Dose-Dependent Enrichments and Improved Redox Status in Tissues

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Bioactive Constituents, Metabolites, and Functions

Dietary supplemental microalgal astaxanthin produced dose-dependent enrichments and improved redox status in tissues of broiler chicks Tao Sun, Ran Yin, Andrew D Magnuson, Samar Amin Tolba, Guanchen Liu, and Xingen Lei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00860 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Journal of Agricultural and Food Chemistry

Dietary supplemental microalgal astaxanthin produced dose-dependent enrichments and improved redox status in tissues of broiler chicks

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under heat stress

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Tao Sun, Ran Yin, Andrew D. Magnuson, Samar A. Tolba, Guanchen Liu,

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and Xin Gen Lei*

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Department of Animal Science, Cornell University, Ithaca, NY, 14853

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*Corresponding author: Xin Gen Lei

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Tel: (607)254-4703

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Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Astaxanthin (AST) is a well-known carotenoid with high antioxidant capacity. This study was to

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evaluate nutritional and metabolic effects of microalgal AST in diets for broiler chicks under

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heat stress. A total of 240 (day-old) Cornish male chicks were divided into 6 cages/treatment (8

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chicks/cage) and fed corn-soy diet supplemented with AST from Haematococcus pluvialis at 0,

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10, 20, 40, and 80 mg/kg for 6 weeks. Heat stress was employed during week 4-6. The

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supplementations led to dose-dependent enrichments (P < 0.05) of AST and total carotenoids in

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plasma, liver, and breast and thigh muscles. There were similar enhancements (P < 0.05) of

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oxygen radical absorbance capacity but decreases or mixed responses (P < 0.05) of glutathione

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concentrations and(or) glutathione peroxidases activities in the tissues. In conclusion,

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supplemental dietary microalgal AST was bioavailable to the chicks and enriched in their tissues

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independent of heat stress, leading to coordinated changes in their endogenous antioxidant

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defense and meat quality.

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KEY WORDS: Astaxanthin, antioxidant, chick, microalgae, fatty acid


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INTRODUCTION Research on microalgal biomass as animal feed has been previously conducted1-3.

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However, there have been only a few studies on microalgal phytochemicals in animal nutrition4,

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Because of its antioxidant, anti-inflammatory, UV-light protective, and coloring properties, AST

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has been used in the food, feed, nutraceutical, and cosmetics industries9-12. The compound is

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often formulated into diets for salmon, trout, shrimp, and lobster12. Rahman et al.13 reported that

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formulated AST at 50 mg/kg in diet showed little impact on growth performance or feed use

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efficiency of juvenile rainbow trout, but increased the antioxidant status, contents of AST and

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total carotenoids, and color in the fish muscles. Meanwhile, plasma activities of catalase (CAT)

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and superoxide dismutase (SOD) were decreased by increasing AST supplementation.

. Astaxanthin (AST) is a xanthophyll carotenoid abundantly distributed in microalgaes6-8.

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Supplemental AST from yeast (Phaffia rhodozyma) at 2.3 and 4.6 mg/kg of diets for broiler

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chicks produced a slight improvement in body weight gain and feed use conversion ratio14, but

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no effect on tissue thiobarbituric acid reactive substances or meat color. Perenlei et al.15

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reported that supplemental AST in broiler diets at 10 and 20 mg/kg improved meat texture, water

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retaining ability, and sensory qualities, and prevented meat protein oxidation during the

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postmortem storage, but exerted no effect on growth performance. In addition, supplemental

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AST protected chick embryos from the glucocorticoid-induced cataract formation16, 17. While

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these reported effects of AST in diets for broilers were apparently inconsistent or conflicting,

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there has been little research on the role of AST in broilers expose to high temperature or under

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heat stress.

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Heat stress is a practical problem to broiler chicks in the summer of Southern States of US

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where a major portion of the US chicks is produced18. Heat stress causes over 128 million dollars



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loss for poultry industry in the US19. Physiologically, heat stress is characterized as a

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thermoregulatory imbalance between the net energy flowing from body to surrounding area and

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the heat generation from animal29. It decreases growth performance and animal product quality20-

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and function23-25. With the known antioxidant activities, AST might be used to protect animals

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from the heat stress-mediated oxidative insults11, 26. Therefore, the objective of this experiment

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was to investigate the bioavailability of microalgal astaxanthin to broiler chicks under normal or

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heat stress condition and its effect on antioxidative status, growth performance, and meat quality

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of these animals.

. Heat stress induces generation of reactive oxygen species (ROS) that impairs cellular structure

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MATERIALS AND METHODS

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Animals, diets, and management

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A total of 240 (day-old) Cornish male broiler chicks were purchased from Moyer’s Chicks

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(Quakertown, PA) and housed in an environmentally-controlled room with cages and randomly

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separated into five treatments (each treatment with six replicates of eight chicks per replicate).

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Chicks were fed with a corn-soybean meal basal diet supplemented with AST from

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Haematococcus Pluvialis (H. Pluvialis, Heliae, Gilbert, AZ) at 0, 10, 20, 40, and 80 mg/kg

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(control, and treatments 1, 2, 3 and 4) for 6 weeks. Supplemental AST for Treatments 1 and 3

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was provided by the defatted H. Pluvialis and Treatments 2 and 4 by the full-fatted H. Pluvialis.

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The two forms of the microalgal biomass were used for the accuracy and convenience of

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supplementing the intended amounts of AST into the diets, minimizing impacts of the

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supplementations on composition of the diets, and fulfilling our interest in exploring the potential

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of defatted microalgal biomass. Light schedule was 2:22 h dark:light cycles for the whole period.


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Animals were given free access to feed and water. All experimental diets were formulated

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according to nutrient requirements of poultry by NRC (1994)27. The nutrient composition of the

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diets with supplemental AST concentrations is shown in Supplemental Table 1. Temperature

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schedule was set according to the industrial guide28 (week 1: 34°C; week 2: 31°C; week 3: 27°C)

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during week 1 to 3. Starting from week 4, heat stress was applied to broilers by rising up room

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temperature 10°F above the recommended temperature (week 4: 32.5°C; week 5: 30°C; week 6:

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28.3°C). Our animal protocol was approved by the Institutional Animal Care and Use Committee

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of Cornell University.

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Blood collection, Tissue Examination, and Biochemical assays

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Growth performance was recorded weekly. At the end of weeks 3 and 6, chicks were

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euthanized by carbon dioxide following cervical dislocation after an 8-h fasting. Blood was

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centrifuged at 3,000 g for 15 min (Beckman GS-6R centrifuge, Brea, CA) and kept on ice before

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analysis. Liver, breast muscle, and legs were removed and weighted, and a portion of each was

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frozen on dry ice and stored at -80oC until analysis. Plasma alanine aminotransferase (ALT)

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activities were determined by Infinity ALT liquid stable reagent kit (Thermo Electron Corp.,

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Waltham, MA). Plasma alkaline phosphatase (AKP) activities were determined using the method

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of Bowers and McComb29. Plasma tartrate-resistant acid phosphatase (TRAP) was determined

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from the method of Lau et al30. Plasma inorganic phosphorus (PIP) was analyzed following the

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method of Gomori31. All samples were tested in duplicates. Glucose assay kit was purchased

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from Sigma Aldrich (St. Louis, MO). Kits for total cholesterol (TC), triglyceride (TG) and non-

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esterified fatty acid (NEFA) were purchased from Wako Chemicals USA (Richmond, VA). Uric

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acid and ALT reagents were obtained from Thermo Scientific, Inc. (Waltham, MA).

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Analyses of astaxanthin and total carotenoid content



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Total AST in diet, plasma, and tissues were extracted using the method of Lopez et al.32 with

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modifications. Samples of tissues (0.5 to 3 g) and plasma (500 µL) were incubated within

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acetone and ethyl acetate for 10 min on ice. Water was then added and centrifuged at 3000 g for

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15 min at 4oC. Upper layer was collected and dried under N2 gas. Residues were dissolved in

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chloroform (HPLC grade) for HPLC-UV analyses. The AST concentrations in the samples were

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measured based on the methods of Sowell et al33, Breithaupt et al34, and Rohrle et al35 with

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modifications. Briefly, AST extraction was eluted isocratically with methanol and acetonitrile

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(50:50) with 0.1% triethylamine (TEA) at a flow rate of 1 mL/min, carried on an Agilent Eclipse

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plus C18 reverse phase column (5 µm, 4.6 x 250 mm) using a Shimadzu HPLC system with a

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LC-10AD micro-plunger pump and a SPD-10 AV vp UV detector. Column temperature was set

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up at 30oC. Mobile phase was sonicated for 15 min before use. All chemicals were HPLC grade

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and solutions were freshly prepared. The detected peaks were identified by comparison with the

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retention times of a standard AST. To validate the results, sample extracts were spiked with the

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standard AST to determine its appearance on the chromatogram in relation to the sample peak

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being identified. Pure AST and β-carotene were purchased from Sigma Aldrich (St. Louis,

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Missouri). The astaxanthin and β-carotene concentrations were calculated by the AUC (area

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under curve) of samples against the AUC of the spiked AST and β-carotene standards in the

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HPLC chromatogram results. The extraction efficiency (recovery) of AST and β-carotene was

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92% and 90%, respectively.

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Oxygen radical absorbance capacity (ORAC) assay

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Oxygen radical absorbance capacity (ORAC) of plasma, liver, and thigh and breast muscles

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were measured using the method of Ou et al36 with modification. Briefly, tissues were

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homogenate and extracted by hexane twice, following with centrifuge at 12000 g for 5 min.


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Supernatants were then combined, and dried under N2 gas. The lipophilic fragment was re-

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dissolved in 7% methylated cyclodextrin, 50% acetone 50% water solution. Extraction residues

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were extracted in 7% acetic acid and 80% methanol solution, sonicated at 37oC for 5 min, and

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incubated at room temperature for 15 min, with shaking frequently. The hydrophilic fragment

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was dissolved in 75 mM phosphate buffer (pH=7.4). Trolox (6-hydroxy-2,5,7,8-

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tetramethylchroman-2-carboxylic acid) was used as the antioxidant activity standard and all

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results were expressed as µΜ of trolox equivalence. Samples and standards were detected under

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350 nm excitation and 450 nm emission (SpectraMax M2, Molecular Devices, Sunnyvale, CA).

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Determination of MDA, GSH, GSSG and antioxidant enzymes

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Malondialdehyde (MDA) levels were determined by the method of McDonald and Hultin37,

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using 2-thiobarbituric acid assay with 1,1,3,3-tetraethoxypropane as the standard. Glutathione

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(GSH) and glutathione disulfide (GSSG), glutathione peroxidase (GPX), glutathione reductase

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(GR), glutathione s transferase (GST) and superoxide dismutase (SOD) in liver, breast and thigh

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was determined using the methods of Anderson38, Flohe and Gunzler39, Massey and William40,

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Guthenberg and Mannervik41, and McCord and Fridovich42, respectively.

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Meat pH, water holding capacity, and texture profile analyses

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Texture profile analysis was used to measure the compression force by compressing meat

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samples with the texturometer (TA.XTplus, Stable Micro Systems, Hamilton, MA). Frozen

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breast and thigh muscle were de-thawed and cut into 2-inch diameter cubes, cooked in oven at

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175oC for 30 min, then subjected to analysis of chewiness, springiness, and hardiness using the

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method from Huidobro et al43. The meat pH was determined using iodacetate method44 with

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modification. Approximately 250 mg of meat was homogenized for 30 sec in 2.5 mL of solution



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composed of 5 mM sodium iodoacetate and 150 mM potassium chloride at pH 7. The mixture

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pH was measured using an Accumet pH probe (AB150, Fisher Scientific, Waltham, MA).

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The water holding capacity (WHC) was determined using a centrifugal method45, 46.

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Approximately 1g of meat was added to a centrifuge tube that contained three pieces of

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Whatman filter paper which were folded into a thimble. The tube and its contents were subjected

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to 7,710 g force for 30 min. The meat samples were separated from the filter paper and weighed

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to determine the WHC.

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Determination of fatty acid profile

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Total lipid was extracted from plasma and tissues according to the method of Folch et al.47 and

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Fristche et al.48 Then, the lipid was methylated using 4% sulfuric acid under 90oCfor 60 min, and

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tridecanoic acid was used as an internal standard. Each fatty acid was identified by comparing its

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retention time and peak area against the individual fatty acid methyl ester standards. Methyl

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esters of fatty acids were analyzed by gas chromatography/mass spectrometry (model HP 5890 A

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with a HP 5970 series mass selective ion monitoring, Hewlett-Packard, Palo Alto, CA)49,50.

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Statistical analyses

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Data were collected using the cage as the experimental unit and were analyzed by one-way

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ANOVA. Mean comparisons were conducted with Duncan’s multiple-ranged method. Data were

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presented as means ± SEM, and the significance level for differences was P < 0.05. The

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correlations between variables were analyzed by Pearson’s correlation, and linear or

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polynominal regression was performed using the Proc General Linear Models procedures of

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SAS (version 9.2, SAS Institute, Cary, NC).

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RESULTS


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Total carotenoid and astaxanthin concentrations

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There were dose-dependent elevations (linear or polynominal, R2 > 0.9, P < 0.05) of total

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carotenoids and AST in the plasma, liver, and breast and thigh muscles with increasing dietary

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AST supplementations (Table 1). The highest concentrations of AST reached 17 µg/mL in the

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plasma and 5.8, 2.2, and 2.1 mg/kg in the liver, breast, and thigh at week 6, respectively. The

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highest concentrations of total carotenoids were 218 µg/mL in the plasma and 53, 12, and 7.0

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mg/kg in the liver, breast and thigh at week 6, respectively.

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Growth performance and plasma health

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Body weight gain, feed intake, and gain:feed ratio were not influenced by AST

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supplementations during the starter period (weeks 1-3) (Table 2). However, during the grower

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period (weeks 4-6), 20 and 80 mg AST/kg treatments decreased (P < 0.05) gain/feed ratio by

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14% and 18% (P < 0.05), respectively, without affecting body weight gain or feed intake.

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Likewise, the four doses of supplemental AST led similar decreases (7 to 11%, P < 0.05) of

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gain/feed ratios compared with the control G: F ratio during the entire period (weeks 1-6), health

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indicators of plasma biochemical assays were largely unaffected at week 3 or 6 by the AST

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inclusion (Supplemental Table 2).

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Antioxidant status

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There were dose-dependent elevations (linear or polynominal, R2 > 0.9, P < 0.05) of ORAC

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in the liver, breast, and thigh with increasing dietary AST supplementations at week 3 and 6

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(Table 3). However, MDA concentrations in these tissues except for the thigh muscle at week 6

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remained similar across the dietary treatments. Meanwhile, GSH and GSSG in the liver were

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affected by the AST supplementations at week 3 but not at week 6. Chicks fed with 40 or 80 mg

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AST/kg had 28% or 45% decrease (P < 0.05) of hepatic GSH compared with the control at week



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3. Breast GSH at both time-points and thigh GSH at week 6 were decreased (P < 0.05) by the

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highest dose of supplemental GST, compared with the controls.

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Antioxidant enzyme activities

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The four doses of supplemental AST enhanced hepatic GR activities by 1.4- to 2.2-fold over

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the controls at week 3 (Table 4). These supplementations caused dose-dependent increases (P

Dose-Dependent Enrichments and Improved Redox Status in Tissues

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