Potential and Metabolic Impacts of Double Enrichments of Docosahexaenoic Acid and 25-Hydroxy Vitamin D₃ in Tissues of Broiler Chickens

Abstract

Background

Chicken may be enriched with 25-hydroxy D₃ [25(OH)D₃] and docosahexaenoic acid (DHA) to enhance the dietary intake of the public.

Objectives

Two experiments (Expt.) were conducted to determine the potential and metabolic impacts of enriching both DHA and 25(OH)D₃ in the tissues of broiler chickens.

Methods

In Expt. 1, 144 chicks (6 cages/treatment and 6 birds/cage) were fed a corn–soybean meal basal diet (BD), BD + 10,000 IU 25(OH)D₃/kg [BD + 25(OH)D₃], BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), or BD + 25(OH)D₃+DHA for 6 wk. In Expt. 2, 180 chicks were fed the BD, BD + DHA-rich microalgal oil (1.5–3.0 g DHA/kg, BD + DHA), BD + DHA + eicosapentaenoic acid (EPA)-rich microalgae (0.3–0.6 g EPA/kg, BD + DHA + EPA), BD + DHA + 25(OH)D₃ [6000 to 12,000 IU/kg diet; BD + DHA + 25(OH)D₃], and BD + DHA + EPA + 25(OH)D₃ for 6 wk.

Results

Supranutrition of these 2 nutrients resulted in 57–62 mg DHA and 1.9–3.3 μg of 25(OH)D₃/100 g of breast or thigh muscles. The DHA enrichment was independent of dietary EPA or 25(OH)D₃, but that of 25(OH)D₃ in the liver was decreased (68%, P < 0.05) by dietary DHA in Expt. 1. Compared with BD, BD + 25(OH)D₃ enhanced (P < 0.05) gene expression related to D₃ absorption (scavenger receptor class B type 1 and Niemann-pick c1 like 1) in the liver and D₃ degradation (cytochrome P450 24A1) in the breast, and decreased mRNA or protein concentrations of vitamin D binding protein in the adipose tissue or thigh muscle. Supranutrition of DHA decreased mRNA concentrations of lipid metabolism-related genes (fatty acid desaturase 1,2, ELOVL fatty acid elongase 5, fatty acid desaturase 2, fatty acid synthase, and sterol regulatory element-binding protein 1).

Conclusions

Both DHA and 25(OH)D₃ were enriched in the muscles up to meeting 50%–100% of the suggested intakes of these nutrients by consuming 2 servings of 100 g of fortified chicken. The enrichments altered gene expression related to lipid biosynthesis and vitamin D transport or storage.

Introduction

Daily dietary intakes of long-chain ω-3 fatty acids, including DHA and EPA by average Americans, are ∼113 mg which is lower than the recommended 250 mg/d [[1], [2], [3]]. Meanwhile, ∼42% of US adults are vitamin D deficient, with serum concentrations of 25-hydroxy D₃ [25(OH)D₃] <20 μg/L. This is due to a lack of sunlight exposure and (or) failure to ingest the recommended daily intake of 800 IU D₃/d [[4], [5], [6], [7], [8]]. It is estimated that 74% of Americans consume animal-sourced foods. Among these, poultry meat is the most consumed with an annual average per capita consumption of 44 kg [9]. However, chicken from commercial production contains minute amounts of DHA or 25(OH)D₃. Thus, bio-fortifying chicken with these bioactive nutrients has been suggested as a strategy to improve their daily intake by the public [[10], [11], [12]].

Previous research has shown enrichments of DHA in chicken ≤84 and 96 mg/100 g of breast and thigh tissues, respectively, which could meet the daily requirement in 2–3 servings [13]. In comparison, much less research has been done to enrich chicken with bioactive vitamin D₃ such as calcidiol, 25(OH)D₃ [12]. Practically, it is not convenient or feasible to expect the public to ingest 2 types of fortified chicken to meet their daily requirements for these nutrients. Thus, it is necessary to pursue a combined fortification of chicken with both DHA and 25(OH)D_3. However, it remains unclear if it is feasible to achieve such a double enrichment and if there is an antagonistic interaction between these 2 nutrient enrichments.

Enrichments of DHA in broiler tissues were found to modulate mRNA concentrations of genes regulating lipid metabolism, such as fatty acid elongation [ELOVL fatty acid elongase 2 (ELOVL2) and ELOVL fatty acid elongase 5 (ELOVL5)], desaturation [fatty acid desaturase 1 (FADS1) and fatty acid desaturase 2 (FADS2)], de novo synthesis [fatty acid synthase (FASN) and sterol regulatory element-binding protein 1 (SREBP1)], and β-oxidation [carnitine palmitoyltransferase 1 (CPT1)] [14,15]. Likewise, 25(OH)D₃ enrichments influenced mRNA concentrations of genes involved in cholecalciferol absorption and transportation [cluster determinant 36 (CD36), Scavenger receptor class B type 1 (SR-B1), Niemann-pick c1 like 1 (NPC1L1), ABC transporter, and vitamin D binding protein (VDBP)], 25-hydroxylation (cytochrome P450 2R1 and cytochrome P450 27A1), 1α-hydroxylation, and 24-hydroxylation [cytochrome P450 24A1 (CYP24A1) and cytochrome P450 3A4 (CYP3A4)] [[16], [17], [18]]. To the best of our knowledge, little research has been conducted to examine interactions between DHA and vitamin D supplementations on gene expression related to lipid and vitamin D metabolisms in the chicken tissues [19].

Therefore, we conducted 2 broiler chicken experiments (Expt.) to determine: 1) if it was feasible to enrich both DHA and 25(OH)D₃ simultaneously in the breast and thigh muscles to concentrations relevant to meeting the recommended daily intakes by the public and 2) if there was a synergistic or antagonistic interaction between supranutritions of DHA and 25(OH)D₃ on gene expression and protein production related to lipid and vitamin D metabolisms in the 2 muscles as well as liver, adipose tissue, and kidney.

Methods

Animal, diets, and management

Our animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee. The DHA-rich microalgal biomass (Aurantiochytrium, 12% DHA in the biomass) used in Expt. 1 was provided by Heliae. The DHA-rich microalgal oil (45% DHA w/v) used in Expt. 2 was provided by Archer Daniels Midland Company. The EPA-rich microalgal biomass (Nannochloropsis sp. CO18, 1.6% EPA in the biomass) used in Expt. 2 was provided by Duke University. The Rovimix HyD Premix [138 mg 25(OH)D₃/kg of premix] used in both experiments was provided by Royal Dutch State Mines (DSM) N.V.

In Expt. 1, Cornish male broiler chicks (1 d old, total = 144) were purchased from Moyer’s Chicks. The chicks were allotted into 4 treatment groups based on body weight (6 cages/diet and 6 birds/cage). Birds were fed 1 of the following 4 diets: a corn–soybean meal basal diet (BD), BD + 10,000 IU 25(OH)D₃/kg of diet [BD + 25(OH)D₃)], BD + 1% DHA-rich microalgal biomass [Aurantiochytrium, 1.2 g DHA/kg diet (BD + DHA)], and BD + 25(OH)D₃ + DHA. The chickens were fed the respective starter (weeks 0–3) and grower (weeks 4–6) diets. The design of supplementing 1% Aurantiochytrium microalgae biomass was based on our previous research in which graded levels of the same biomass were fed to provide 1.2–4.8 g DHA/kg diet for enriching DHA in the tissues of chickens [13,20]. Because the inclusion of >1% biomass (1.2 g DHA/kg diet) in that study adversely impacted animal performance, we chose the 1% level in this study. The supplemental level of 10,000 IU 25(OH)D₃/kg diet was chosen because supplementing ≤7000 IU of D₃/kg diet for broiler chickens improved growth performance and gait score [21]. Thus, we chose a slightly higher level, but lower than the toxic level, in the hope of better matching the fast-growing chickens nowadays than in the past [8].

In Expt. 2, 180 Cornish male broiler chicks were purchased from the same supplier as in Expt. 1 and allotted into the following 5 treatment groups (6 cages/diet and 6 birds/cage): BD, BD + DHA (0.33% and 0.66% of the DHA-rich microalgal oil to provide 1.5 and 3.0 g DHA/kg diet for weeks 0–3 and 4–6, respectively), BD + DHA + EPA (1.9 and 3.8% of EPA-rich Nannochloropsis sp. CO18 to provide 0.3 and 0.6 g EPA/kg diet for weeks 0–3 and 4–6, respectively), BD + DHA + 25(OH)D₃ [6000 and 12,000 IU of 25(OH)D₃/kg diet for weeks 0–3 and 4–6, respectively], and BD + DHA + EPA + 25(OH)D₃ (a combination of all 3 supplements at the doses used in the previous diets). Likewise, chickens were fed respective starter (weeks 0–3) and grower (weeks 4–6) diets for each treatment.

In Expt. 2, we used a more DHA-concentrated microalgal oil as the source of dietary DHA supplementation. This change was made to avoid the following 2 concerns: 1) the lower DHA concentrations than intended in the supplemental diets in Expt. 1, probably because of oxidation loss of DHA during relatively long storage and 2) the negative effects of the microalgal biomass at high concentrations (>1%) on animal performance (as indicated above). The concentrations of supplemental 25(OH)D₃ in Expt. 2 were based on the results of Expt. 1. Notably, we doubled the DHA and 25(OH)D₃ supplementation concentrations from the starter to grower diets in Expt. 2 to be in line with industrial practice for enriching these nutrients in the final product at minimal cost and maximal overall enrichment efficiency by avoiding potential feedback inhibition of the enrichments via homeostatic regulation [[22], [23], [24], [25], [26], [27]]. A combination of dietary supplementations of DHA and EPA (at a ratio of 5:1) was tested to explore if both long-chain ω-3 fatty acids could be enriched in the chicken tissues.

In both experiments, all diets were supplemented with adequate cholecalciferol (at 1.5-fold of the National Research Council (NRC) recommendation, 300 IU/kg) [28]. Nutrient compositions and fatty acid profiles of all experimental diets (Expts. 1 and 2) are presented in Supplemental Tables 1–6. Both experiments lasted for 6 wk. Birds had free access to food and water and were housed in a temperature-controlled room (gradually decreased from 34°C to 18°C between days 0 and 42) with 22:2 h of light and dark at the Cornell University Poultry Research Farm.

Growth performance and sample collection

The effects of diets on growth performance in both experiments were determined and reported by us previously [29]. Briefly, the body weights of individual birds were recorded at weeks 0, 3, and 6. Feed intake was measured daily as feed disappearance by weighing trays before and after adding fresh feed. Mortality was recorded daily. At the ends of weeks 3 and 6, 2 birds/cage with representative body weights of the cage were killed via asphyxiation using carbon dioxide. Blood was drawn via heart puncture using heparinized needles to prepare plasma samples that were stored immediately at –20 °C until analysis. Liver, breast (pectoralis major), thigh (iliotibialis lateralis), adipose tissue (abdominal fat pad), and kidney samples were removed, snap-frozen in liquid nitrogen, and stored at –80°C until analysis.

Fatty acid profiles

Total fatty acids from liver, breast, and thigh muscles, and adipose tissue were extracted, methylated, and quantified by gas chromatography (Agilent 6890N, Agilent Technologies) fitted with a flame ionization detector and a fused-silica capillary column coated with CP-97 SIL 88 (100 m × 0.25 mm i.d., 0.2 mm film thickness) (Varian Inc), as described in previous studies [30,31]. An internal standard (200 μL of 18.7 mM tridecanoic acid) was added into per gram of tissue sample before the extraction procedure.

Analyses of 25(OH)D₃

25(OH)D₃ in liver and muscle samples was extracted and purified using a silica solid phase extraction (SPE) column (0.5 g, 3 mL, Mega Bond Elut, Agilent) as previously described [32]. The 25(OH)D₃ isolated fractions were resuspended in the mobile phase (80% methanol in water) and injected into a Vydac 201TP54 column (5 μm, 4.6 × 250 mm, and 300 Å) mounted on an HPLC-UV-DAD (Agilent 1100 HPLC system). Samples were analyzed at 0.42 mL/min with a column temperature of 10°C, and 25(OH)D₃ was detected using DAD (210–330 nm), quantified by UV (265 nm), and calibrated using 25(OH)D₃ as a standard (Millipore; catalog number 679102) and 25(OH)D₂ as an internal standard (Enzo Life Sciences; catalog number BML-DM101-001).

Gene expression analysis

qPCR was performed on liver, breast, and thigh muscles, adipose tissue and kidney samples to determine the abundances of mRNA using beta-actin (β-Actin) as a reference. Supplemental Table 7 shows the primer sequences used for all the determined genes in both experiments. Total mRNA was isolated using TRIzol reagent (Life Technologies) according to an established method [33] and reverse transcribed using a cDNA reverse transcription kit. qPCR analysis was performed using a 7900HT Fast RT-PCR System (Applied Biosystems). Relative mRNA abundance was calculated using the Δ cycle threshold (ΔCt) method [34].

Western blot analysis

Amounts of VDBP in the liver and muscle samples were determined by Western blot as described in previous studies [35]. The primary antibody against VDBP (Rabbit) was purchased from Proteintech, and β-Actin (Rabbit, Bio-Rad Technology) was used as a reference to normalize the relative amounts of VDBP.

Statistical analysis

In both experiments, the biochemical and molecular data were obtained from 2 representative chickens per cage and the cage averages were used as the experimental units (n = 6). Data from the 2 experiments were analyzed for treatment effects by differences in least-square means using the fit model program of JMP Pro (version 11; SAS Institute). Differences in treatment means were compared using Tukey’s honest significant difference test. Data were presented as mean ± SEM, and P < 0.05 was assumed to be statistically significant.

Results

Experiment 1

Enrichments of DHA and 25(OH)D₃

As mentioned above, the effects of diets on growth performance of chickens, including average body weight gain, daily feed intake, and gain/feed ratio in this and the next experiments were reported by us previously [29]. In the present study, we found that the final body weight of birds fed BD + 25(OH)D₃ was greater (P < 0.05) than that fed the BD, whereas diets exerted no effects on mortality (Supplemental Table 8). As shown in Figure 1A, birds fed BD + DHA and BD + 25(OH)D₃ + DHA had higher (P < 0.05) concentrations of DHA (5- to 12-fold at week 3 and 2.5–5-fold at week 6) and lower (P < 0.05) concentrations of arachidonic acid (ARA, 12%–26% at week 3 and 4%–52% at week 6) in the liver and breast and thigh muscles than those fed BD (Supplemental Tables 9 and 10). As shown in Figure 1B, birds fed BD + 25(OH)D₃ had higher concentrations (P < 0.05) of 25(OH)D₃ in the liver (68%) than those fed BD. However, the increases in the breast (72% and 51%) and thigh (65% and 8%) muscles resulted from feeding BD + 25(OH)D₃ or BD + 25(OH)D₃ + DHA over BD were not statistically significant. Feeding BD + 25(OH)D₃ + DHA resulted in less (P < 0.05) 25(OH)D₃ deposition in the liver compared with feeding BD + 25(OH)D₃. However, the differences in the breast and thigh muscles between these 2 diets were not statistically significant.

FIGURE 1.

Effects of supplementation of DHA-rich microalgal biomass and 25(OH)D₃ on tissue DHA (A) and 25(OH)D₃ (B) concentrations in broiler chickens at week 6 in Experiment 1. Data are expressed as mean ± SEM, n = 6. Bars without sharing a common letter differ, P < 0.05. BD = corn–soybean basal diet; BD + 25(OH)D₃ = BD + 10,000 IU 25(OH)D₃/kg of diet; BD + DHA = BD + 1% DHA-rich microalgal biomass; BD + 25(OH)D₃ + DHA = BD + 10,000 IU 25(OH)D3 /kg of diet + 1% DHA-rich microalgal biomass. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D₃.

Gene expression

Compared with BD, BD + 25(OH)D₃ and BD + DHA enhanced (P < 0.05) mRNA concentrations of CD36 (40% to 2.1-fold) and CYP24A1 (40% to 2.8-fold) in liver, muscles, and (or) kidney. In addition, feeding BD + 25(OH)D₃ elevated (P < 0.05) mRNA concentrations of hepatic scavenger receptor class B type 1 (SRB1) (40% to 2.7-fold), CYP3A4 (40%), and NPC1L1 (90% to 2.6-fold), but down-regulated (P < 0.05) mRNA concentrations of VDBP (27%–62%) in the adipose tissue and thigh muscle, respectively, compared with BD. There were antagonistic interactions (P < 0.01) between supplemental DHA and 25(OH)D₃ on mRNA concentrations of genes related to 25(OH)D₃ absorption (SRB1 and NPC1L1) and degradation (CYP24A1) in the liver and breast muscle (Figure 2 and Supplemental Table 11). However, diets exerted nonsignificant effects on mRNA concentrations of other vitamin D metabolism-related genes in any tissue (Supplemental Table 12).

FIGURE 2.

Effects of supplementation of DHA-rich microalgal biomass and 25(OH)D₃ on relative mRNA concentrations of vitamin D metabolism-related genes in different tissues of broiler chickens at week 6 in Experiment 1, n = 6. Values between 0 and 1 indicate a decrease, whereas values >1 indicate an elevation in relative mRNA concentrations compared with the control (BD). BD = corn–soybean basal diet; BD + 25(OH)D₃ = BD + 10,000 IU 25(OH)D₃/kg of diet; BD + DHA = BD + 1% DHA-rich microalgal biomass; BD + 25(OH)D₃ + DHA = BD + 10,00IU 25(OH)D₃/kg diet + 1% DHA-rich microalgal biomass. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D₃.

Protein production

Compared with BD, BD + 25(OH)D₃ decreased (P < 0.05, 45%) the protein level of VDBP in the thigh muscle (Figure 3). However, diets showed no such effect on the protein concentrations of VDBP in the liver or breast muscle.

FIGURE 3.

Effects of supplementation of DHA-rich microalgal biomass and 25(OH)D₃ on VDBP protein concentrations in the chicken liver (A), breast muscle (B), and thigh (C) muscle. Data are expressed as mean ± SEM, n = 6. Bars without sharing a common letter differ, P < 0.05. Sample bands shown were from 2 chickens per treatment. BD = corn–soybean basal diet; 25(OH)D₃ = BD + 10,000 IU 25(OH)D₃/kg of diet; BD + DHA = BD + 1% DHA-rich microalgal biomass; BD + 25(OH)D₃ + DHA = BD + 10,000 IU 25(OH)D₃/kg diet + 1% DHA-rich microalgal biomass. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D₃; VDBP, vitamin D binding protein.

Experiment 2

Enrichments of DHA and 25(OH)D3

Detailed effects of diets on the growth performance of chickens were published by us previously [29]. In Supplemental Table 8, we reported here a final body weight improvement (2770 compared with 2520, P < 0.05) in the birds fed BD + DHA + EPA compared with those fed the BD, but no difference in mortality among diets. Compared with BD, all other diets enhanced (P < 0.05) DHA concentrations in the liver and breast and thigh muscles (5- to 19-fold at week 3; 4- to 18-fold at week 6) (Figure 4; Supplemental Tables 13 and 14). Supplemental 25(OH)D₃ and (or) EPA into the BD + DHA diet decreased DHA deposition in the breast (12%–23%) and thigh muscle (5%–21%), but these decreases were not statistically significant. The adipose tissue had no detectable DHA in any group of chickens but showed slight decreases in total fatty acid contents from week 6 (59–62 mg/g) to week 3 (62–67 mg/g). Likewise, concentrations of ARA in the breast and thigh muscles were decreased by the 4 diets containing DHA (24%–48% at week 3 and 17%–44% at week 6) compared with BD (Supplemental Tables 13 and 14). No EPA was detected in any of the assayed tissues. As shown in Figure 4B, birds fed BD + DHA or BD + DHA + EPA diets had no difference in the 25(OH)D₃ concentrations in any of the 3 assayed tissues compared with those fed BD, whereas the addition of 25(OH)D₃ alone or in combination with EPA increased the 25(OH)D₃ concentrations in all 3 tissues (≤70%) compared with the other 3 diets.

FIGURE 4.

Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and 25(OH)D₃ on tissue concentrations of DHA (A) and 25(OH)D₃ (B) in broiler chickens at week 6 in Experiment 2. Data are expressed as mean ± SEM, n = 6. Bars without sharing a common letter differ, P <0.05. BD = corn–soybean basal diet; DHA = BD + 3.0 g DHA oil/kg; DHA + EPA = BD + DHA + 0.6 g/kg Nannochloropsis sp. CO18; DHA + 25(OH)D₃ = BD + DHA + 12,000 IU 25(OH)D₃; DHA + EPA + 25(OH)D₃ = BD + DHA + EPA (0.6 g/kg Nannochloropsis sp. CO18) + 12,000 IU 25(OH)D₃/kg. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D_3.

Gene expression

At week 3, diets exerted no significant effect on mRNA concentrations of vitamin D metabolism-related genes. In contrast, BD + DHA decreased (P < 0.05) mRNA concentrations of ELOVL5 (50%–62% in liver and adipose tissue), FADS2 (48% in the adipose tissue), FASN (34%–53% in the adipose and thigh muscle), SREBP1 (4%–32% in the liver and adipose tissue), CPT1 (45% in the breast muscle), and ethanolamine phosphotransferase 1 (23% in the thigh muscle) compared with BD (Figure 5 and Supplemental Table 15). Supplemental EPA in the BD + DHA diet decreased (P < 0.05) hepatic mRNA concentrations of SREBP1 (44%). Effects of the experimental diets on mRNA concentrations of other lipid and vitamin D-related genes in different tissues at week 3 are shown in Supplemental Table 16. At week 6, BD + DHA upregulated (P < 0.05) mRNA concentrations of CYP24A1 (60% in the liver and breast muscle), VDBP (10% in the breast muscle), and down-regulated (P < 0.05) mRNA concentrations of FADS1 (52% in the liver), FADS2 (38%–64% in the liver, breast, thigh, and adipose tissue), ELOVL5 (49%–67% in the adipose tissue and breast muscle), and SREBP1 (50% in the liver) compared with BD (Figure 6 and Supplemental Table 17). However, supplemental EPA or 25(OH)D₃ into the BD + DHA diet produced only numerical increases in mRNA concentrations of CYP24A1 (7%–28%), SREBP1 (14%–43%), and ELOVL5 (9%–36%) and decreases in mRNA concentrations of FADS1 (4%–38%), and FADS2 (3%–100%) in all assayed tissues (Supplemental Table 17). Likewise, no diets altered mRNA concentrations of other lipid and vitamin D-associated genes in different tissues at week 6 (Supplemental Table 18).

FIGURE 5.

Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and 25(OH)D₃ on relative mRNA concentrations of vitamin D and fatty acid metabolism-related genes in different tissues of broiler chickens at week 3 in Experiment 2, n = 6. Values between 0 and 1 indicate a decrease, whereas values >1 indicate an elevation in mRNA expression compared with the control (BD). BD = corn–soybean basal diet; BD + DHA = BD + 1.5 g of DHA oil/kg; BD + DHA + EPA = BD + DHA + 0.3 g/kg Nannochloropsis sp. CO18; BD + DHA + 25(OH)D₃ = BD + DHA + 6000 IU 25(OH)D₃/kg; BD + DHA + EPA + 25(OH)D₃ = BD + DHA + EPA (0.3 g/kg Nannochloropsis sp. CO18) + 6000 IU 25(OH)D₃/kg. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D₃.

FIGURE 6.

Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and 25(OH)D₃ on relative mRNA concentrations of vitamin D and fatty acid metabolism-related genes in different tissues of broiler chickens at week 6 in Experiment 2, n = 6. Values between 0 and 1 indicate a decrease, whereas values >1 indicate an elevation in mRNA expression compared with the control (BD). BD = corn–soybean basal diet; BD + DHA = BD + 3.0 g DHA oil/kg; BD + DHA + EPA = BD + DHA + 0.6 g/kg Nannochloropsis sp. CO18; BD + DHA + 25(OH)D₃ = BD + DHA + 12,000 IU25(OH)D₃/kg; BD + DHA + EPA + 25(OH)D₃ = BD + DHA + EPA (0.6 g/kg Nannochloropsis sp. CO18) + 12,000 IU25(OH)D₃ /kg. Abbreviations: BD, basal diet; 25(OH)D₃, 25-hydroxy D₃.

Protein production

Compared with BD, the 4 treatment diets showed no effect on the protein concentrations of VDBP in the liver or breast and thigh muscles (Figure 7).

FIGURE 7.

Effects of supplementation of DHA-rich microalgal oil, EPA-rich biomass, and 25(OH)D₃ on VDBP protein level in the chicken liver (A), breast muscle (B), and thigh muscle (C) at week 6 in Experiment 2. Data are expressed as mean ± SEM, n = 6. BD = corn–soybean basal diet; BD + DHA = BD + 3.0 g DHA oil/kg; BD + DHA + EPA = BD + DHA + 0.6 g/kg Nannochloropsis sp. CO18; BD + DHA + (25(OH)D₃ = BD + DHA + 12,000 IU 25(OH)D₃/kg; BD + DHA + EPA + 25(OH)D₃ = BD + DHA + EPA (0.6 g/kg Nannochloropsis sp. CO18) + 12,000 IU 25(OH)D₃/kg. Abbreviations: β-Actin, beta-actin; BD, basal diet; 25(OH)D₃, 25-hydroxy D₃; VDBP, vitamin D binding protein.

Discussion

We conducted 2 consecutive experiments to enrich chicken tissues with both DHA and 25(OH)D₃ to improve the public status of these nutrients through ingesting chicken. Overall, our enrichments reached 57–62 mg DHA and 1.9–3.3 μg or 228–396 IU of 25(OH)D₃/100 g of the breast and thigh muscles, respectively. These dual enrichments are not only different from past studies fortifying chicken with solely DHA [[36], [37], [38], [39], [40]] but also relevant to improving human nutrition. The European Food Safety Authority guidelines suggest that food must contain 40 mg EPA + DHA/100 g to be considered a source of ω-3 PUFA [41]. Our achieved concentrations of DHA in the breast and thigh muscles will meet or exceed the criteria. Meanwhile, the dietary guidelines for Americans 2015–2020 by the USDA recommended a daily intake of 250 mg EPA + DHA and ∼600 IU of vitamin D₃ for maintaining important metabolic functions and skeletal health in humans [2,3]. Consuming 2 servings of 100 g of chicken fortified with both DHA and 25(OH)D₃ in the present study will meet 46%–50% of the suggested intake of EPA + DHA and >100% of the suggested intake of vitamin D₃, respectively. Thus, our enriched chicken will be a meaningful and novel animal food source of DHA and 25(OH)D₃ for improving intakes and status of these 2 nutrients simultaneously in the US public. It is equally important to point out that supranutritions of DHA and 25(OH)D₃ in the present study exerted no negative effects on growth performance [29] or mortality of chickens. In fact, these high levels of supplementation enhanced body weights, body weight gains, and (or) feed use efficiency [10,42]. There was a decrease in the hepatic 25(OH)D₃ enrichment by adding DHA-rich microalgal biomass into the BD + 25(OH)D₃ diet in Expt. 1. However, such effects were not observed in Expt. 2. The differences in the interaction between these 2 nutrients in these experiments might be attributed to the sources and concentrations of DHA: a whole microalgal biomass rich in DHA was used in Expt. 1, whereas a concentrated and extracted microalgal DHA oil was used in Expt. 2. Supplemental dietary EPA did not increase tissue concentrations of EPA. In fact, no EPA was detected in any of the tissues of chickens fed any of the diets, suggesting that dietary EPA might be fully converted into DHA in the chicken tissues [43]. Meanwhile, concentrations of ARA were decreased in all tissues by supplemental DHA or the resultant DHA enrichments. The decreases might be attributed to enzymatic competition between ω-3 and ω-6 fatty acids for the same enzymes, which was associated with the down-regulation of gene expression of Δ5 and Δ6 desaturases and elongases (ELOVL2 and ELOVL5) [[44], [45], [46], [47], [48]].

When the BD + 25(OH)D₃ diet enhanced the hepatic and breast muscle mRNA concentrations of CD36, SRB1, NPC1L1, and CYP3A4 in Expt. 1, the increases were mitigated by supplemental DHA to the BD + 25(OH)D₃ diet. This type of antagonistic effect of DHA is consistent with the decrease of the hepatic 25(OH)D₃ enrichment and the previously reported decrease in intestinal absorption and bioavailability of vitamin D [49,50] by supplemental DHA. An elevated mRNA level of CYP24A1 in the tissues of birds fed BD + 25(OH)D₃ might reflect an induced degradation of 25(OH)D₃ to 24,25(OH)D₃ and down-regulation of 1α-hydroxylation activity through a feedback mechanism [51] to control the supranutrition of 25(OH)D₃. Furthermore, the down-regulated gene expression or protein production of VDBP in the adipose tissue or thigh muscle of the birds fed BD + 25(OH)D₃ was likely related to its role in buffering against the toxicity of excess vitamin D intake [52,53]. The BD + DHA diet elevated hepatic FASN mRNA concentrations [54]. Conversely, mRNA concentrations of hepatic desaturases (FADS1 and FADS2) and elongase (ELOVL5) were decreased by the BD + DHA, BD + DHA + EPA, or BD + DHA + 25(OH)D₃ diets. Njoroge et al. [46] and Matsuzaka et al. [55] reported similar decreases in Δ5-desaturase and Δ6-desaturase mRNA concentrations by supplementing PUFA in human and mouse diets. Dietary PUFA was reported to inhibit hepatic SREBP-1 activity [56], whereas mRNA concentrations of hepatic SREBP-1 were decreased by the DHA-supplemented diets in this study.

One of the limitations of this work was derived from the use of 2 different sources and the actual DHA concentrations in diets between the 2 experiments. These differences precluded us from a clear view of how supplemental DHA into the BD + 25(OH)D₃ diet decreased the 25(OH)D₃ deposition in Expt. 1. Another limitation was the lack of appropriate antibodies against many avian proteins for us to explore mechanisms beyond gene expression. The third limitation was the intriguing variations in adiposity and its responses to dietary DHA supplementation between the 2 experiments. Specifically, the BD + DHA diet in Expt. 1 decreased relative adiposity compared with the BD or other diets, whereas no such effects were seen by supplemental DHA alone or in combination with EPA in Expt. 2 [57,58]. It remains unclear to us if there was a differential effect of dietary DHA on adiposity upon the adiposity level because the bird adipose tissue had lower FA concentrations in Expt. 2 (59–67 mg/g) than in Expt. 1 (72–96 mg/g). The differences in adiposity between the 2 experiments might be related to the differences in the final body weights of the birds. Furthermore, there was also an unexpected or unexplainable decrease in total fatty acid contents in the adipose tissue at week 6 (59–62 mg/g) compared with at week 3 (62–67 mg/g) in Expt. 2. Another intriguing scenario was the lack of statistically significant changes in many genes related to vitamin D and fatty acid metabolism in various tissues in response to the dietary treatments when these diets caused apparent changes or shifts in the involved pathways. That lack might be due to the relatively small sample sizes, the interval of the treatments, and (or) the presumed responses that might be more at the protein or function level than at the gene transcript level.

In conclusion, our study has demonstrated the feasibility in enriching both DHA and 25(OH)D₃ in the breast and thigh muscles to nutritionally relevant levels for improving intakes of these nutrients by the public. The DHA enrichment was independent of dietary supplementations of EPA or 25(OH)D₃, the supplemental 25(OH)D₃-mediated 25(OH)D₃ enrichment and related gene expression seemed to be negatively affected by supplemental DHA. There was an undesired decrease in ARA concentrations by the DHA enrichment in the tissues.

Author contributions

The authors’ responsibilities were as follows – XGL: designed and supervised the research; SK, ADM, TS: conducted the animal trial and collected data; SK, ADM: performed statistical analyses and wrote the paper; ZS, ADM, SK: performed the 25(OH)D₃ analysis; XGL; revised and edited the manuscript and has primary responsibility for its final content; and all authors have read and approved this submission.

Funding

This study is funded in part by a DOE MAGIC grant (DE-EE0007091) and a USDA grant (2019-69012-29905).

Conflict of interest

XGL is the Editor of the Journal of Nutrition and played no role in the evaluation of the manuscript. All other authors have no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: