Effects of supplementing microalgae in laying hen diets on productive performance, fatty-acid profile, and oxidative stability of eggs

 C 2015 Poultry Science Association Inc.

Effects of supplementing microalgae in laying hen diets on productive performance, fatty-acid profile, and oxidative stability of eggs T. Ao,1 L. M. Macalintal, M. A. Paul, A. J. Pescatore, A. H. Cantor, M. J. Ford, B. Timmons, and K. A. Dawson Alltech-University of Kentucky Nutrition Research Alliance, Lexington, Kentucky 40546, USA

SUMMARY The purpose of this trial was to investigate the effects of adding graded levels of dehydrated, whole-cell microalgae (All-G-RichTM , Alltech Inc.) in layer diets on the performance, egg and shell quality, yolk color, oxidative stability, and fatty-acid concentration of eggs. Six replicate groups of 5 Hy-Line W-36 laying hens were assigned to each of 4 dietary treatments. Treatments consisted of feeding a corn-soybean meal control diet alone or supplemented with 1.0, 2.0, or 3.0% All-G-RichTM during a 32-wk production period. Fatty-acid content of egg yolk was analyzed after feeding the experimental diets for 4 wk. The docosahexaenoic acid (DHA) content in egg yolk was linearly increased (P < 0.01) with increasing levels of algae in the diet. There was no effect of dietary treatments on production performance of layers in the entire trial period. Egg weight and eggshell quality were not affected by dietary treatments. Supplements of 2 or 3% All-G-RichTM significantly increased redness (a∗ ) and decreased lightness (L) of egg yolk. Yolk fatty-acid oxidation, as measured by thiobarbituric acid reactive substances (TBARS), was not affected by treatments in eggs stored up to 30 d at 4◦ C. Based on these data, adding All-G-RichTM in layer diets can produce DHA-enriched eggs with no negative impact on egg quality. Key words: layer hens, microalgae, DHA, egg quality, performance 2015 J. Appl. Poult. Res. 00:1–7 http://dx.doi.org/10.3382/japr/pfv042

DESCRIPTION OF PROBLEM There is an increasing demand for functional foods for human consumption that provide various benefits in addition to the nutrients. Eggs can be enriched with certain nutrients through dietary manipulation to create specialty products that could possibly provide health benefits for humans [1–3]. Omega (n)-3 fatty acids, es1

Corresponding author: [email protected]

pecially DHA and eicosapentaenoic acid (EPA), have been indicated to play important roles during pregnancy and early infant development [4,5]. In adults, high levels of dietary DHA and EPA have been associated with lower rates of coronary heart disease, arrhythmias, atherosclerosis and inflammation, diabetes, and cancers of the breast, prostate, and colon [6–10]. Enrichment of eggs with n-3 fatty acids by feeding plant seeds such as flaxseed or oil [11,12] and fish oil [13–15] has been widely investigated. It has

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Primary Audience: Nutritionist, Researchers, Feed Formulators, Egg Producers

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MATERIALS AND METHODS Birds, Diets, and Housing R W-36 laying hens were housed in Hy-Line layer cages (25 × 41 cm) in an environmentally controlled room with temperature maintained at 20◦ C (+/−5◦ C) and light provided at 16 h per day. Feed and water were provided on an ad libitum basis. Records of daily egg production, bi-weekly feed consumption, and mortality were maintained. The body weight of layers was measured periodically. All procedures were approved by the University of Kentucky Institute of Animal Care and Use Committee. A corn-soybean meal diet was used for all the layers before the trial. The nutrient levels for all treatment diets were approximately equal and designed to meet or exceed that recommended for commercial production by Hy-Line International [27]. A commercial source of dehydrated, whole-cell microalgae, All-G-RichTM , previously termed “Alltech SP1” (Schizochytrium

limacinum CCAP 4087/2), which contains 64% fat, 16% DHA, and 11% CP was supplied by Alltech Inc., Nicholasville, KY. The ingredients and calculated composition of nutrients are presented in Table 1. The treatment diets were formulated to be isocaloric and isonitrogenous, containing with 2.85 Mcal/kg of ME and 16.5% CP, respectively. Experimental Design and Sampling A total of 120 laying hens (45-wk-old) were used in this trial. Dietary treatments consisted of feeding a corn-soybean meal control diet alone or supplemented with 1.0, 2.0, or 3.0% All-GRichTM for a period of 32 wk. A randomized, complete-block design was utilized with blocks based on location of the cages within the room. Six replicate groups of 5 hens were randomly allotted to each of the 4 treatments. Analysis of Fatty Acid, Haugh Unit, Yolk Color, and Percent Shell To analyze fatty-acid concentration in egg yolk, 3 eggs per replicate group were taken after feeding treatment diets for 4 weeks [26]. The yolks were separated from whites, and then 3 egg yolks were pooled and homogenized. Yolk homogenate was freeze-dried first, and then used for fatty-acid analysis by gas chromatography (GC) [28]. Feed samples pooled from multiple sub-samples of each experimental diet were finely ground before analysis. Fatty-acid methyl esters (FAME) were synthesized using alkaline boron trifluoride in methanol at 105◦ C for 45 minutes. The FAME was finally extracted with hexane for GC analysis. Six egg samples were also taken after 25 wk of treatment for measurement of egg, yolk and shell weights, Haugh units, and yolk color. Individual egg weight and albumen height were measured and used to calculate Haugh units. Yolk color was analyzed by using ColorFlex EZ spectrophotometer (HunterLab, Inc, Reston, VA 20190). Values are reported in L∗ a∗ b∗ color notation system with L axis representing lightness, a∗ axis representing the redness, and b∗ axis representing the yellowness. Eggshells were washed, dried and weighed to calculate percent shell. Oxidative stability of yolks of whole eggs stored at

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been reported that flaxseed and oil were effective in enriching eggs with linolenic acid (LNA, 18:3 n-3), but not EPA and DHA [16,17]. This is because the poultry have a limited ability to metabolically convert LNA into EPA and DHA [3]. Dietary supplementation of fish oil has been shown to enrich EPA and DHA levels in eggs [15,18]. However, concern about off-flavors of eggs due to dietary fish oil has limited the wide acceptance of this ingredient into layer diets [14,19–21]. Microalgae produced through fermentation contains high levels of DHA [22,23] and could be potentially used in animal diets [24,25]. Herber and Van Elswyk [26] reported that dietary inclusion of marine algae increased DHA content of egg yolk. However, egg production and egg weight were negatively affected by adding high-level microalgae in the diet. Fats with more polyunsaturated fatty acids are more susceptible to oxidation. Therefore, the impact of enrichment with DHA on oxidative stability of egg yolk needs to be determined. The aim of this study was to investigate the effects of adding graded levels of microalgae in layer diets on productive performance, fatty-acid profile, and oxidative stability of eggs.

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Table 1. Ingredients and nutrient composition of treatment diets. Diet1 +1% All-G-RichTM

+2% All-G-RichTM

+3% All-G-RichTM

62.14 24.00 3.00 2.00 0.00 6.70 1.37 0.42 0.12 0.25

62.00 23.60 3.00 1.54 1.00 6.70 1.37 0.42 0.12 0.25

61.62 23.30 3.00 1.19 2.00 6.70 1.40 0.42 0.12 0.25

61.42 22.91 3.00 0.78 3.00 6.70 1.40 0.42 0.12 0.25

Calculated nutrient concentrations 2.85 AMEn , Mcal/kg CP,% 16.5 Ca,% 4.1 Available P,% 0.36 TSAA,% 0.67 Lysine,% 0.87

2.85 16.5 4.1 0.36 0.67 0.87

2.85 16.5 4.1 0.36 0.67 0.87

2.85 16.5 4.1 0.36 0.67 0.87

Corn Soybean meal (48% CP) Oyster shell Soy oil All-G-RichTM Limestone Dicalcium phosphate Salt DL-methionine Vitamin-mineral premix2

Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM . 2 Supplied per kg diet: 11,025 I.U. vitamin A, 3,528 I.U. vitamin D3 , 33 I.U. vitamin E, 0.91 mg vitamin K, 2 mg thiamin, 8 mg riboflavin, 55 mg niacin, 18 mg Ca pantothenate, 5 mg vitamin B-6, 0.221 mg biotin, 1 mg folic acid, 478 mg choline, 28 μg vitamin B-12, 50 mg zinc, 45 mg iron, 60 mg manganese, 5 mg copper, and 0.3 mg selenium. 1

4◦ C in commercial egg flats for 0, 15, and 30 d was evaluated by measuring TBARS according to Sinnhuber and Yu [29]. For each replicate group, yolks from 4 eggs were separated and pooled. The TBARS concentration, using a molar extinction coefficient of 152,000 M−1 cm−1 for the chromophore, was expressed as mg malondialdehyde (MDA) per 100 g egg yolk.

DHA content numerically increased with the increasing levels of algae in the diet.

Statistical Analysis

Hen-day egg production, feed intake, and feed conversion from week 45 to 77 are listed in Table 3. No effect of dietary All-G-RichTM on these parameters was observed. However, it is usually difficult to find differences in performance variables when small numbers of birds are used in treatments with small dietary changes. Body weight of layer hens was assayed at the age of 65 and 77 weeks, respectively. No difference of body weight among all treatment groups was detected (Table 3). Herber and Van Elswyk [26] observed that hens fed a diet supplemented with 4.8%, but not 2.4%, marine algae had decreased egg production compared with hens fed a control diet without algae.

Data were analyzed by ANOVA for a randomized, complete-block design [30] using the Linear Model of Statistix V. 9 [31]. Mean differences were determined using Fisher’s protected least significant difference test. Significance was accepted at P < 0.05. Linear effects were tested using polynomial contrasts.

RESULTS AND DISCUSSIONS The major fatty-acid content in treatment diets are listed in Table 2. Dietary palmitic acid and

Hen Productive Performance and Body Weight

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Control

Ingredient,%

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Palmitic acid Stearic acid Oleic/Elaidic acid Linoleic/Linolelaidic acid a-Linolenic acid Docosahexaenoic acid (DHA)

Control

+1%All-G-RichTM

+2%All-G-RichTM

+3%All-G-RichTM

All-G-RichTM

6.17 1.68 9.01 18.11 1.13 ND

9.02 1.48 9.03 20.53 1.29 1.08

12.98 1.54 9.49 21.97 1.34 2.58

15.33 1.33 7.70 18.96 1.10 4.26

32.8 1.08 ND ND ND 16.32

1 Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM .

Table 3. Effects of dietary treatments on the production performance and body weight of laying hens from wk 45 through 77∗ . Diet1 Control +1%All-G-RichTM +2%All-G-RichTM +3%All-G-RichTM Pooled SEM P-value

Egg production (HDP,%) Feed intake (g/hen/d) Feed conversion (kg/dz) Bird weight of wk 65 (kg) Bird weight of wk 77 (kg)

77.6 116 1.81 1.80 1.88

73.7 110 1.80 1.73 1.92

78.3 110 1.69 1.76 1.77

76.5 113 1.77 1.77 1.84

1.9 1.8 0.06 0.03 0.04

0.39 0.09 0.48 0.59 0.10

∗

Data are means of 6 replicates of 5 hens. Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM . 1

Table 4. Effects of dietary treatments on egg characteristics.1 Diet2 Egg characteristic

Control

+1%All-G-RichTM

+2%All-G-RichTM

+3%All-G-RichTM

Pooled SEM

P-value

Egg weight (g) Percent yolk (%) Percent shell (%) Haugh unit L Color a∗ Color b∗ Color

66.3 28.9 8.64 74.1 63.9a 12.7b 59.5

66.8 29.6 8.60 70.7 64.0a 12.7b 60.7

66.4 28.4 8.35 72.0 62.7b 13.6a 62.6

65.8 29.0 8.23 74.8 63.0b 13.8a 62.8

1.12 0.58 0.19 1.83 0.24 0.35 0.97

0.93 0.55 0.38 0.40 0.004 0.006 0.08

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Data are means of 6 replicates of 6 eggs sampled after feeding treatment diets for 25 week. Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM . a,b Means within a row with no common superscription differ (P < 0.05). 2

Egg Characteristics Table 4 lists the egg weight, percent yolk, percent shell, Haugh unit, and yolk color of hens fed the experimental diets. There was no significant effect of dietary All-G-RichTM on egg and yolk weight, percent shell, and Haugh units. A previous study showed that the egg and yolk weight were both significantly reduced by dietary inclusion of high-level (4.8%) marine algae [26]. The a∗ color value of egg yolk from hens fed diets supplemented with 2 or 3% All-

G-RichTM was higher (P < 0.01) than that from hens fed diets with no or 1% All-G-RichTM addition. The L color value of egg yolk from layers fed diets supplemented with 2 or 3% AllG-RichTM was lower (P < 0.01) than that from layers fed diets with no or 1% All-G-RichTM . Similar results were reported by Herber-McNeill and Van Elswyk [32]. Zeller et al. [23] pointed out that Schizochytrium sp. algae contained carotenoids, specifically canthaxanthin and beta-carotene, which are major sources for yolk pigmentation.

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Production parameter

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Table 5. Effects of dietary treatments on TBARS values (mg MDA/100 g) of egg yolk homogenate after storage.1 Diet2 Days in storage

Control

+1% All-G-RichTM

+2% All-G-RichTM

+3% All-G-RichTM

Pooled SEM

P-value

0 15 30

8.1 8.7 9.9

7.7 9.0 9.7

8.8 8.9 9.8

7.8 8.9 10.4

0.51 0.15 0.24

0.73 0.39 0.27

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Data are means of 6 replicates of 4 eggs sampled after feeding treatment diets for 25 weeks. Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM .

2

Lipid Oxidative Stability of Egg Yolk

Total Lipids and Egg Fatty Acids The content of total lipid and major fatty acids in egg yolk are shown in Table 6. There was no effect of dietary treatments on total lipid content

Table 6. Effects of dietary treatments on the concentration of total lipids and major fatty acids of egg yolk.1 Diet2 Items Total lipid,% Major fatty acids (mg/100 g of yolk) C16:0 Palmitic C16:1n7 Palmitoleic C18:0 Palmitic C18:1n7C Vaccenic C18:1n9C Oleic C18:2n6 Linoleic C20:4n6 Arachidonic C22:6n3 Docosahexaenoic (DHA) 1

+1% +2% +3% Pooled Linear P-value contrast Control All-G-RichTM All-G-RichTM All-G-RichTM SEM 30.5

29.3

30.0

29.6

0.51

0.43

7631 563 2844 435a 11503 5557a 608a 248d

7402 519 2745 404a 11064 5183a,b 469b 509c

7901 615 2650 426b 11255 5066b 403c 717b

8005 611 2725 385c 11304 4555c 336d 776a

172 27 84 5.9 213 135 15 16

0.13 0.11 0.48 0.01 0.57 0.01 0.00 0.00

ns ns ns ∗∗

ns ∗∗ ∗∗ ∗∗

Data are means of 6 replicates of one yolk sample pooled from 3 eggs. Diet: control = corn-soybean meal basal diet with no All-G-RichTM ; +1% All-G-RichTM = basal diet with 1% All-G-RichTM ; +2% All-G-RichTM = basal diet with 2% All-G-RichTM ; +3% All-G-RichTM = basal diet with 3% All-G-RichTM . a–d Means within a row with no common superscription differ (P < 0.05). ∗∗ significant linear contrast: P < 0.01. ns: not significant linear contrast: P > 0.05. 2

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Thiobarbituric acid reactive substances (TBARS) are formed during oxidation of unsaturated fatty acids. These substances include MDA and other similar compounds. Therefore, lipid oxidative stability of egg yolk can be evaluated based on the TBARS value. In the current study, there was no significant difference of TBARS value of egg yolk among treatments after storage of eggs up to 30 d (Table 5). This indicates that dietary supplementation of up to 3% All-GRichTM does not affect oxidative stability of eggyolk lipids. However, Hayat et al. [33] reported that feeding flaxseed in a layer diet led to an increase in TBARS value in egg yolk. King et al. [34] observed increased TBARS and peroxide values of egg yolk when fish oil was included in the diet. Song et al. [35] showed that TBARS values were significantly increased in plasma and

tissue lipids of rats fed DHA-containing oil. In contrast to these previous studies using flaxseed or fish oil, no increase in oxidation of yolk lipid was observed in the present study. It is possible that adequate levels of antioxidant nutrients in the diet (e.g., vitamin E and Se) were sufficient to prevent oxidation of the polyunsaturated fatty acids in the eggs. Furthermore, as more algae was used in the diet, the eggs not only contained more polyunsaturated fatty acids, but also more pigments, as indicated by the color change of the yolks. Bendich [36] and Burton [37] demonstrated carotenoids have antioxidant activity.

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CONCLUSIONS AND APPLICATIONS 1. All-G-RichTM can be used in laying-hen diets to produce DHA-enriched, high-quality eggs without increasing susceptibility to oxidation.

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in egg yolk. Hayat et al. [33] reported that dietary supplementation of flaxseed affected fatty-acid composition, but not total lipid content of egg yolk. Dietary supplementation of graded levels of All-G-RichTM linearly increased DHA content, which is consistent with the data reported by Herber and Van Elswyk [26] and Sefer et al. [38]. For human health considerations, DHA can serve as a source of both EPA and DHA, because human tissue can reconvert DHA to EPA [39,40]. The vaccenic acid, linoleic acid and arachidonic acid concentrations in egg yolk were linearly decreased due to dietary increasing levels of All-GRichTM . There was no difference among dietary treatments for other major fatty acids.

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