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

Background: Although polyunsaturated fatty acids are in the spotlight due to their physiological effects on inflammation and stress of livestock animals, the biological roles of their derivatives, termed lipid mediators, have been little reported in laying hens. Results: In this study, two hundred 33-week-old laying hens were fed 0, 0.9, 1.8, or 3.6% (w/w) dietary flaxseed (Lintex170) with a commercial basal diet for 4 weeks to determine the physiological effects of dietary flaxseed, an omega-3-rich ingredient, on host inflammation or stress states regarding lipid mediator profiles, and also its impact on laying performance. The physiological changes in the omega-6 to omega-3 ratio, lipid mediator profiles, serum proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6) levels, serum corticosterone levels, and the ratio of heterophils to lymphocytes were monitored. Supplementing dietary flaxseed greatly reduced the omega-6 to omega-3 fatty acid ratio from 25.85 to 4.16 in eggs and from 19.23 to 4.08 in serum samples between groups fed with 0% and 3.6% dietary flaxseed after the experimental period. In addition, the lipid mediator profiles of laying hens were modulated by supplementation with flaxseed, mainly resulting in enrichment of omega-3 fatty acid-derived lipid mediators. Furthermore, the level of proinflammatory cytokines TNF-alpha decreased when fed 3.6% (w/w) dietary flaxseed. Two stress indices, corticosterone in the serum and the heterophil to lymphocyte ratio showed significant reductions in laying hens fed 3.6% (w/w) dietary flaxseed. Additionally, overall laying performance indices were significantly improved by supplementary flaxseed. Conclusion: Taken together, these findings suggest that the decreased omega-6 to omega-3 ratio and enrichment of omega-3-derived lipid mediators induced by dietary flaxseed may contribute to reducing the stress state in laying hens, improving will be applied to develop antiinflammatory and antistress feed additives for the poultry industry.


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 [1]. 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. Among them, linoleic acid (LA), an omega-6 fatty acid and alpha-linolenic acid (ALA), an omega-3 fatty acid are essential fatty acids, which must be obtained from the diet, and omega-3 fatty acids cannot be converted to omega-6 fatty acids and vice versa in mammals and poultry. When they enter the body, they undergo bioconversion by common elongases and desaturases, which result in the conversion of LA to arachidonic acid (AA) and ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). 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 membraneincorporated enzymes such as lipoxygenases (LOXs), cyclooxygenases (COXs) and cytochrome P450 (CYP450) [2]. Lipid mediators have known roles in the onset and resolution of inflammation and their systemic profiles can be nutritionally controllable depending on the quantity of ingested precursors such as omega-6 and omega-3 fatty acids [3]. In general, omega-6-derived lipid mediators exert proinflammatory actions, whereas omega-3-derived lipid mediators exert anti-inflammatory actions [4]. Meanwhile, cytokines are regulators of host responses to various states, including inflammation. Some cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6), act as promoters of inflammation and are therefore called proinflammatory cytokines [5]. They play key roles in the acute phase response. However, when they persist in their actions so that inflammation is not resolved, chronic inflammation occurs and results 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 [6][7][8] and obesity [9]. Recently, the contribution of the network between lipid mediators and cytokines in immune homeostasis has been investigated. In general, it has been reported that omega-6 derived lipid mediators increase proinflammatory cytokines, while omega-3-derived lipid mediators raise the level of antiinflammatory cytokines and suppress that of proinflammatory cytokines [10]. For these reasons, 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 but low levels of omega-3 fatty acids due to the high dependence of omega-6-rich resources, such as corn and soybean, to manufacture cooking oils and animal feeds. It was reported that the ratio of omega-6 to omega-3 in modern diets has increased to approximately 20:1 from 1:1 in the past [11]. Since the fatty acid composition in the inner body has also been influenced by this omega-6 and omega-3 fatty acid imbalance in the diet, the incidence of chronic inflammatory diseases could be increased [12]. Various studies have found that omega-3 fatty acids counter the effects of chronic inflammatory diseases associated with cardiovascular diseases [13][14][15], central nervous system diseases [16], diabetes [17], obesity [18] and even several cancers [19][20][21]. Similarly, the omega-6 to omega-3 fatty acid ratio of animal feed is also obviously distorted. One of the rational inferences would be that livestock animals might suffer from problems such as chronic inflammation during feeding, which increase stress and adverse effects on their performance. 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 for humans and animals because it contains abundant amounts of ALA, representing approximately 53% of total fatty acids [22]. However, whole flaxseed has been known to contain antinutritional factors such as mucilage, cyanogenic glycosides, phytic acid and trypsin inhibitors [23], which could induce adverse effects on digestion and absorption and even cause diarrhea [24]. One of the common treatments available to reduce these side effects of flaxseed is extrusion [25].
Currently, there are various reports about omega-3 fatty acid-fortified livestock products through the supplementation of omega-3 fatty acid-rich resources such as flaxseed [26][27][28].
However, it is difficult to find any report on the physiological effects of feeding omega-3 fatty acid-rich resources to livestock animals. In this study, the effect of flaxseed supplementation in laying hens not only to improve the omega-6 to omega-3 fatty acid ratio in eggs, but also its effects on animal physiology and health, mainly by profiling lipid mediators after feeding was investigated. Associated with the regulation of inflammation by lipid mediators and cytokines, it was hypothesized that supplementing omega-3 fatty acids to laying hens could improve their performance through the alleviation of 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.

Animals, Treatments, and Management
The experiment involving laying hens (n = 200, 33 weeks old, Lohmann Brown-Lite) was 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 divided randomly into 4 groups with 5 hens per cage (48 cm ⅹ 45 cm ⅹ 45 cm, width ⅹ depth ⅹ height) and 10 cages per group. Fifty hens were assigned to each of the following diet treatments for 4 weeks: 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 21.8 (C), 8.1 (T1), 5.2 (T2), and 2.9 (T3), calculated by comparison with the FAME 37 peak pattern (Table 2). Feed was offered 600 g per cage daily and fresh water was offered ad libitum during the experimental period.

Sample and Data Collection
Performance was assessed for each cage. The number of eggs and their weights were recorded daily. Abnormal eggs, such as broken, cracked or shell-less eggs, were also noted daily. Feed intake was measured every week, and then the weekly feed conversion ratio [feed consumed (g)/egg mass (g)] was calculated. Egg samples [10 eggs/(group × week)] were randomly selected and analyzed for albumen height, Haugh unit, eggshell thickness and egg fatty acid profile. Albumen height was measured with a micrometer. The Haugh unit was calculated with egg weight using the following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W 0.37 ), where H = albumen height (mm) and W = egg weight (g) [29]. The value of eggshell thickness was determined by averaging measurements taken at 3 different locations on the egg (air cell, equator, and sharp end) by using a dial pipe gage. 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 week 0, week 2 and week 4. They were collected in 2 tubes: BD Vacutainer SST TM Ⅱ Advance Tubes (Becton Dickinson, Le Pont de Claix, France) for sampling serum and V-Tube TM EDTA K3 Tubes (AB MEDICAL, Republic of Korea) for measuring complete blood cell count. Blood samples were centrifuged at 6,000 rpm for 10 min, and supernatant serum was transferred to 1.5 mL plastic tubes and stored at -20°C until use. Blood samples for complete blood cell count were stored at 4°C until use.

Gas Chromatography for Fatty Acid Profile
The fatty acid profiles were analyzed 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 using the direct methylation method [30] with some modifications.
Briefly, 0.5 mg of tridecanoic acid was added to samples in 15 mL glass tubes, followed by the addition of 5.3 mL of methanol and 700 μL of 10 N KOH. Tubes were incubated in a water bath at 55°C for an hour and a half with brief vortexing every 20 min. Then, tubes were chilled at RT, and 580 μL of 24 N H2SO4 was added. Incubation and chilling steps were repeated as before. 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 analyzed with a gas chromatography-flame ionization detector (GC-FID, Agilent 7890B, Santa Clara, CA, USA) with SP-2560 (100 m × 0.25 mm, L × I.D; 0.2 μm, df, Sigma-Aldrich, St. Louis, MO, USA). FAME 37 was used as a reference for peak identification. The running condition 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).

Ultra-performance Liquid Chromatography for Serum Lipid Mediator Profile
Lipid mediators in serum were separated by the solid-phase extraction method [31] 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.

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

Complete Blood Cell Count for Heterophils to Lymphocytes (H/L) Ratio
To assess the stress state of hens, the ratios of H/L 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 slide glasses. Then, the smears were stained with Wright's stain. White blood cells, such as heterophils, lymphocytes, eosinophils, and monocytes were identified with a compound microscope (Axio Scope.A1, Carl Zeiss, Germany) and the ratio of H/L was calculated.

Statistical Analysis
Using SAS 9.3, significant differences among 4 groups were determined by one-way

Flaxseed Altered Serum Lipid Mediator Profile
Exploiting the multiple-reaction monitoring (MRM) method based on UPLC-MS/MS, the profiles of lipid mediator profiles in serum samples of laying hens at week 4 were investigated.
As a result, a total of 91 lipid mediators were identified in serum samples (Table S1).
Partial least square discriminant analysis (PLS-DA) was performed to observe the dissimilarities of the lipid mediators profile among groups (Fig. 1A). 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%).

Flaxseed Decreased Proinflammatory Cytokine Levels in Serum
To evaluate 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 levels of TNF-alpha, IL-1beta, or IL-6 in serum samples at week 4. However, when comparing values of week 0 and week 4 per group, the levels of TNF-alpha (p-value: 0.043) were significantly decreased in the T3 group.

Flaxseed Suppressed Serum Corticosterone Levels and Lowered the H/L Ratio
To investigate the effect of flaxseed supplementation on stress state, two stress indices, serum corticosterone level (Table 5) and H/L ratio (Table 6) were examined. During the experiment, the T3 group showed a significant reduction in serum corticosterone levels compared to Week 0 (p-value: 0.046) (Table 5). Similarly, the T2 (p-value: 0.015) and T3 (p-value<0.001) groups also showed significantly decreased H/L ratios compared to Week 0 ( Table 6). The results clearly showed that flaxseed supplementation decreased both stress indices.

Performance and Egg Quality
By supplementing flaxseed, all treatment groups (T1, T2, and T3) significantly improved both hen-day egg production (week 1 to 2 p-value: 0.003 for Trt, 0.003 for Quad; week 2 to 3 p-value: 0.029 for Trt, 0.005 for Lin; week 3 to 4 p-value: <0.001 for Trt, <0.001 for Lin, 0.002 for Quad) and egg mass production (week 1 to 2 p-value: 0.001 for Trt, <0.001 for Quad; week 2 to 3 p-value: 0.021 for Trt, 0.014 for Lin, 0.048 for Quad; week 3 to 4 p-value: <0.001 for Trt, <0.001 for Lin) in the second, third and fourth weeks compared to the C group.
Additionally, average egg weight was elevated in all treatment groups during the overall period (week 0 to 1 p-value: 0.005 for Trt, 0.015 for Lin, 0.004 for Quad; week 1 to 2 p-value: <0.001 for Trt, 0.001 for Quad; week 2 to 3 p-value: 0.015 for Quad; week 3 to 4 p-value: 0.014 for Trt, 0.003 for Quad). Although there was no significant difference among the group in terms of feed intake during the experiment, the feed conversion ratio improved over the overall period (week 0 to 1 p-value: 0.034 for Trt; Week 1 to 2 p-value: <0.001 for Trt, <0.001 for Quad; Week 2 to 3 p-value: 0.029 for Quad; Week 3 to 4 p-value: 0.026 for Trt, 0.006 for Quad).
Collectively, dietary supplementation with flaxseed appeared to have positive effects on the performance of the laying hens. In particular, hen-day egg production and egg mass production linearly increased with the level of flaxseed addition. The average egg weight and feed conversion ratio were greatest in the T2 group, which showed a quadratic effect of flaxseed supplementation.
Meanwhile, the indices of egg quality (albumen height, Haugh unit, and eggshell thickness) showed no notable improvement with flaxseed supplementation despite albumen height (p-value: 0.026 for Trt, 0.003 for Lin) and Haugh unit (p-value: 0.027 for Trt, 0.003 for Lin) being significantly greater in week 2 (Table 8).

Discussion
As PUFAs have been studied for their important roles in controlling inflammation, the imbalance in the omega-6 to omega-3 PUFA ratio in the diet due to excessive omega-6 fatty acids has been suspected to be the main cause of disease involving chronic inflammation [34].
Thus, restoration of the balance is a relevant issue, and sufficient intake of omega-3 fatty acidrich diets is often recommended as a solution for health.
Meanwhile, in the livestock industry field, corn, an ingredient containing high contents of omega-6 fatty acids, is mainly used as an energy source in animal feed. Thus, the ratio of livestock products such as meat, eggs, and milk is generally unbalanced, which can adversely affect the health of consumers. Hence, a variety of studies have tried to produce omega-3 fatty acids-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 milks with an improved omega-6 to omega-3 fatty acid ratio [35][36][37]. However, contrary to improving the quality of livestock products for consumers, few studies have investigated physiological changes in livestock when their ratio of omega-6 to omega-3 ratios is balanced.
In particular, lipid mediator profiles of livestock are rarely studied despite their strong biological impact on chronic inflammation. In the case of laying hens, owing to their high stocking density, their breeding environment is poor, which makes them chronically exposed to inflammatory stimuli.
The present study aims to validate the hypothesis that dietary supplementation with flaxseed,

Conclusions
In conclusion, the results in this study imply that dietary supplementation of flaxseed, as an omega-3-rich feed ingredient, to laying hens could alleviate inflammation and stress states by adjusting the lipid mediator profile strengthen anti-inflammatory lipid mediators originating from omega-3 fatty acids, which consequently improved overall laying performance. Therefore, it is inferred that the balance of omega-6 and omega-3 ratio can improve laying performance by downregulating inflammation and stress state.

Consent for publication
Not applicable.

Availability of data and material
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary information. Additional data related to this paper may be available from the corresponding authors.

Competing interests
The authors declare that they have no competing interests.