative proinflammatory cytokines in serum, corticosterone levels, and the heterophil to lymphocyte ratio (H/L) for stress indices and the performance of laying hens were monitored and analyzed after supplementation with dietary flaxseed.

2. Materials and methods

2.1. Materials

The basal diet used in this study was commercial laying hen feed product provided by a domestic feed manufacturing company (Cargill Agri Purina, Inc., Seoul, Republic of Korea). Dietary flaxseed was obtained from a commercial product, Lintex170 (HANYOU BNF Co., Ltd, Seoul, Republic of Korea) which was developed by Seoul National University and HANYOU BNF Co., Ltd and manufactured by HANYOU BNF Co., Ltd. The product comprises 85% extruded flaxseed and 15% starch source; 17% of the product is ALA.

The nutrient compositions of the basal diet and dietary flaxseed used in this study are listed in Table 1. The lipid mediator standards were as follows: 14, 15-epoxyeicosatrienoic acid (EET)-d11, 5(S)hydroxyeicosatetraenoic acid (HETE)-d8, leukotriene B₄ (LTB₄)-d4, prostaglandin D₂ (PGD₂)-d4 and AA-d8 (Cayman Chemical, Ann Arbor, MI, USA). The fatty acid methyl ester standard (Supelco® 37 Component FAME Mix, FAME 37), internal standard (tridecanoic acid, C13:0), potassium hydroxide, and sulfuric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade water, acetonitrile, isopropyl alcohol, and hexane were purchased from J.T. Baker (Avantor Performance Material, Inc., Center Valley, PA, USA). A Strata-x-33-μm polymerized solid reverse-phase extraction column (Cat no.8B-S100-UBJ) was purchased from Phenomenex (Torrance, CA, USA).

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.

2.2. Animals, treatments, and management

The experiments associated with laying hens (n = 200, 33 weeks old, Lohmann Brown-Lite, initial body weight (kg): 1.89 ± 0. 05) were conducted at the Seoul National University Animal Farm (Pyeongchang, Republic of Korea). The protocol for this experiment was reviewed and approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-180219-1). The hens were randomly selected from 2000 hens of experimental animal farms and divided randomly into four groups with five hens per cage (48 cm × 45 cm × 45 cm, width × depth × height) and 10 cages per group. Fifty hens were assigned to each of the following diet treatments for 4 weeks. The experimental diets were prepared by mixing each assigned proportion (w/w) of Lintex170 (extruded flaxseed product) into the basal diet as follows: (1) commercial basal diet (C), (2) basal diet with 0.9% (w/w) Lintex170 (T1), (3) basal diet with 1.8% (w/w) Lintex170 (T2), and (4) basal diet with 3.6% (w/w) Lintex170 (T3). The ratio of omega-6 to omega-3 of each feed was 21.82 (C), 8.04 (T1), 5.28 (T2), and 2.94 (T3), calculated by comparison with the FAME 37 peak pattern (Table 2). Each experimental diet was offered (600 g per cage) daily, and fresh water was offered ad libitum during the experimental period. The hen house was maintained at approximately 25 °C, was lit for 10 h/day, and auto-ventilated by a built-in Information and Communications Technology (ICT) management automation system during the experimental period.

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.

2.3. Sample and data collection

The performance of each cage was also assessed. The number of eggs and their weights were recorded daily. Abnormal eggs, such as broken, cracked, or shell-less eggs, were also recorded daily. Feed intake was measured weekly, and the weekly feed conversion ratio [feed consumed (g)/egg mass (g)] was calculated. Egg samples [10 eggs/(group × week)] were randomly selected and analysed for albumen height, Haugh unit, eggshell thickness, and egg fatty acid profile. Albumen height was measured using a micrometre. The Haugh unit was calculated with egg weight by using the following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W^0.37), where H is albumen height (mm) and W is egg weight (g) (Eisen et al. 1962). Eggshell thickness was determined by averaging measurements taken at three locations on the egg (air cell, equator, and sharp end) by using a dial pipe gauge. After these procedures, 1 g of egg yolk was separated and collected in a glass tube to measure the egg fatty acid profile.

Blood samples were collected from 10 randomly selected hens per group at Weeks 0, 2, and 4. They were collected in two tubes: BD Vacutainer SST^TM I Advance Tubes (Becton Dickinson, Le Pont de Claix, France) for sampling serum and V-Tube^TM EDTA K3 Tubes (AB MEDICAL, Republic of Korea) to measure complete blood cell count. Blood samples were centrifuged at 3,887 × g for 10 min, and the supernatant serum was transferred to 1.5 mL plastic tubes and stored at −20 °C until use. Complete blood cell counts were immediately determined after collecting whole blood samples as quickly as possible using a hematology analyser (Advia 2120i, Siemens; Erlangen, Germany).

2.4. Gas chromatography for fatty acid profile

The fatty acid profiles were analysed using feed, egg, and serum samples (feed, 1 g × 3 replicates; egg, 1 g × 5 replicates; serum, 500 μL × 5 replicates). Fatty acids were extracted and methylated in one tube via the direct methylation method (O'Fallon et al. 2007) with some modifications. Briefly, tridecanoic acid (0.5 mg) was added to the samples in 15 mL glass tubes, followed by the addition of 5.3 mL of methanol and 700 μL of 10 N KOH. The tubes were incubated in a water bath at 55 °C for 1.5 h with brief vortexing every 20 min. Next, the tubes were chilled at RT, and 580 μL of 24 N H₂SO₄ was added. The incubation and chilling steps were repeated. Finally, 3 mL of hexane was added, and the sample was vortexed for 5 min and centrifuged at 3,000 rpm for 5 min. The upper phase was transferred to a GC vial (Agilent, Santa Clara, CA, USA) and analysed with a gas chromatography-flame ionisation detector (GC-FID, Agilent 7890B, Santa Clara, CA, USA) with SP-2560 (100 m × 0.25 mm, L × I.D; 0.2 μm, d_f, Sigma-Aldrich, St. Louis, MO, USA). FAME 37 was used as a reference for peak identification. The operating conditions of GC-FID followed the FAME 37 manual (oven temperature: 140 °C, 5 min; ramp: 240 °C at 4 °C/min and hold for 28 min; injector and detector temperature: 260 °C; split ratio: 1:30; injection volume: 1 μL).

2.5. Ultra-performance liquid chromatography for serum lipid mediator profile

Lipid mediators in serum were separated by the solid-phase extraction method (Kortz et al. 2014) using a Strata-x-33-μm polymerized solid reverse-phase extraction column. Briefly, the columns were activated with 3.5 mL of methanol followed by the same amount of water for equilibration. Samples were prepared by adding 10 ng of each deuterated internal standard to 1 mL of serum with 200 μL of methanol and 800 μL of distilled water. Samples were then loaded onto columns and washed with 10% methanol (3.5 mL) in distilled water. Finally, the samples were eluted using 1 mL of methanol. These samples were concentrated using a Speed-Vac concentrator (Labconco, Kansas City, MO, USA), resuspended in 90 μL of solvent A (70:30:0.02, water:acetonitrile:acetic acid, v:v:v) and stored at −80 °C until use.

The Ultra-Performance Liquid Chromatography (UPLC) analyses and tandem mass spectrometry (MS/MS) analyses followed reported methods (Lee et al. 2016a). The Skyline 21.1 software package (MacCoss Laboratory, Seattle, WA, USA) was used to determine the peak area of each lipid from raw data. The extracted peak areas were normalized by internal standard.

2.6. Enzyme-linked immunosorbent assay for serum proinflammatory cytokine and corticosterone levels

The levels of serum TNF-alpha, IL-1beta, IL-6 and corticosterone were determined using Week 0 and Week 4 serum samples (5 replicates per group) by using ELISA kits specific for chicken TNF-alpha, IL-1beta, IL-6, and corticosterone (CUSABIO, Wuhan, China) and following the manufacturer’s method.

2.7. Complete blood cell count for heterophils to lymphocytes (H/L) ratio

For assessing the stress state of hens, the H/L ratios were measured using the complete blood cell count method. First, 10 μL blood samples in EDTA K3 tubes were collected to make blood smears on glass slides. The smears were stained with Wright’s stain. White blood cells, such as heterophils, lymphocytes, eosinophils, and monocytes, were identified using a compound microscope (Axio Scope.A1, Carl Zeiss, Germany), and the ratio of H/L was calculated.

2.8. Statistical analysis

Using SAS 9.3, significant differences among the four groups were determined by one-way ANOVA with post-hoc Tukey Honest Significant Difference tests. For the comparison of the before and after flaxseed supplementation in each group, paired two-sided Student’s t-tests were conducted with data from Weeks 0 and 4. The fixed effect (Trt) in this model was a flaxseed (Lintex 170) treatment. Orthogonal polynomial contrasts were also used to validate the linear (Lin) and quadratic (Quad) effects when the level of Lintex 170 supplementation increased (* p < 0.05; ** p < 0.01; *** p < 0.001). Variability in the data is shown as pooled SEM. Partial least squares discriminant analysis (PLS-DA) plots of quantified lipid mediators and variable importance in projection (VIP) plots of 15 lipid mediators (VIP scores Top 15) were performed using the MetaboAnalyst 4.0 website (Chong et al. 2018) to determine the discriminant lipid mediators.

3. Results

3.1. Flaxseed improved ratio of omega-6 to omega-3

Flaxseed supplementation improved the omega-6 to omega-3 ratios in egg samples (Table 3). The omega-6 to omega-3 ratio decreased with increasing flaxseed concentration. The average ratios in eggs at week 4 for the different treatment groups were 9.81 (T1), 6.87 (T2) and 4.16 (T3), compared with 25.85 in the basal diet (C). The content of each PUFA component was also similar between egg and serum samples. Likewise, the average ratios in serum samples were 8.33 (T1), 5.66 (T2), and 4.08 (T3), as compared with the value of 19.23 in the basal diet (C) (Fig. S1).

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).

3.2. Flaxseed altered serum lipid mediator profile

Exploiting the multiple-reaction monitoring method based on UPLC-MS/MS, the profiles of lipid mediators in serum samples of laying hens at week 4 were investigated. A total of 91 lipid mediators were identified in the serum samples (Table S1).

Partial least square discriminant analysis (PLS-DA) was performed to observe the dissimilarities of the lipid mediators among the groups (Figure 1(a)). PLS-DA plot analysis revealed clustering among groups according to flaxseed concentration in the corresponding PLS-DA score plot including Component 1 (19.4%) and Component 2 (11.7%). Figure 1(b) shows the VIP plot of 15 lipids (VIP scores of Top 15) that were differentially regulated among the four groups, which shows that individual lipid mediator species, such as hydroxyoctadecatrienoic acid (HOTrE), prostaglandin F3 alpha (PGF3α), and hydroxydocosahexaenoic acid (HDoHE), mainly contributed to the clustering among groups.

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).

Next, the concentrations of serum PUFAs and lipid mediators among groups were analysed by one-way ANOVA and orthogonal polynomial contrasts to determine the effects of flaxseed on the host lipid profile. In the case of PUFAs, the level of AA, an omega-6 PUFA, was lower in the flaxseed-fed groups (T1, T2, and T3) than in the C group (Figure 2(a)), whereas the levels of EPA and DHA were higher in the T3 group (Figure 2(b,c)). In addition, concentrations of several lipid mediators were altered by flaxseed treatment. Levels of lpid mediators derived from the omega-3 fatty acids, ALA (9-HOTrE, p = 0.004 for Trt, <0.001 for Lin) (Figure 3(a)), EPA (PGF-3α, p = 0.038 for Trt) (Figure 3(b)) and DHA (4-HDoHE, p = 0.022 for Trt, 0.006 for Lin) (Figure 3(c)), increased linearly with flaxseed addition. Additionally, the levels of one ALA-derived lipid mediator, 13-HOTrE (p = 0.014 for Quad) (Fig. S2); two EPA-derived lipid mediators, 5-HEPE (p = 0.045 for Lin) (Fig. S3A) and 9-HEPE (p = 0.023 for Lin) (Fig. S3B); and three DHA-derived lipid mediators, 8-HDoHE (p = 0.013 for Trt, 0.017 for Lin, and 0.022 for Quad) (Fig. S4A), and 14-HDoHE (p = 0.026 for Lin) (Fig. S4B), and 16-HDoHE (p = 0.018 for Lin) (Fig. S4C), were also increased by dietary supplementation with flaxseed.

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.

3.3. Flaxseed decreased proinflammatory cytokine levels in serum

For evaluating the effect of dietary flaxseed-induced changes in lipid mediator profiles on inflammatory processes, cytokine levels were examined in serum samples from laying hens (Table 4). No significant differences among groups were observed at any level of TNF-alpha, IL-1beta, or IL-6 in serum samples at week 4. However, when comparing the values of Weeks 0 and 4 per group, the levels of TNF-alpha (p = 0.043) were significantly decreased in the T3 group.

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.

3.4. Flaxseed suppressed serum corticosterone levels and lowered the H/L ratio

In the investigation of the effect of flaxseed supplementation on stress state, two stress indices, serum corticosterone level and H/L ratio, were examined (Table 4). During the experiment, the T3 group had significantly lower serum corticosterone levels than in Week 0 (p = 0.046). Similarly, the T2 (p = 0.015) and T3 (p < 0.001) groups also showed significantly decreased H/L ratios compared with those in Week 0. The results showed that flaxseed supplementation decreased both stress indices.

3.5. Performance and egg quality

By supplementing with flaxseed, laying performance such as average egg weight and feed conversion ratio, was changed (Table 5). Average egg weight was elevated in all treatment groups during the overall period (p < 0.001 for Trt, 0.022 for Lin, and <0.001 for Quad). Although no significant difference was observed among the groups in feed intake during the experiment, the feed conversion ratio improved over the entire period (p < 0.001 for Trt, 0.018 for Lin, < 0.001 for Quad). The average egg weight and feed conversion ratio were the highest in the T2 group, which showed a quadratic effect of flaxseed supplementation. Collectively, dietary supplementation with flaxseed was observed to have positive effects on the performance of laying hens. Additionally, indices of egg quality (Haugh unit and eggshell thickness) showed no notable improvement with flaxseed supplementation (Table 5).

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.

4. Discussion

Because PUFAs have been studied for their crucial roles in controlling inflammation, the imbalance in the omega-6 to omega-3 PUFA ratio in modern human diets due to excessive content of omega-6 fatty acids has been suspected to be a possible cause of many diseases involving chronic inflammation (Subash-Babu and Alshatwi 2018). Thus, restoration of balance is a relevant issue, and sufficient intake of omega-3 fatty acid-rich diets is often recommended as a solution for the maintenance of a healthy life.

In the livestock industry, corn, an ingredient with a high content of omega-6 fatty acids, is mainly used as an energy source in animal feed (Ayaşan et al. 2020). Thus, the omega-6 to omega-3 PUFA ratio of livestock products such as meat, eggs, and milk is generally unbalanced, which could adversely affect the health of consumers. Hence, studies have attempted to produce omega-3 fatty acid-fortified livestock products, and dietary supplementation with omega-3 fatty acid-rich feed ingredients, such as flaxseed, has resulted in the successful production of meats, eggs, and milk with an improved omega-6 to omega-3 fatty acid ratio (Anjum et al. 2013; Lee et al. 2021; Rico et al. 2021). However, although studies on livestock products with a balanced omega-6 to omega-3 PUFA ratio from the view of consumers have been conducted, few studies have reported the physiological effects of omega-6 to omega-3 PUFA ratio-modulated feeds from the view of livestock health. Specifically, lipid mediator profiles of livestock have rarely been studied, despite their strong biological impact on chronic inflammation.

Laying hens are prone to exposure to inflammatory stimuli and stress because of the practices used: high stocking density, intensive production cycles, and relatively harsh breeding environments. Therefore, this study aimed to evaluate the potential availability of dietary flaxseed as a functional feed additive by modulating lipid mediator profiles and alleviating the chronic inflammation and stress states in laying hens associated with animal welfare problems. With the goal of mimicking stress conditions caused by dense stocking, the stocking density (432 cm²/hen) was set to be higher than that in other studies on laying hens (Erensoy et al. 2021; Liebers et al. 2019; Wang et al. 2021).

The supplementation levels of dietary flaxseed used in this study were set up to implement the FAO-recommended human nutrition omega-6 to omega-3 ratio (4:1) (FAO 2010). In a preliminary experiment, laying hens were fed with flaxseed supplementation at various concentrations for two weeks and 3.6% (w/w) dietary flaxseed was suggested as an optimum treatment level because the omega-6 to omega-3 ratio reached nearly 4:1 in Week 2 after feeding (data not shown). Considering a report that more than 5% dietary flaxseed had undesirable effects such as growth depression in chicks (Feng et al. 2003), from the optimum concentration of 3.6% (w/w), a half [1.8% (w/w)] and a quarter [0.9% (w/w)] amounts of dietary flaxseed were tested to determine linear and quadratic effects on laying hens.

As shown in Table 3, the supplementation of flaxseed resulted in a decrease in the omega-6 to omega-3 fatty acid ratios in laying hens as the omega-3 fatty acid content increased. Notably, the 3.6% (w/w) dietary flaxseed supplement group (T3) stably maintained a 4:1 ratio of omega-6 to omega-3 fatty acid in both eggs and sera after 4 weeks. Similar results were observed in a prior study by our research group (Lee et al. 2016b). When flaxseed oil (ALA, 55%) was supplemented in the diet of laying hens, 0.5% and 1.0% (w/w) of flaxseed oil treatment groups showed an omega-6 to omega-3 ratio of approximately 4:1 and 2:1 in eggs, respectively, in the second week after feeding. In addition, ALA-rich flaxseed oil treatment prominently increased not only ALA but also long-chain omega-3 fatty acids such as EPA and DHA in eggs. Consistent with the prior result, this study showed increases in the proportion of ALA (egg: 0.3–2.5; serum: 0.4–2.4) and DHA (egg: 0.5–2.0; serum: 0.5–2.0) in both eggs and sera at the fourth week after feeding (Fig. S1).

Notably, the results in the prior and present studies showed that the omega-6 to omega-3 fatty acid ratio in laying hens changed proportionally with supplementation levels of flaxseed sources. These findings suggest that the fatty acid composition and omega-6 to omega-3 ratio in livestock can be controlled by appropriate feed resources.

PUFAs are released from the cell membrane when the expression of phospholipase A₂ is induced by inflammatory stimuli (e.g. lipopolysaccharide); next, the free form of PUFAs is transformed into a series of lipid mediators by COXs, LOXs, and CYP450 (Mouchlis and Dennis 2019). Other human studies have reported that dietary omega-3 fatty acid increases the omega-3 fatty acid content of erythrocyte membranes (Drobnic et al. 2017; Jacek et al. 2020), and dietary flaxseed probably also modulates the fatty acid composition of erythrocytes, which might have a direct effect on the alteration of lipid mediator profile in laying hens.

From a total of 91 lipid mediators identified by UPLC-MS/MS, nine lipid mediators (9-HOTrE, 13-HOTrE, PGF3α, 5-HEPE, 9-HEPE, 4-HDoHE, 8-HDoHE, 14-HDoHE, and 16-HDoHE) showed significant alterations among the groups by both one-way ANOVA and contrast analysis. Notably, these lipid mediators originated from omega-3 fatty acids. Thus, it can be inferred that dietary flaxseed enriched the lipid mediators whose precursors were omega-3 fatty acids. Although few studies have investigated the lipid mediator profile and its shift by diet in laying hens, the finding is consistent with other studies using humans fed with fish oil (Lundstrom et al. 2013) or mice fed with flaxseed oil (Sawane et al. 2019).

Omega-3 fatty acids and their derivatives have been reported to exert anti-inflammatory effects (Ren et al. 2007). For example, the ALA-derived lipid mediators, 9-HOTrE and 13-HOTrE, the levels of which were increased by flaxseed supplementation in this study, have anti-inflammatory effects by inactivating the nod-like receptor family, the pyrin domain containing 3 (NLRP3) inflammasome (Kumar et al. 2016; Yang et al. 2017; Zahradka et al. 2017). Likewise, EPA-derived lipid mediators (PGF3α, 5-HEPE, 9-HEPE) and DHA-derived lipid mediators (4-HDoHE, 8-HdoHE, 14-HDoHE and 16-HDoHE) have been identified as anti-inflammatory lipid metabolites that counteract proinflammatory signals in other animals such as mice, rats and monkeys (Devassy et al. 2016; Nagatake et al. 2018; Onodera et al. 2017; Wang et al. 2017; Yang et al. 2014). 5-HEPE is particularly recognized as a potent inducer of regulatory T cells that play a pivotal role in regulating excessive immune responses (Onodera et al. 2017).

In addition to changes in lipid mediator profiles, serum levels of several representative proinflammatory cytokines and stress indices were investigated to determine whether dietary flaxseed affects the host’s inflammation and stress state, as many studies have demonstrated the crosstalk between inflammation and stress such as stress-stimulated production of proinflammatory cytokines, through the activation of the nuclear factor kappa B (NF-κB) signalling pathway (Chen et al. 2018).

As a result, although the serum levels of proinflammatory cytokines such as IL-1beta and IL-6 showed only ambiguous tendencies among the groups, the serum level of TNF-alpha and two stress indices (corticosterone, H/L ratio) in laying hens (Kaab et al. 2018) were significantly reduced by dietary flaxseed supplementation, which indicates its potential effects on alleviating inflammation and the stress state of laying hens. The decreased concentration of the proinflammatory cytokine, TNF-alpha, by dietary omega-3 fatty acid is also consistent with a prior study (Flock et al. 2014). Collectively, the results confirmed our original hypothesis because dietary supplementation of omega-3 fatty acid (ALA) from flaxseed could increase the omega-3 derived anti-inflammatory lipid mediators and reduce the strass indices in laying hens.

The flaxseed-fed hens also showed an improvement in overall laying performance. Slight increases in metabolizable energy (Mcal/kg; C: 2.70, T1: 2.72, T2: 2.74, T3: 2.79) and fat content (g/kg feed; C: 2.50, T1: 2.78, T2: 3.07, T3: 3.64) may positively affect laying performance, due to the extra addition of the extruded flaxseed product to the basal diets in test groups (T1 to T3). These differences were considered acceptable to validate the effect of feed additives in other studies (Aguillon-Paez et al. 2020; Damaziak et al. 2021; Tamiru et al. 2021). Compositional differences in other calculated nutrients between the control group and test groups, such as crude protein, crude fibre, crude ash, calcium, phosphorus, and amino acids were all less than 1% of the total feed. Thus, what is inferred is that dietary flaxseed contributes to improving the performance of laying hens by enriching omega-3-derived lipid mediators and alleviating inflammation and stress states.

5. Conclusions

The results of this study imply that dietary supplementation of flaxseed to laying hens could enable the production of omega-3 PUFA-fortified eggs and alleviate inflammation and stress indices by upregulating the anti-inflammatory lipid mediators originating from omega-3 fatty acids, which consequently improved overall laying performance. Therefore, a conclusion is that adjusting the physiological balance between omega-6 and omega-3 PUFA would benefit productivity and the health of farm animals. This report is the first report demonstrating changes in lipid mediator profiles by a feed additive in laying hens. Moreover, this study suggests that flaxseed might be used as a new category of functional feed additive with anti-inflammatory and anti-stress effects in the livestock industry associated with animal welfare issues. In addition, research on differentially regulated lipid mediators depending on dietary supplementation of lipid resources would expand understanding of the mechanisms underlying the regulation of immune homeostasis in livestock animals.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Ministry of Agriculture, Food and Rural Affairs: [Grant Number 316005-5].