The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions Defatted Microalgae-Mediated Enrichment of n–3 Polyunsaturated Fatty Acids in Chicken Muscle Is Not Affected by Dietary Selenium, Vitamin E, or Corn Oil Ling Tao, Tao Sun, Andrew D Magnuson, Tahir R Qamar, and Xin Gen Lei Abstract Background: We previously showed enrichments of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in broiler chicks fed defatted microalgae. Objectives: The aims of this study were to determine 1) if the enrichments affected meat texture and were enhanced by manipulating dietary corn oil, selenium, and vitamin E concentrations and 2) how the enrichments corroborated with hepatic gene expression involved in biosynthesis and oxidation of EPA and DHA. Methods: Day-old hatching Cornish Giant cockerels (n = 216) were divided into 6 groups (6 cages/group and 6 chicks/cage). Chicks were fed 1 of the 6 diets: a control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg (4CO), 4CO + 10% microalgae (defatted Nannochloropsis oceanica; 4CO+ MA), 4CO+ MA – 2% corn oil (2CO+MA), 2CO+MA + 75 IU vitamin E/kg (2CO+MA+E), 2CO+MA + 0.3 mg Se/kg (2CO+MA+Se), and 2CO+MA+E + 0.3 mg Se/kg (2CO+MA+E+Se). After 6 wk, fatty acid profiles, DHA and EPA biosynthesis and oxidation, gene expression, lipid peroxidation, antioxidant status, and meat texture were measured in liver, muscles, or both. Results: Compared with the control diet, defatted microalgae (4CO+MA) enriched (P < 0.05) DHA and EPA by ≤116 and 24 mg/100 g tissue in the liver and muscles, respectively, and downregulated (41–76%, P < 0.01) hepatic mRNA abundance of 4 cytochrome P450 (CYP) enzymes (CYP2C23b, CYP2D6, CYP3A5, CYP4V2). Supplemental microalgae decreased (50–82%, P < 0.05) lipid peroxidation and improved (16–28%, P < 0.05) antioxidant status in the liver, muscles, or both. However, the microalgae-mediated enrichments in the muscles were not elevated by altering dietary corn oil, vitamin E, or selenium and did not affect meat texture. Conclusion: The microalgae-mediated enrichments of DHA and EPA in the chicken muscles were associated with decreased hepatic gene expression of their oxidation, but were not further enhanced by altering dietary corn oil, vitamin E, or selenium. J Nutr 2018;148:1547–1555. Keywords: antioxidant enzymes, broiler chicken, DHA/EPA, meat texture, microalgae Introduction Omega-3 (n–3) FAs mostly comprise α-linolenic acid (ALA), EPA, and DHA. Enhancing the consumption of EPA and DHA has been shown to improve fetal brain and retina development and to reduce the risks of cardiovascular disease and Alzheimer disease (1). Although fish oil is a rich source of EPA and Supported in part by a Department of Energy/USDA Biomass Research and Development Initiative grant (201110006-30361), a DOE MAGIC grant (DEEE0007091), and Cornell University (Hatch grants NYC-127419 and NYC127302). Author disclosures: LT, TS, ADM, TRQ, and XGL, no conflicts of interest. Supplemental Tables 1–6 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Address correspondence to XGL (e-mail: xl20@cornell.edu). DHA (2), several factors, including decreasing amounts of sea fish, the high cost of production, and smell, may render it an uneconomical or unsustainable dietary source of EPA and DHA. Nuts, seeds, and vegetable oils are rich in ALA, but the conversion rate from ALA to EPA or DHA in the human body Abbreviations used: ALA, α-linolenic acid; CYP, cytochrome P450; ELOVL, elongase; FADS, fatty acid desaturase; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GST, glutathione S-transferase; PTGS, prostaglandin-endoperoxide synthase; SOD, superoxide dismutase; 2CO+MA, control diet – 2% corn oil + 10% microalgae; 2CO+MA+Se, control diet – 2% corn oil + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+E+Se, control diet – 2% corn oil + 75 IU vitamin E/kg + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+Se, control diet –2% corn oil + 0.3 mg Se/kg + 10% microalgae; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO + MA, control diet + 10% microalgae. © 2018 American Society for Nutrition. All rights reserved. Manuscript received March 9, 2018. Initial review completed April 6, 2018. Revision accepted July 2, 2018. First published online September 10, 2018; doi: https://doi.org/10.1093/jn/nxy164. 1547 Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 Department of Animal Science, Cornell University, Ithaca, NY 1548 Tao et al. Methods Animals, diets, and management. All of the procedures involving the use of animals were approved by the Cornell University Institutional Animal Care and Use Committee. Day-old hatchling Cornish Giant cockerels (n = 216) were divided into 6 treatment groups (6 replicate cages/treatment and 6 chicks/cage) and fed 1 of the following diets: 1) a control diet containing 4% corn oil (Dyets), 25 IU vitamin E as d,l-α-tocopherol/kg (Dyets), and 0.2 mg Se as sodium selenite/kg (Dyets) (4CO); 2) 4CO + 10% microalgae [defatted Nannochloropsis oceanica (45% crude protein, 3.8% ether extract; Cellana] (4CO+MA); 3) 4CO+MA – 2% corn oil (2CO+MA); 4) 2CO+MA + 75 IU vitamin E/kg (2CO+MA+E); 5) 2CO+MA + 0.3 mg Se/kg (2CO+MA+Se); and 6) 2CO+MA + 75 IU vitamin E/kg + 0.3 mg Se/kg (2CO+MA+E+Se). The nutrient composition of defatted N. oceanica is shown in Supplemental Table 1. Starter (weeks 1–3) and grower (weeks 4–6) diets are described in Supplemental Tables 2 and 3, respectively, and were formulated to meet the essential nutrient requirements by chicks during each growth phase (26). Because of the high concentrations of n–6 FAs of corn oil and their strong effect on tissue distribution of n–3 FAs (24), we actually used the same corn-oil concentration (1.5%) in the 4CO+MA and 2CO+MA diets during the starter period to normalize the background of chicks for showing a direct or early-phase, instead of an adapted, response to the change in corn-oil amount. The FA compositions of the diets are shown in Supplemental Table 4. The experiment lasted 6 wk. Chicks had free access to feed and water and were housed under a 22-h light–2-h dark lighting cycle. Body weights were recorded at the beginning of the study and at the end of each week. Feeders were weighed and fresh feed was added daily. At the end of week 6, 3 chicks per cage were killed via asphyxiation with carbon dioxide, and the liver, breasts, and thighs were collected. A subset of tissue samples were snap-frozen in liquid nitrogen and stored at –80°C until analysis. The remaining samples were sealed in plastic bags and frozen for lipid and meat quality analyses. Vitamin E analysis. Vitamin E was measured following the method described by Siluk et al. (27), with slight modifications. Briefly, 0.1-g samples of liver were weighed, cut, and homogenized in 500 µL PBS. Vitamin E, d,l-α-tocopherol (T3251; Sigma Chemical Co.), was used as a standard. The extraction was performed by mixing 200 µL tissue homogenate or standard with 200 µL water, 400 µL ethanol, and 800 µL hexane. After centrifuging at 3500 × g for 10 min at 4°C, 600 µL of the hexane layer from each sample was transferred into new glass tubes, evaporated under nitrogen gas, and diluted in 500 µL 100% methanol. The mobile phase used for tocopherol consisted of 100% methanol with a flow rate of 1 mL/min through a high-performance liquid chromatograph (Agilent 1000 series; Agilent Technologies) equipped with an Agilent ZOBRAX Eclipse C18 column (Agilent Technologies). Tocopherol was detected with excitation and emission wavelengths of 292 and 330 nm, respectively. Gene expression analysis. RT-PCR was performed on snapfrozen liver samples from the control (4CO) and the microalgaeadded (4CO+MA) groups to estimate mRNA abundance using GAPDH as a reference gene. Total RNA was isolated and was reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems). The resultant cDNA (30 ng) was added into a 10-μL total reaction volume containing SYBR Green PCR master mix (Applied Biosystems) and forward and reverse primers (Supplemental Table 5). Real-time PCR analysis was performed using a Viia7 Sequence Detection System (Applied Biosystems). Each sample was analyzed in duplicate for both the target and reference genes. Relative mRNA abundance was determined using the  cycle threshold method. Lipid peroxidation and FA profiles. Total lipids were extracted according to Folch et al. (28). Lipid peroxidation was quantified by the TBARS assay as described by Iba (29), with modifications. Briefly, samples were homogenized in lysis buffer (50 mM HEPES, 10 mM NaCl, 5 mM EDTA, 1% Triton, pH 7.6). Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 is low (0.01–7%) (3, 4). It is therefore necessary to identify sustainable and bioavailable dietary sources of EPA and DHA. Microalgae have recently received attention as a rich source of n–3 FAs (5). Feeding chicks with microalgae is an attractive way to enrich n–3 FAs because they have high elongase and desaturase activities for EPA and DHA biosynthesis, and chicken is consumed in large quantities (40 kg/person per year) in the United States (6, 7) and elsewhere in the world. Yan and Kim (8) reported that supplementation of the microalgae Schizochytrium JB5 enhanced DHA concentrations and decreased the n–6 to n–3 FA ratio in chicken breast. Defatted microalgae are a byproduct from biofuel processing. After oil extraction, the residual biomass contains good amounts of proteins, carbohydrates, minerals, vitamins, and PUFAs (9– 11). We previously showed that feeding broiler chicks graded amounts of the defatted microalgae Nannochloropsis oceanica resulted in dose-dependent enrichments of total n–3 FAs, EPA, and DHA in plasma, liver, breast, and thigh (10). However, the molecular and biochemical mechanisms underlying microalgaeinduced EPA and DHA accumulation remain unclear. Body concentrations of n–3 FAs are affected by biosynthesis and oxidation. Tissue EPA and DHA may be synthesized from ALA through elongation and desaturation (12, 13) by elongases 2 and 5 (ELOVL2 and 5), and fatty acid desaturases 1 and 2 (FADS1 and 2), respectively (14, 15). Synthesized EPA and DHA are prone to oxidation due to the presence of unsaturated bonds in the molecules (16, 17). Phospholipase A2 group 2A (PLA2G2A) cleaves FAs from phospholipids, which can be further oxidized through lipoxygenases [i.e., arachidonate 5-lipoxygenase (ALOX5)], cyclooxygenases [i.e., prostaglandin-endoperoxide synthase (PTGS) 1, PTGS2], and cytochrome P450 (CYP) enzymes (18–21). Therefore, activating biosynthesis and protecting EPA and DHA from oxidation are critical to maintaining their concentrations in the body. Selenium and vitamin E are 2 key antioxidant micronutrients often supplemented in chick diets (22). Indeed, vitamin E supplementation in cockerel diets decreased lipid peroxidation, whereas the addition of selenium enhanced glutathione peroxidase activity (23). However, to our knowledge, the effects of supplementing vitamin E and selenium on the microalgaemediated enrichments of EPA and DHA in chickens have not been explored. Corn oil is a commonly used fat source for broiler chick diets, but it contains high concentrations of n–6 FAs (50%) with little n–3 FAs (24). It is unknown if lowering the amount of corn oil in diets could favorably modify n– 3 FA concentrations and the n–6-to-n–3 FA ratio in chicken meat. Lipid oxidation has deleterious effects on meat quality (9). Oxidized meat is characterized by off-flavors, rancidity, discoloration, altered muscle texture, and a shortened shelf-life. Apart from environmental factors (light and oxygen exposure, temperature, pH, etc.), the degree of lipid unsaturation and presence or absence of antioxidants are important factors influencing lipid oxidation (25). It is unclear how dietary microalgae as well as vitamin E and selenium in the presence of microalgae affect lipid oxidation and meat texture, including chewiness, springiness, hardness, and cooking loss. Therefore, this study was performed to determine the following: 1) whether the microalgae-mediated enrichments of DHA and EPA in chick liver and muscle were associated with the expression of key genes in the liver related to DHA and EPA biosynthesis and oxidation and 2) whether the enrichments affected the chick meat texture and could be further enhanced by supplementing less corn oil or additional vitamin E or selenium in their diets. Homogenate was centrifuged at 15,000 × g for 5 min. For each sample, 100 μL supernatant was mixed with 200 μL reaction reagent (15% trichloroacetic acid, 0.4% 2-thiobarbituric acid, 0.06% BHT). The mixture was then heated at 95°C for 10 min. After cooling on ice, the solution was read at 532 nm. Protein was determined by bicinchoninic acid assay following the protocol provided by Thermo Fisher Scientific, Inc. Lipid peroxidation was expressed as the malondialdehyde equivalent (micromoles per milligram of protein). FAs from feed and liver, breast, and thigh tissues were extracted and analyzed following previously described protocols (10). Total glutathione concentration and antioxidant enzyme activity. Total glutathione concentration was measured as previously described (31). Glutathione-S-transferase (GST) activity was measured according to the protocols published by Ahokas et al. (32) and Boyland and Chasseaud (33). The glutathione peroxidase (GPX) activity assay was previously described by Paglia and Valentine (34) and Lawrence et al. (35). Glutathione reductase (GR) and superoxide dismutase (SOD) activities were measured according to the technical bulletin provided by Sigma. FIGURE 1 Effects of supplemental defatted microalgae, selenium, and vitamin E and decreasing corn oil content on chicken hepatic vitamin E concentrations. Values are means ± SEs, n = 5 chicks/treatment. Labeled means without a common letter differ, P < 0.05. 2CO+MA, control diet – 2% corn oil + 10% microalgae; 2CO+MA+E, control diet – 2% corn oil + 75 IU vitamin E/kg + 10% microalgae; 2CO+MA+E+Se, control diet – 2% corn oil + 75 IU vitamin E/kg + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+Se, control diet – 2% corn oil + 0.3 mg Se/kg + 10% microalgae; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). Statistical analysis. GraphPad Prism 6.0 (GraphPad Software) was used for statistical analyses. One-way ANOVA with Tukey’s posttest was performed to compare differences between treatments containing >2 groups. Student’s t test (unpaired, equal variance) was used to compare differences between 2 groups. The cage (6 chicks/cage) was set as the experimental unit for growth performance. The rest of the experiments used individual birds (from different cages) as the experimental unit. Treatment differences were considered significant at P < 0.05. Results Growth performance and vitamin E concentrations. There was no difference in body-weight gain, feed intake, or gain to feed ratio of chicks among the 6 treatments (Supplemental Table 6). Hepatic vitamin E concentrations were higher (P < 0.05) in the 2 groups supplemented with extra vitamin E than in the other 4 groups (Figure 1). Dietary microalgae or selenium supplementation did not affect hepatic vitamin E concentrations. FA compositions of the liver, breast, and thigh. The microalgae-containing diets had measurable EPA (45–61 mg/100 g) and lower n–6 to n–3 FA ratios than the control diet (Supplemental Table 4). At week 6, the microalgae supplementation (4CO+MA diet) decreased (P < 0.05) the concentration of ALA (from 31 to 19 mg/100 g) and increased (P < 0.05) the concentrations of EPA (from undetectable to 36 mg/100 g) and DHA (from 30 to 80 mg/100 g) in the liver compared with the control (4CO) (Table 1). Lowering dietary corn oil from 4% to 2% did not change the microalgaemediated ALA decrease, but increased (P < 0.05) EPA and DHA concentrations by 57% and 52%, respectively. Lowering the corn oil amount also decreased (P < 0.05) the n–6 to n–3 FA ratio from 8.5 to 4.7. Supplementation of vitamin E or selenium or both did not further enhance liver concentrations of EPA or DHA. Compared with the control diet (4CO), dietary microalgae supplementation (4CO+MA) increased (P < 0.05) concentrations of EPA from undetectable to 7.4 mg/100 g breast and 10 mg/100 g thigh and DHA from undetectable to 9.3 mg/100 g breast and from 3.3 to 14 mg/100 g thigh (Table 1). In contrast, decreasing corn oil or adding extra vitamin E or selenium did not further enhance the microalgaemediated EPA or DHA enrichment. Although lowering the dietary corn-oil amount (2CO+MA compared with 4CO+MA) decreased (P < 0.05) the n–6 to n–3 FA ratio from 16 to 11 in both muscles, supplementing extra vitamin E, selenium, or both did not produce a further decrease in that ratio in either muscle. Liver gene expression related to PUFA synthesis and oxidation. Compared with the control diet (4CO), supplemental microalgae (4CO+MA) did not alter the mRNA abundance of PUFA synthesis–related enzyme genes (ELOVL2 and 5, FADS1 and 2) in the liver of chicks (Table 2). However, the supplementation resulted in decreases of 41–76% (P < 0.01) of the mRNA abundance of 4 PUFA oxidation–associated CYP enzyme genes (CYP2C23b, CYP2D6, CYP3A5, and CYP4V2). Lipid peroxidation, antioxidant status, and meat texture. Supplemental microalgae (4CO+MA) decreased (P < 0.05) malondialdehyde contents in the liver, breast, and thigh by 50– 82%, compared with the control (4CO) (Figure 2). Lowering dietary corn oil or supplementing extra vitamin E or selenium or both did not further decrease malondialdehyde concentrations in any tissue. Compared with the control (4CO), supplemental microalgae (4CO+MA) enhanced (P < 0.05) liver GST activity Enrichment of n–3 fatty acids in chicken tissues 1549 Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 Meat texture analysis. After breast and thigh tissues were defrosted at 4°C for 24 h, the samples were cut into 1-cm3 cubes. Three cubes from each sample were cooked in an oven at 175°C for 30 min. Cubes were weighed before (W0 ) and after (W1 ) cooking to calculate the cooking loss (%): (W1 – W0 )/W0 × 100. Chewiness, springiness, and hardness were analyzed by using a TA.XTplus Texture Analyzer (Stable Micro Systems Ltd.). The parameters were set as follows: pre- and posttest speed, 5.0 mm/s; distance, 5.0 mm; trigger type, auto; and trigger force, 10 g (30). The results were analyzed using the built-in texture profile analysis module. TABLE 1 Effects of supplemental defatted microalgae, selenium, and vitamin E and decreasing corn oil content on FA composition in chicken liver, breast, and thigh at week 61 Diet FAs, mg/100 g tissue 4CO+MA 2CO+MA 2CO+MA+E 2CO+MA+Se 2CO+MA+E+Se SEM P 1096 698 1400 61.7a 31.2b NDa 30.4a 1340b 21.7c 1175 662 1280 136b 19.4a 35.9b 80.2b 1150a,b 8.54b 1089 485 1090 194c 15.7a 56.5c 122c 892a 4.67a 1187 773 1320 157c 15.8a 45.9b 95.4b,c 891a 6.52a,b 944 377 984 166c 12.7a 53.9c 99.6b,c 818a 5.02a 1073 463 1110 182c 16.8a 63.9c 101b,c 931a 5.17a 81.0 110 133 11.2 3.57 4.88 7.72 98.5 0.64 0.48 0.20 0.35 <0.0001 0.003 <0.0001 <0.0001 0.012 <0.0001 Breast SFAs MUFAs PUFAs n–3 ALA EPA DHA n–6 n–6:n–3 290 325 349 11.1a 11.1 NDa NDa 338 31.1c 428 500 527 31.5b 14.9 7.36b 9.25b 496 15.7b 243 274 242 19.5a,b 6.55 5.28b 7.67b 223 11.1a,b 265 325 305 18.5a,b 9.20 3.36b 5.96b 286 15.8b 293 358 288 23.6a,b 9.55 6.34b 7.70b 264 11.2a,b 271 299 256 22.6a,b 7.78 7.01b 7.85b 234 10.2a 51.6 69.3 61.1 3.3 2.0 0.9 1.0 58.1 0.9 0.27 0.38 0.07 0.01 0.14 0.0004 <0.0001 0.06 <0.0001 Thigh SFAs MUFAs PUFAs n–3 ALA EPA DHA n–6 n–6:n–3 661b 836b 990 36.5 33.2b NDa 3.27a 905 25.9c 531a,b 744a,b 825 46.6 22.2a,b 10.2b 14.2b 744 15.5b 357a 510a 495 36.9 13.7a 8.91b 14.3b 437 11.3a 323a,b 354a,b 373 29.2 9.00a 8.08b 12.1b 334 11.2a 412a,b 485a,b 453 35.4 13.1a 9.57b 12.7b 405 11.4a 418a 486a,b 526 41.3 16.3a,b 11.7b 13.4b 466 10.7a 74.6 129.6 134.4 7.4 5.0 1.2 1.5 119.4 0.7 0.07 0.21 0.05 0.77 0.069 0.0003 0.001 0.04 <0.0001 1 Values are means, n = 5–6 chicks/treatment. Labeled means without a common superscript letter differ, P < 0.05. ALA, α-linolenic acid; ND, not detectable (<1 mg/100 g); 2CO+MA, control diet – 2% corn oil + 10% microalgae; 2CO+MA+E, control diet – 2% corn oil + 75 IU vitamin E/kg + 10% microalgae; 2CO+MA+E+Se, control diet – 2% corn oil + 75 IU vitamin E/kg + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+Se, control diet – 2% corn oil + 0.3 mg Se/kg + 10% microalgae; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). by 16%, breast GST and GR activities by 25% and 28%, thigh reduced glutathione (GSH) content by 17%, and thigh activities of GR, GPX, and SOD by 21%, 16%, and 22%, respectively (Figure 3). The 2CO+MA+Se diet enhanced (P < 0.05) the breast chewiness over the control and other diets (Figure 4). The same diet and the 2CO+MA+E+Se diet also improved (P < 0.05) the thigh chewiness over the control diet. The thigh springiness was improved (P < 0.05) by the microalgae supplementation (4CO+MA), whereas the breast springiness was decreased (P < 0.05) by the 2CO+MA+E diet compared with the controls. None of the dietary treatments affected cooking loss or hardness of either the breast or thigh muscle. Discussion Adding 10% defatted microalgal biomass to a commonly used broiler chick diet enriched EPA and DHA in the breast and thigh muscles. This effect is comparable to our previous study that found an accumulation of 4–7 mg EPA or DHA/100 g breast or thigh tissue (10). Corn oil is the major fat component in diets for chicks. Given its low concentration of n–3 FAs (1.6%), 1550 Tao et al. high concentration (50%) of n–6 FAs, and high n–6 to n–3 FA ratio (32:1), corn oil is likely to account for the high n–6 FA and low n–3 FA deposition in chicken tissues (24). The present results show that lowering corn oil from 4% to 2% further enhanced microalgae-mediated enrichment of DHA and EPA in the liver and improved n–6 to n–3 PUFA ratios in the breast and thigh muscles. Because chick growth performance and meat quality were not impaired by decreasing the corn-oil amount, this approach could be applied to further enhance the DHA and EPA enrichment and to improve the n–6 to n–3 PUFA ratios in poultry meat. Considering the antioxidant potency of vitamin E and selenium supplemented in animal feed (36), we anticipated that adding these 2 micronutrients alone or in combination could further elevate the microalgae-mediated enrichment of EPA and DHA. Because this hypothesis was not supported by our data, it is possible that the defatted microalgae contained sufficient antioxidant components or activities (37, 38) to protect the DHA and EPA from oxidation or peroxidation. An important finding from this study is the downregulation of cytochrome P450 family gene expression in the liver by the defatted microalgae supplementation. Mechanistically, tissue PUFA accumulation can be affected by the balance between biosynthesis and oxidation. However, the supplemental Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 4CO Liver SFAs MUFAs PUFAs n–3 ALA EPA DHA n–6 n–6:n–3 TABLE 2 Effects of supplemental microalgae on hepatic PUFA biosynthesis and oxidation–associated gene expressions in chickens1 Percentage2 Group and gene symbol Gene name PUFA synthesis ELOVL2 ELOVL5 FADS1 FADS2 Fatty acid elongase 2 Fatty acid elongase 5 Fatty acid desaturase 1 Fatty acid desaturase 2 +20 −9.1 −23 −9.1 0.44 0.45 0.14 0.07 PUFA oxidation ALOX5 CYP1A1 CYP2C23b CYP2D6 CYP3A5 CYP4V2 PLA2G2A PTGS1 PTGS2 Arachidonate 5-lipoxygenase Cytochrome P450, family 1, subfamily A, polypeptide 1 Cytochrome P450, family 2, subfamily C, polypeptide 23b Cytochrome P450, family 2, subfamily D, member 6 Cytochrome P450, family 3, subfamily A, member 5 Cytochrome P450 family 4, subfamily V, member 2 Phospholipase A2, group IIA Prostaglandin-endoperoxide synthase 1 Prostaglandin-endoperoxide synthase 2 +10 −33 −76 −55 −41 −50 +10 +20 0 0.55 0.14 <0.01 <0.01 <0.01 <0.01 0.87 0.37 0.76 P 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). 2 Percentage increase (+) or reduction (–) in gene expression in the 4CO+MA group compared with the 4CO group (n = 5 chicks/treatment). microalgae did not affect the mRNA levels of enzyme genes involved in PUFA synthesis, including desaturases and elongases. We previously observed that FADS2 (-6 desaturase) was upregulated by microalgae (∼2-fold change) (9). Reasons for this discrepancy remain unclear but are likely due to various small differences in experimental conditions. Meanwhile, the microalgae supplementation could have caused changes in these biosynthetic enzymes at the protein level, which should be tested in the future when the required reagents are available. In contrast, supplemental microalgae downregulated the expression of CYP2C23b, CYP2D6, CYP3A5, and CYP4V2 genes involved in the oxidation of DHA and EPA and other PUFAs (21). This downregulation should presumably attenuate the respective enzyme activities and help stabilize EPA and DHA, thereby enhancing their net enrichments. To the best of our knowledge, there has been no previous report of microalgal effects on CYP gene expression in chicks. Thus, our findings provide a novel mechanism for the microalgae-mediated DHA and EPA enrichment in the tissue of chicks. Another interesting explanation for the microalgae-mediated enrichment of DHA and EPA are the 41–76% decreases in the formation of malondialdehyde, an end product of lipid peroxidation, in the 3 assayed tissues of chicks fed the microalgae-supplemented diets compared with the control diet. This agrees with other studies describing anti–lipid peroxidation effects of microalgae (39, 40). Notably, this FIGURE 2 Effects of supplemental defatted microalgae, selenium, and vitamin E and decreasing corn oil content on lipid peroxidation of chicken liver (A), breast (B), and thigh (C). Values are means ± SEs, n = 4–5 chicks/treatment. Labeled means without a common letter differ, P < 0.05. MDA, malondialdehyde; 2CO+MA, control diet – 2% corn oil + 10% microalgae; 2CO+MA+E, control diet – 2% corn oil + 75 IU vitamin E/kg + 10% microalgae; 2CO+MA+E+Se, control diet – 2% corn oil + 75 IU vitamin E/kg + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+Se, control diet – 2% corn oil + 0.3 mg Se/kg + 10% microalgae; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). Enrichment of n–3 fatty acids in chicken tissues 1551 Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 1 1552 Tao et al. Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 FIGURE 3 Effects of supplemental defatted microalgae on antioxidant activities of chicken liver (A, D, G, J, M), breast (B, E, H, K, N), and thigh (C, F, I, L, O). Values are means ± SEs, n = 5 chicks/treatment. Labeled means without a common letter differ, P < 0.05. GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GST, glutathione Stransferase; SOD, superoxide dismutase; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). Enrichment of n–3 fatty acids in chicken tissues 1553 Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 FIGURE 4 Effects of supplemental defatted microalgae, selenium, and vitamin E and decreasing corn oil content on the texture of chicken breast (A, C, E, G) and thigh (B, D, F, H). Values are means ± SEs, n = 6 chicks/treatment. Labeled means without a common letter differ, P < 0.05. 2CO+MA, control diet – 2% corn oil + 10% microalgae; 2CO+MA+E, control diet – 2% corn oil + 75 IU vitamin E/kg + 10% microalgae; 2CO+MA+E+Se, control diet – 2% corn oil + 75 IU vitamin E/kg + 0.3 mg Se/kg + 10% microalgae; 2CO+MA+Se, control diet – 2% corn oil + 0.3 mg Se/kg + 10% microalgae; 4CO, control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg; 4CO+MA, control diet + 10% microalgae (10% defatted Nannochloropsis oceanica). Acknowledgments We thank Michael Burke, Matthew Barcus, Hui Xia Li, and Zhi Luo for their technical support. The authors’ responsibilities were as follows—XGL: designed the study and had primary responsibility for the content; LT, TS, ADM, and TRQ: conducted the experiments; LT, TS, and ADM: analyzed the data; LT and XGL: wrote the manuscript; and all authors: read and approved the final manuscript. References 1. Swanson D, Block R, Mousa SA. 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A validated liquid chromatography method for the Downloaded from https://academic.oup.com/jn/article/148/10/1547/5094773 by guest on 03 February 2023 presumed function of microalgae was independent of the supplementations of vitamin E and/or selenium in the present study. Supplemental microalgae also elevated GSH concentrations and GR, GPX, GST, and SOD activities in the muscle tissues. This implies that the supplemental microalgae might have attenuated lipid oxidation by modulating glutathionerelated pathways. Similarly, Vijayavel et al. (41) proposed that lowering the naphthalene-induced oxidative stress by Chlorella vulgaris in albino rats resulted from the upregulation of both enzymatic and nonenzymatic antioxidants such as GPX and GSH. The microalgae-mediated enrichments of EPA and DHA in both breast and thigh muscles did not produce consistent effects on their texture properties. Although supplemental microalgae alone enhanced the thigh springiness, and adding extra selenium to the diet with microalgae and lowered corn oil improved the chewiness of both breast and thigh, the underlying mechanism remains to be elucidated. In addition, we did not observe changes in hardness or cooking loss of either muscle by any dietary treatment. These findings are consistent with results showing that adding algae or antioxidants to diets for chicks did not alter cooking loss or meat tenderness (42). In summary, this study shows that supplemental defatted N. oceanica biomass enriched EPA and DHA in the chicken liver, breast, and thigh. The enrichment was associated with the downregulation of PUFA oxidation–related gene expression, attenuated lipid peroxidation, and enhanced antioxidant activities in the liver, muscles, or both. 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