Skip to main content

Supranutrition of microalgal docosahexaenoic acid and calcidiol improved growth performance, tissue lipid profiles, and tibia characteristics of broiler chickens



Docosahexaenoic acid (DHA) and calcidiol could be enriched in chicken for improving public nutrition and health. It remains unclear if supranutritional levels of DHA and calcidiol impair growth performance or metabolism of broiler chickens. This study was to determine singular and combined effects of high levels of supplemental DHA-rich microalgal biomass or oil and calcidiol on growth performance, concentrations of triglycerides, cholesterol, and nonesterfied fatty acids in plasma, liver, breast, and thigh, and biophysical properties of tibia.


In Exp. 1, 144 day-old Cornish chicks were divided into 4 groups (6 cages/treatment, 6 birds/cage), and were fed a corn-soybean meal basal diet (BD), BD + 10,000 IU calcidiol/kg (BD + Cal), BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), and BD + Cal + DHA for 6 weeks. In Exp. 2, 180 day-old chicks were divided into 5 groups, and were fed: BD, BD + DHA (0.33% to 0.66% oil, 1.5 to 3.0 g DHA/kg), BD + DHA + EPA (1.9% to 3.8% eicosapentaenoic acid-rich Nannochloropsis sp. CO18, 0.3 to 0.6 g EPA/kg), BD + DHA + calcidiol (6000 to 12,000 IU/kg diet), and BD + DHA + EPA + Cal for 6 weeks.


Birds fed BD + Cal diet in Exp. 1 and BD + DHA + EPA diet in Exp. 2 had higher (P < 0.05) body weight gain (10%–11%) and gain:feed ratio (7%), and lower (P < 0.05) total cholesterol and triglyceride concentrations in plasma (18%–54%), liver (8%–26%), breast (19%–26%), and thigh (10%–19%), respectively, over the controls. The two diets also improved (P < 0.05) tibial breaking strength (8%–24%), total bone volume (2%–13%), and (or) bone mineral density (3%–19%) of chickens.


Supranutrition of dietary calcidiol and DHA alone or together did not produce adverse effects, but led to moderate improvements of growth performance, lipid profiles of plasma and muscle, and bone properties of broiler chickens.


Biofortifications of chicken with bioactive nutrients such as DHA and calcidiol [25(OH)D3] have been viewed as an effective strategy to produce health-promoting meat for human consumption [1, 2]. Relatively low to moderate inclusion levels of calcidiol (1600 to 2800 IU/kg diet) [3, 4] and DHA-rich microalgal biomass or oil (0.55 to 2.55 g DHA/kg diet) [5,6,7] in broiler diets caused no negative effects on growth performance, lipid profile of tissues, or bone strength. In contrast, a high inclusion of calcidiol (27,600 IU/kg diet) in broiler diet decreased body weight by 5% [8], and a high inclusion of DHA-rich microalgal biomass (6.8 g DHA/kg diet) decreased growth performance by 19% and breast muscle weight by 21% [9]. However, these past studies were focused on fortifying chicken with DHA [10] and calcidiol singularly [4]. Little research was attempted to enrich chicken simultaneously with these two nutrients or to look out for potential adverse effects of a combined high supplementation of these two nutrients on growth performance, lipid metabolism, and bone integrity of chickens.

To fill in the gap of knowledge, we conducted two experiments to examine those effects of supplementing high levels of these two bioactive nutrients in broiler chickens. In the first experiment, we supplemented 1% DHA-rich Aurantiochytrium sp. biomass (1.2 g DHA/kg) as the source of DHA in diets for the enrichment of DHA according to our previous findings [11]. In a subsequent experiment, we used a commercial source of DHA-rich microalgal oil (1.5 to 3.0 g DHA/kg) as the source of DHA, along with an EPA-rich Nannochloropsis sp. CO18 biomass (0.3 to 0.6 g EPA/kg). In both experiments, we supplemented a feed grade of synthetic calcidiol (6000 to 12,000 IU/kg) in vitamin D-adequate diets (300 IU/kg) as the source of bioactive cholecalciferol.

Materials and 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) and oil (45% DHA in the oil) were provided by Heliae (Gilbert, AZ, USA) and Archer Daniels Midland Company (ADM, Decatur, IL, USA), respectively. The EPA-rich microalgal biomass (Nannochloropsis sp. CO18, 1.6% EPA in the biomass) and calcidiol (Rovimix HyD Premix, 138 mg calcidiol/kg of premix) were provided by Duke University (Beaufort, NC, USA) and Royal DSM N.V. (DSM, Parsippany, NJ, USA), respectively. In Exp. 1, a total of 144 day-old Cornish male broiler chicks were purchased from Moyer’s Chicks (Quakertown, PA, USA) and housed in a temperature-controlled unit at Cornell University Poultry Research Farm. Chicks were allotted into 4 treatment diets (6 cages/treatment, 6 birds per cage). Birds were fed 1 of the 4 diets: a corn-soybean meal basal diet (BD), BD + 10,000 IU calcidiol/kg of diet (BD + Cal), BD + 1% DHA-rich microalgal biomass (Aurantiochytrium, 1.2 g DHA/kg diet; BD + DHA), and BD + Cal + DHA. The supplemental dietary level of DHA was based on the results of our previous study in which grade levels of the same microalgal biomass were used for enriching DHA in tissues of chickens [11]. The 10,000 IU of calcidiol/kg was chosen based on the safe range of supplementations reported in literature for the enrichments of calcidiol or testing of toxicity [12, 13]. In the BD and all treatment diets, cholecalciferol was supplemented at 1.5-fold (300 IU/kg) of the recommendation by National Research Council (NRC, 1994) [14].

In Exp. 2, a total of 180 day-old Cornish male broiler chicks were purchased from same supplier as in Exp. 1 and allotted into 5 treatment diets (6 cages/diet, 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 0–3 and 4–6 weeks, 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 0–3 and 4–6 weeks, respectively); BD + DHA + Cal (6000 and 12,000 IU of calcidiol/kg diet for 0–3 and 4–6 weeks, respectively); and BD + DHA + EPA + Cal (a combination of all 3 supplements at the doses used in diets of 2 supplements). Different from the design of Exp. 1, we used the commercial source of microalgae DHA oil, with more concentrated DHA than the microalgal biomass, to provide higher supplementations of DHA for a better enrichment outcome. We also intended to determine if supplemental both DHA and EPA (at a ratio of 5:1) could enrich both in the chicken tissues. The design for doubling the supplementation of DHA, EPA, and calcidiol from the starter diet to the grow diet was for a high enrichment efficiency of these nutrients by avoiding a potential feedback or homeostatic regulation [15, 16]. Likewise, the BD and all treatment diets were supplemented with cholecalciferol at 1.5-fold (300 IU) of the NRC recommendation [14]. All other nutrients in all diets used in both experiments were formulated to meet the nutrient requirements for broilers by NRC [14]. Compositions of starter and finisher diets used in both experiments are presented in Additional file 1: Table S14. Both experiments lasted for 6 weeks, Birds had free access to feed and water and received a lighting schedule of 22 h light and 2 h dark throughout.

Growth performance and sample collections

During both experiments, body weights of individual birds were recorded at week 3 and week 6. Feed disappearance of cages were recorded weekly to calculate feed intakes. Chicken health and mortality were checked daily. At the end of week 3 and week 6, 2 birds per cage were euthanized via asphyxiation with carbon dioxide. Blood was drawn from heart puncture by using heparinized needles to prepare plasma samples that were stored at –20 °C until analysis. Liver, breast, thigh, and tibia samples were removed and stored at –20 °C for later analyses.

Laboratory analyses

Concentrations of non-esterified fatty acids (NEFAs), total cholesterol (TC), triglycerides (TGs), and phospholipids (PL) in plasma, liver, breast, and thigh samples were determined using commercially available kits (Wako Chemicals, Richmond, VA, USA) as described in previous studies [17, 18]. In Exp. 1, tibia bone (week 6) characteristics were determined following the protocol described previously [19]. Briefly, soft tissues were removed manually from the bone. The length, width, and depth were measured at the center of the shaft for both tibias and averaged for each bird. Bone breaking strength was measured on the right tibia with the use of an Instron 5965 (Instron Corporation, Norwood, MA, USA) equipped with a 5-kN load cell and a cross-head speed of 20 mm/min. Bluehill 3 Testing Software (Instron Corporation, Norwood, MA, USA) was used to perform flexure tests with a 38-mm supported length. Maximum slope, maximum load, and energy to maximum load were recorded for each tibia. In Exp. 2, characteristics of tibia (week 6) bone were determined using Micro-CT using the method described by Sharma et al. [20]. Briefly, tibia bones were thawed at 4 °C and cleaned of all soft tissues, and analyzed by Skyscan X-ray microtomography (Bruker MicroCT, Billerica, MA, USA). The X-ray source was set at 75 kV and 133 µA. The pixel size was fixed at 25 µm, the rotation angle of 0.4o was applied at each step, and 4 images per rotation were captured. A series of 2D images were captured, which were later used to reconstruct a 3D image using N-Recon (Brucker MicroCT, Billerica, MA, USA). Microtomography was performed on the distal epiphyses of the tibia, and a part of the distal supracondylar region was selected as a volume of interest wherein all bone sections (cortical bone and trabecular bone) were present. Percentage bone volume and bone mineral density (BMD) were measured from the whole total volume of interest, cortical bone, and trabecular bone sections. From trabecula bone, trabecular thickness, trabecular separation, and degree of anisotropy were also measured.

Statistical analysis

Data from Exp. 1 and 2 were analyzed by two-way (2 by 2 factorial arrangement of treatments) and one-way analysis of variance (ANOVA) using a completely randomized design, respectively. Data were presented as mean ± SEM and P < 0.05 was assumed to be statistically significant. Means of different treatment groups were compared using Duncan’s multiple range test. Pen was served as an experimental unit (n = 6).


Growth performance

In Exp. 1, there was no difference in the body weight gain (BWG) or feed intake of chicks among the 4 treatment diets at week 3 (Additional file 1: Table S5). Compared with those fed BD, birds fed BD + Cal had 11% higher (P < 0.05) BWG, and 7% higher (P < 0.05) gain:feed ratio at week 6 (Table 1). Although birds fed BD + DHA had 6% higher BWG and 8% higher feed intake (8%) than those fed BD, the differences were not statistically significant. Moreover, birds fed BD + Cal + DHA also showed non-significantly higher BWG (9%) and feed intake (12%) compared with birds fed BD at week 6 (Table 1).

Table 1 Effects of supplementation of calcidiol, DHA-rich microalgal biomass or oil, and EPA-rich microalgal biomass on growth performance of broiler chickens in Exp. 1 and 2 (0–6 weeks)

In Exp. 2, birds fed BD + DHA + EPA had 17%–27% higher BWG and 6%–25% higher gain:feed ratio than those fed the other diets at week 3, but the differences were not statistically significant (Additional file 1: Table S5). Compared with birds fed the BD, birds fed BD + DHA + EPA had 10% higher (P < 0.05) BWG and 14% higher (P < 0.05) feed intake at week 6 (Table 1). Birds fed the BD + DHA diet had 3% higher BWG and gain:feed ratio, compared with birds fed BD, but the differences were not statistically significant (Table 1). Moreover, birds fed BD + DHA + EPA + Cal had 15% higher (P < 0.05) feed intake, compared with those fed BD. No difference in the BWG, feed intake, or gain:feed ratio was shown in birds fed BD + DHA + Cal, compared with those fed BD (Table 1).

Plasma and tissue lipid profiles

In Exp. 1, there was no difference in concentrations of plasma TGs, TC, and NEFAs among all 4 diets at week 3 (Table 2). At week 6, birds fed BD + Cal had 22% and 29% lower (P < 0.05) plasma TC concentrations compared with birds fed BD + DHA or BD alone, respectively. In Exp. 2, diets produced no significant effects on any of the lipid profiles in plasma or tissues at week 3 (Table 3). However, at week 6, birds fed BD + DHA + EPA had lower (P < 0.05) concentrations of TGs (plasma 54%; liver 18%; breast 24%; thigh 19%), TC (plasma 18%; liver 8%; breast 19%; thigh 9%), and NEFA (plasma 12%; liver 26%; breast 26%; thigh 13%), than birds fed BD, respectively (Table 4).

Table 2 Effects of supplementation of calcidiol and DHA-rich microalgal biomass on plasma lipid profile of broiler chickens in Exp. 1
Table 3 Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and calcidiol on plasma and tissue lipid profiles of broiler chickens at week 3 in Exp. 2
Table 4 Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and calcidiol on plasma and tissue lipid profiles of broiler chickens at week 6 in Exp. 2

Tibia bone health

In Exp. 1, tibia from birds fed BD + Cal had 8%–24% greater (P < 0.05) breaking strength than that of birds fed the other diets at week 6 (Table 5). However, there was no difference in other measured variables among the 4 treatment diets. In Exp. 2, birds fed BD + DHA + Cal had 19% higher (P < 0.05) BMD than birds fed BD + DHA diet and 13% higher (P < 0.05) total bone volume compared with birds fed BD and BD + DHA diets at week 6 (Table 6). Diets produced no significant effects on other measured variables including cortical BMD, cortical percentage bone volume, trabecular BMD, trabecular percentage bone volume, trabecular thickness, trabecular separation, or degree of anisotropy.

Table 5 Effects of supplementation of calcidiol and DHA-rich microalgal biomass on tibia bone properties of broiler chickens at week 6 in Exp. 1
Table 6 Effects of supplementation of DHA-rich microalgal oil, EPA-rich microalgal biomass, and calcidiol on tibia bone properties of broiler chickens at week 6 in Exp. 2


Two consecutive experiments were conducted in the present study to determine responses of growth performance, tissue lipid profiles, and bone characteristics of broiler chickens to supranutrition of DHA and calcidiol during the full starter and grower periods. The sources and concentrations of these two nutrients were chosen based on our previous findings [9, 11] and literature [12, 13] to enrich them in the chicken tissues singularly or together. Indeed, supplementing these two nutrients into diets at the doses used in the present study led to 4–19-fold increases in DHA and 44%–52% increase in calcidiol contents of the liver, breast, and thigh (Kalia et al., manuscript in preparation). The most relevant finding for the broiler industry is that the high dietary supplemental levels of DHA and calcidiol exerted no negative effects on growth performance [8, 21], tissue lipid profiles, or bone characteristics. In contrast, those high supplementations actually exhibited moderate benefits to several measures. To our best knowledge, this was the first attempt to evaluate such effects of the two bioactive nutrients supplemented simultaneously at higher doses, although prior trials determined the effects of calcidiol [3, 4] and DHA-rich microalgal biomass or product [6, 22] singularly.

The enhanced BW, BWG, and gain:feed ratio by feeding birds with BD + Cal over BD in Exp. 1 were consistent with the reported improvements in body weight and feed efficiency resulted from feeding high doses of calcidiol to broiler chickens [13, 23] and breeders [24]. Interesting, birds fed BD + DHA + Cal had an increased feed intake and numerical improvements in BW and BWG. In Exp. 2, supplementing EPA into the BD + DHA diet further improved BW, BWG, and FI of broilers to be significantly higher than those of birds fed BD. In contrast, supplementing EPA into the BD + DHA + Cal diet improved only FI. This implies a potential benefit of supplemental EPA and a unique interaction between EPA and DHA and calcidiol on growth performance of chickens [25,26,27]. Long et al. [5] and Ribeiro et al. [28] reported that high doses of n-3 fatty acids in broiler diets improved BWG and feed conversion ratio. However, supplementing calcidiol alone decreased feed intake of chickens in two studies [13, 29]. Meanwhile, a number of studies indicated that broiler chickens or breeders responded well to dietary supplementations of 1400 to 2800 IU of cholecalciferol/kg diet [3, 4, 30] and 0.55 to 2.55 g DHA/kg diet [5,6,7]. Notably, Yarger et al. [8] found no evidence of renal calcification caused by supplemental 27,600 IU of calcidiol/kg, suggesting that our supplementation doses of calcidiol (6000 to 12,000 IU/kg) were within the safe range. Intriguingly, feeding broilers with 5520 IU of calcidiol/kg increased breast meat yield [31].

Because DHA and calcidiol are fat-soluble, bioactive nutrients that play major roles in regulating lipid metabolism [32, 33], we determined how the supranutrition of them intended for their enrichments in the chicken affected lipid profiles of plasma and tissues of chickens. Such effects will have both animal and human health implications. Whereas substantial changes in the plasma and tissue lipid profiles may reflect a metabolic shift to impair overall health status of broilers, the decreases in lipid contents of the tissues (muscles) may render chicken a more desired animal-sourced protein for human nutrition to reduce risks of chronic diseases. In Exp. 1, birds fed BD + Cal had lower plasma TC concentrations than those fed BD at week 6. This decrease agreed with the reported reduced plasma cholesterol levels in rodents fed high doses of calcidiol [34, 35]. Because 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the major rate-limiting step in cholesterol biosynthesis [34], these supplemental calcidiol-mediated cholesterol decreases might be due to an inhibition of the enzyme by calcidiol [34, 35]. In Exp. 2, supplementing EPA into the BD + DHA diet, compared with BD, caused consistent decreases of concentrations of TGs in the plasma, liver, and breast and thigh muscles, concentrations of TC in the plasma, and concentrations of NEFAs in the breast muscle at week 6. At the same time, supplementing DHA into BD decreased plasma concentrations of NEFAs and the combined supplementations of DHA, EPA, and calcidiol decreased plasma concentrations of TC compared with BD. Previous studies [5, 36] showed hypocholesterolemic and hypotriacylglycerolemic effects of EPA and DHA via inhibiting squalene epoxidase enzyme [36, 37] and reducing very low-density lipoprotein (VLDL) synthesis and secretion [5, 38, 39], respectively. These regulations may be used to explain the decreases of TGs and TC by supplemental DHA and EPA in our study. The lack of any treatment effects on plasma and tissue lipid profiles of chickens at week 3 suggests that a longer time than 3 weeks was required for the dietary supplementations of DHA, EPA, and calcidiol to alter lipid metabolism in the broilers.

It is practically relevant to show an elevated energy at maximum load of the tibia bone from chickens fed BD + Cal compared with those fed BD in Exp. 1. This elevation implies that adding extra 10,000 IU of calcidiol/kg into the BD contained 300 IU of vitamin D3 improved tibia bone strength. Improvements in tibia bone strength and bone volume were produced by supplementing calcidiol at 2760 IU/kg [40] and 12,000 IU/kg [13], respectively, in the poultry diet. It will be interesting to find out if these improvements are still detectable when 10,000 to 12,000 IU calcidiol/kg is added to commercial diets supplemented with > 2000 IU of vitamin D3/kg. The lack of supplemental DHA alone or with calcidiol effect on tibia bone characteristics in Exp. 1 are consistent with outcomes of DHA supplementation on bone structural integrity and strength in several earlier studies [41,42,43]. In Exp.eriment 2, supplementing high levels of calcidiol into the BD + DHA diets appeared to improve total bone volume and total BMD, implying synergistic potential of DHA and calcidiol in improving bone mineral metabolism [44, 45] and reducing risk of bone fracture [46]. Because broiler chickens are among the most fast-growing animals and are susceptible to tibial dyschondroplasia (TD) that reduces the stability of leg bones and deteriorates the quality of meat from the legs [3], superanutrition of DHA and calcidiol may help not only enrichments of chicken with those nutrients but also prevention of TD and associated losses.


Feeding broiler chickens with supranutritional levels of DHA, either from microalgal biomass or oil, and synthetic calcidiol from day 1 to day 42 of age produced no adverse effects on growth performance, plasma and tissue lipid profiles, or tibia characteristics. Instead, some of these singular or combined supplementations led to moderate beneficial responses of the three types of measures. Supplementing low levels of EPA into the BD + DHA diet or the BD + DHA + Cal diet resulted in rather consistent improvements in a number of those measures. There was a synergistic potential between supranutritions of DHA and calcidiol in improving tibial traits. Overall, it seems to be not only safe but also metabolically beneficial to supplement high levels of dietary DHA and calcidiol, much higher than the nutrient requirements, for biofortifying chicken with these bioactive nutrients.

Data Availability

All data generated or analyzed during this study are available from the corresponding author upon reasonablerequest.



Basal diet


Bone mineral density


Body weight gain


Calcidiol (25(OH)D3)


Docosahexaenoic acid


Eicosapentaenoic acid


Feed intake


Non-esterified fatty acid




Total cholesterol


Tibial dyschondroplasia




  1. Patel A, Desai SS, Mane VK, Enman J, Rova U, Christakopoulos P, et al. Futuristic food fortification with a balanced ratio of dietary ω-3/ω-6 omega fatty acids for the prevention of lifestyle diseases. Trends Food Sci Technol. 2022;120:140–53.

    Article  CAS  Google Scholar 

  2. Neill HR, Gill CIR, McDonald EJ, McRoberts WC, Pourshahidi K. The future is bright: Biofortification of common foods can improve vitamin D status. Crit Rev Food Sci Nutr. 2021.

    Article  PubMed  Google Scholar 

  3. Ledwaba MF, Roberson KD. Effectiveness of twenty-five-hydroxycholecalciferol in the prevention of tibial dyschondroplasia in ross cockerels depends on dietary calcium level. Poult Sci. 2003;82:1769–77.

    Article  CAS  PubMed  Google Scholar 

  4. Wideman RF, Blankenship J, Pevzner IY, Turner BJ. Efficacy of 25-OH vitamin D3 prophylactic administration for reducing lameness in broilers grown on wire flooring. Poult Sci. 2015;94:1821–7.

    Article  CAS  PubMed  Google Scholar 

  5. Long SF, Kang S, Wang QQ, Xu YT, Pan L, Hu JX, et al. Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass trait, antioxidant status, and fatty acid profile of broilers. Poult Sci. 2018;97:1881–90.

    Article  CAS  PubMed  Google Scholar 

  6. Moran CA, Keegan JD, Vienola K, Apajalahti J. Broiler tissue enrichment with docosahexaenoic acid (DHA) through dietary supplementation with aurantiochytrium limacinum algae. Food Nut Sci. 2018;9;1160–73.

  7. Khan IA, Parker NB, Lohr CV, Cherian G. Docosahexaenoic acid (22:6 n-3)-rich microalgae along with methionine supplementation in broiler chickens: effects on production performance, breast muscle quality attributes, lipid profile, and incidence of white striping and myopathy. Poult Sci. 2021;11:865–74.

    Article  CAS  Google Scholar 

  8. Yarger JG, Quarles CL, Hollis BW, Gray RW. Safety of 25-hydroxycholecalciferol as a source of cholecalciferol in poultry rations. Poult Sci. 1995;74:1437–46.

    Article  CAS  PubMed  Google Scholar 

  9. Sun T, Tolba SA, Magnuson AD, Lei XG. Excessive Aurantiochytrium acetophilum docosahexaenoic acid supplementation decreases growth performance and breast muscle mass of broiler chickens. Algal Res. 2022;63:102648.

    Article  Google Scholar 

  10. Gatrell S, Lum K, Kim J, Lei XG. Potential of defatted microalgae from the biofuel industry as an ingredient to replace corn and soybean meal in swine and poultry diets. J Anim Sci. 2014;92:1306–14.

    Article  CAS  PubMed  Google Scholar 

  11. Tolba SA, Sun T, Magnuson AD, Liu GC, Abdel-Razik WM, Gamal MF, et al. Supplemental docosahexaenoic-acid-enriched microalgae affected fatty acid and metabolic profiles and related gene expression in several tissues of broiler chicks. J Agric Food Chem. 2019;67:6497–507.

    Article  CAS  PubMed  Google Scholar 

  12. Adhikari R, White D, House JD, Kim WK. Effects of additional dosage of vitamin D3, vitamin D2, and 25-hydroxyvitamin D3 on calcium and phosphorus utilization, egg quality and bone mineralization in laying hens. Poult Sci. 2021;99:364–73.

    Article  Google Scholar 

  13. El-Safty SA, Galal A, El-Gendi GM, El-Azeem NAB, Ghazaly MA, Abdelhady AYM. Effect of 25-hydroxyvitamin D supplementation, ultraviolet light and their interaction on productive performance, bone characteristics, and some behavioral aspects of broiler chicks. Ann Agric Sci. 2022;67:72–8.

    Article  Google Scholar 

  14. National Research Council. Nutrient Requirements of Poultry. 9th rev. ed. Washington: National Academy Press; 1994.

  15. Cachaldora P, Garcia-Rebollar P, Alvarez C, Blas JCD, Mendez J. Effect of type and level of fish oil supplementation on yolk fat composition and n-3 fatty acids retention efficiency in laying hens. Br Poult Sci. 2006;47:43–9.

    Article  CAS  PubMed  Google Scholar 

  16. Feng J, Long S, Zhang HJ, Wu SG, Qi GH, Wang J. Comparative effects of dietary microalgae oil and fish oil on fatty acid composition and sensory quality of table eggs. Poult Sci. 2020;99:1734–43.

    Article  CAS  PubMed  Google Scholar 

  17. Sun T, Yin R, Magnuson AD, Tolba SA, Liu G, Lei XG. Dose-dependent enrichments and improved redox status in tissues of broiler chicks under heat stress by dietary supplemental microalgal astaxanthin. J Agric Food Chem. 2018;66:5521–30.

    Article  CAS  PubMed  Google Scholar 

  18. Magnuson AD, Liu G, Sun T, Tolba SA, Xi L, Whelan R, et al. Supplemental methionine and stocking density affect antioxidant status, fatty acid profiles, and growth performance of broiler chickens. J Anim Sci. 2020;98:skaa092.

  19. Manor ML, Derksen TJ, Magnuson AD, Raja F, Lei XG. Inclusion of dietary defatted microalgae dose-dependently enriches ω-3 fatty acids in egg yolk and tissues of laying hens. J Nutr. 2020;149:942–50.

    Article  Google Scholar 

  20. Sharma MK, White D, Chen C, Kim WK, Adhikari P. Effects of the housing environment and laying hen strain on tibia and femur bone properties of different laying phases of Hy-Line hens. Poult Sci. 2021;100:100933.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Chou SH, Chung TK, Yu B. Effects of supplemental 25-hydroxycholecalciferol on growth performance, small intestinal morphology, and immune response of broiler chickens. Poult Sci. 2009;88:2333–41.

    Article  CAS  PubMed  Google Scholar 

  22. Yan L, Kim IH. Effects of dietary ω-3 fatty acid-enriched microalgae supplementation on growth performance, blood profiles, meat quality, and fatty acid composition of meat in broilers. J Appl Anim Res. 2013;4:392–7.

    Article  CAS  Google Scholar 

  23. Fritts CA, Waldroup PW. Effect of source and level of vitamin D on live performance and bone development in growing broilers. J Appl Poult Res. 2003;12:45–52.

    Article  CAS  Google Scholar 

  24. Atencio A, Edwards HM, Pesti GM. Effect of the level of cholecalciferol supplementation of broiler breeder hen diets on the performance and bone abnormalities of the progeny fed diets containing various levels of calcium or 25-hydroxycholecalciferol. Poult Sci. 2005;84:1593–603.

    Article  CAS  PubMed  Google Scholar 

  25. Wei H-K, Zhou Y, Jiang S, Tao Y-X, Sun H, Peng J, et al. Feeding a DHA-enriched diet increases skeletal muscle protein synthesis in growing pigs: association with increased skeletal muscle insulin action and local mRNA expression of insulin-like growth factor 1. Br J Nutr. 2015;110:671–80.

    Article  CAS  Google Scholar 

  26. Betiku OC, Barrows FT, Ross C, Sealey WM. The effect of total replacement of fish oil with DHA-Gold® and plant oils on growth and fillet quality of rainbow trout (Oncorhynchus mykiss) fed a plant-based diet. Aqua Nutr. 2016;22:158–69.

    Article  CAS  Google Scholar 

  27. Liu B, Jiang J, Yu D, Lin G, Xiong YL. Effects of supplementation of microalgae (Aurantiochytrium sp.) to laying hen Diets on fatty acid content, health lipid Indices, oxidative stability, and quality attributes of meat. Food. 2020;

  28. Ribeiro T, Lordelo MM, Alves SP, Bessa RJB, Costa P, Lemos JPC, et al. Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3 long-chain polyunsaturated fatty acids. Br Poult Sci. 2013;54:753–65.

    Article  CAS  PubMed  Google Scholar 

  29. Garcia AFQM, Murakami AE, Duarte CRA, Rojas ICV, Picoli KP, Puzotti MM. Use of vitamin D3 and its metabolites in broiler chicken feed on performance, bone parameters, and meat quality. Asian-Aust J Anim Sci. 2013;26:408–15.

    Article  CAS  Google Scholar 

  30. Atencio A, Edwards HM Jr, Pesti GM, Ware GO. The vitamin D3 requirements of broiler breeders. Poult Sci. 2006;85:674–92.

    Article  CAS  PubMed  Google Scholar 

  31. Vignale K, Greene ES, Caldas JV, England JA, Boonsinchai N, Sodsee P, et al. 25-hydroxycholecalciferol enhances male broiler breast meat yield through the mTOR pathway. J Nutr. 2015;145:855–63.

    Article  CAS  PubMed  Google Scholar 

  32. Mansoori A, Sotoudeh G, Djalali M, Eshraghian MR, Keramatipour M, Nasli-Esfahani E, et al. Effect of DHA-rich fish oil on PPARγ target genes related to lipid metabolism in type 2 diabetes: A randomized, double-blind, placebo-controlled clinical trial. J Clin Lipidol. 2015;9:770–7.

    Article  PubMed  Google Scholar 

  33. Surdu AM, Pinzariu O, Ciobanu DM, Negru AG, Cainap SS, Lazea C, et al. Vitamin D and its role in the lipid metabolism and the development of atherosclerosis. Biomedicines. 2021;9:172.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gupta AK, Sexton RC, Rudney H. Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells. J Lipid Res. 1989;30:379–86.

    Article  CAS  PubMed  Google Scholar 

  35. Quach HP, Dzekic T, Bukuroshi P, Pang KS. Potencies of vitamin D analogs, 1α-hydroxyvitamin D3, 1α-hydroxyvitamin D2 and 25-hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo. Biopharm Drug Dispos. 2018;39:196–204.

    Article  CAS  PubMed  Google Scholar 

  36. Froyland L, Vaagenes H, Asiedu DK, Garras A, Lie O, Berge RK. Chronic administration of eicosapentaenoic acid and docosahexaenoic acid as ethyl esters reduced plasma cholesterol and changed the fatty acid composition in rat blood and organs. Lipids. 1996;31:169–78.

    Article  CAS  PubMed  Google Scholar 

  37. Bahety P, Nguyen THV, Hong Y, Zhang L, Chan ECY, Ee PLR. Understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling. Eur J Nutr. 2017;56:29–43.

    Article  CAS  PubMed  Google Scholar 

  38. Grimsgaard S, Bonaa H, Hansen J-B, Nordoy A. Highly purified eicosapentaenoic acid and docosahexaenoic acid in humans have similar triacylglycerol-lowering effects but divergent effects on serum fatty acids. Am J Clin Nutr. 1997;66:649–59.

    Article  CAS  PubMed  Google Scholar 

  39. Bernstein AM, Ding EL, Willett WC, Rimm EB. A meta-analysis shows that docosahexaenoic acid from algal oil reduces serum triglycerides and increases HDL-Cholesterol and LDL-cholesterol in persons without coronary heart disease. J Nutr. 2011;142:99–104.

    Article  CAS  PubMed  Google Scholar 

  40. Chen C, Turner B, Applegate TJ, Litta G, Kim WK. Role of long-term supplementation of 25-hydroxyvitamin D3 on laying hen bone 3-dimensional structural development. Poult Sci. 2020;99:5771–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sirois I, Cheung AM, Ward WE. Biomechanical bone strength and bone mass in young male and female rats fed a fish oil diet. Prostaglandins Leukot Essent Fatty Acid. 2003;68:415–21.

    Article  CAS  Google Scholar 

  42. Damsgaard CT, Molgaard C, Matthiessen J, Gyldenlove SN, Lauritzen L. The effects of n-3 long-chain polyunsaturated fatty acids on bone formation and growth factors in adolescent boys. Pediatric Res. 2012;71:713–9.

    Article  CAS  Google Scholar 

  43. Anez-Bustillos L, Cowan E, Cubria MB, Villa-Camacho JC, Mohamadi A, Dao DT, et al. Effects of dietary omega-3 fatty acids on bones of healthy mice. Clin Nutr. 2019;38:2145–54.

    Article  CAS  PubMed  Google Scholar 

  44. Zhao B, Nemere I. 1,25(OH)2D3-mediated phosphate up-take in isolated chick intestinal cells: effect of 24,25(OH)2D3, signal transduction activators, and age. J Cell Biochem. 2002;86:497–508.

    Article  CAS  PubMed  Google Scholar 

  45. Bar A. Calcium homeostasis and vitamin D metabolism and expression in strongly calcifying laying birds. Comp Biochem Physiol A Mol Integr Physiol. 2008;151:477–90.

    Article  CAS  PubMed  Google Scholar 

  46. Ammann P, Rizzoli R. Bone strength and its determinants. Osteoporos Int. 2003;14:13–8.

    Article  Google Scholar 

Download references


We thank Dr. Nelson Ward of DSM for providing calcidiol and Dr. John Less of ADM for providing DHA oil.


This work was funded in part by a DOE MAGIC grant (DE-EE0007091), USDA grant (2019-69012-29905), and Cornell University (Hatch grants NYC-127302).

Author information

Authors and Affiliations



XL designed the research from project conception to study oversight and edited the paper. SK, AM, TS, and GL conducted the animal trial and collected data. SK performed statistical analyses and wrote the paper. WK supervised the tibia analysis. ZJ supervised the cultivation of EPA-rich Nannochloropsis sp. CO18. All authors have read and approved this submission.

Corresponding author

Correspondence to Xin Gen Lei.

Ethics declarations

Ethics approval and consent to participate

The current study was conducted at Cornell University Poultry Research Farm. Animal research protocols were approved by the Cornell University Institutional Animal Care and Use Committee.

Consent for publication

Not applicable.

Competing interest

All the authors declared no conflict of interest.

Supplementary Information

Additional file 1:

Table S1. Composition of experimental diets used in starter period (Exp. 1). Table S2. Composition of experimental diets used in finisher period (Exp. 1). Table S3. Composition of experimental diets used in starter period (Exp. 2). Table S4. Composition of experimental diets used in finisher period (Exp. 2). Table S5. Effects of supplementation of calcidiol, DHA-rich microalgal biomass or oil, and EPA-rich microalgal biomass on body weight, feed intake, and gain: feed ratio in broiler chickens in Exp. 1 and 2 (0–3 weeks).

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kalia, S., Magnuson, A.D., Sun, T. et al. Supranutrition of microalgal docosahexaenoic acid and calcidiol improved growth performance, tissue lipid profiles, and tibia characteristics of broiler chickens. J Animal Sci Biotechnol 14, 27 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: