Mortierella alpina feed supplementation enriched hen eggs with DHA and AA

Abstract

Eggs enriched with long-chain polyunsaturated fatty acids (PUFAs) are considered an important nutrition source. Mortierella alpina is a food-grade oleaginous fungus with a high level of PUFAs, especially arachidonic acid (AA) and eicosapentaenoic acid (EPA). In this study, hens were randomly assigned to three diet groups: control, 5% and 10% M. alpina. Our data indicated that EPA, being converted to docosahexaenoic acid (DHA), and AA were accumulated in the yolk after 10 days of feeding. The amount of DHA tripled and AA doubled compared with the control eggs. The ratio of ω-6 to ω-3 PUFAs in those eggs decreased from approximately 13 : 1 to 8 : 1. These results suggest that M. alpina may represent a valuable source for producing functional eggs enriched with DHA and AA.

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Introduction

Generally, ω-3 polyunsaturated fatty acids (ω-3 PUFAs), especially eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are considered to be important for human health. ω-3 PUFAs play an important role in the prevention and treatment of inflammatory diseases, coronary heart disease (CHD), hypertension, and cancer.^1,2 Calder has reported that long chain ω-3 fatty acids play important roles in growth, optimal function, development, and maintenance of health.³ The health benefits are mainly ascribed to EPA and DHA rather than to the shorter chain ω-3 PUFAs, namely α-linolenic acid (ALA, 18:3n-3). Arachidonic acid (AA, 20:4n-6) is one of ω-6 PUFAs. DHA and AA are the two major long-chain PUFAs in human milk. Their importance for infants has been demonstrated by numerous clinical trials.⁴

ω-3 and ω-6 PUFAs both are essential fatty acids, however, a dietary intake ratio of ω-6 to ω-3 PUFAs 1 : 1 is considered desirable. In modern diet especially the western diet this ratio is commonly as high as 15 : 1 to 20 : 1.⁵ Dietary imbalance would lead to metabolic disturbances.⁶ Therefore it is important to increase the intake of ω-3 PUFAs, primarily EPA and DHA, to reach a balanced ratio of PUFAs.

Fish, fish oil or food enriched with EPA and DHA has been consumed to increase the intake in ω-3 PUFAs. Hen eggs are widely considered as an excellent food source, containing essential nutrients for people of all ages. Importantly, it has been demonstrated that the level and type of PUFAs in eggs can be easily modified by adding ω-3 PUFAs to the diet of the laying hens.⁷ Studies have revealed that dietary lipids can be deposited into the egg yolk without compromising the performance and egg quality of the flock.^8,9 Hence, hen egg is an interesting food product to be subjected to ω-3 PUFA enrichment. Eggs can be enriched with various sources of ω-3 PUFAs. When flaxseed or other plant sources rich in ALA are supplemented in the diet of hens, eggs are mainly enriched with ALA, but not EPA or DHA, and are called ‘ALA-enriched’ eggs.^10,11 We should note that the presence of DHA, rather than ALA, offers more of the positive effects of ω-3 PUFAs.¹² The conversion efficiency of ALA to the longer chain ω-3 PUFAs metabolites is limited in laying hens. “Fishy” off-flavors were perceived when adding 10% of flaxseed.¹³ When hens were fed fish oil rich in EPA or DHA, egg yolk lipids were mainly enriched with DHA.¹⁴ In such “DHA-enriched” eggs, EPA is rarely detected and seems to be converted to DHA before it is deposited.⁷ However, fish oil added to the diet of hens cause undesirable off-flavors, with oxidation products developing in the enriched eggs. A negative impact of feeding a high level (>1.5%) of fish oil on the egg sensory quality has been observed.¹⁵ Another concern is that feeding fish oil may raise cholesterol content in yolk in laying hens, which increases the risk of CHD after those cholesterol-rich eggs were consumed by human body.¹⁴ The increase in DHA upon fish oil supplementation was accompanied by a decrease in AA.¹⁶ Recently, microalgae, as the primary natural producers of long chain ω-3 PUFAs, have been investigated by many research groups. The PUFAs profile of eggs from hens fed those microalgae was very similar to that of eggs from hens fed fish oil.¹⁷ Autotrophic microalga Nannochloropsis gaditana can serve as a good supplementation source to improve ω-3 PUFAs levels in hen eggs.¹⁸ Similarly to eggs from hens fed a diet enriched with fish oil, in microalga-enriched eggs, EPA was also barely accumulated, as it was preferentially converted to DHA before being deposited in yolk phospholipids. In the above three strategies to produce eggs enriched with ω-3 PUFAs, AA did not increase.

Mortierella alpina can produce PUFAs, including ω-6 and ω-3 PUFAs. A thorough safety evaluation of M. alpina has been completed,¹⁹ thus M. alpina is thought to be a suitable oleaginous microorganism.²⁰ However, no research was published in which M. alpina was used as a feed supplement for laying hens to enrich their eggs with PUFAs, due to its low production of ω-3 PUFAs. In order to enhance EPA production, the ω-3 fatty acid desaturase gene was overexpressed and an EPA increase was observed in recombinant M. alpina (CCFM442, CGMCC No. 10259) in our laboratory.²¹ No DPA nor DHA was in the wild and the recombinant M. alpina strains. Here, we use this recombinant M. alpina to feed hens in order to enrich the eggs with long-chain ω-3 PUFAs and AA. The aim of this study was to investigate the impact of using M. alpina as a supplement on PUFAs enrichment in egg yolks.

Materials and methods

M. alpina biomass and standard diet

The M. alpina CCFM442 was obtained from our laboratory.²¹ Heated-air dried biomass was obtained and its composition was analyzed. The standard diet was a wheat-corn-soybean based diet bought from Lvkemao Animal Husbandry Co. Ltd (Zhumadian, China). The composition of the M. alpina biomass and standard diet is shown in Table 1. Furthermore, the fatty acid contents of M. alpina biomass and standard diet was analyzed and shown in Table 2.

Table 1 Composition of heated-air dried M. alpina (%, wet base) and the standard diet (%, wet base, unless specified otherwise)^a

	M. alpina	Standard diet
a Values are means and their standard error of 3 analysis per sample.
Metabolisable energy (MJ kg^−1)		11.28 ± 0.85
Moisture	26.38 ± 1.92	11.26 ± 0.44
Crude protein	8.69 ± 0.37	14.36 ± 0.47
Lysine	0.91 ± 0.02	3.01 ± 0.01
Methionine	0.27 ± 0.00	1.26 ± 0.03
Crude fat	10.56 ± 0.19	3.51 ± 0.02
Crude ash	N.D.	10.69 ± 1.28
Calcium	N.D.	3.57 ± 0.21
Phosphorus	N.D.	0.54 ± 0.06
Sodium	N.D.	0.29 ± 0.02
Crude fiber	N.D.	4.23 ± 0.15

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Table 2 Composition of fatty acids (expressed as percentage of total fatty acids) in M. alpina and the standard diet^a

Fatty acids	M. alpina (%)	Standard diet (%)
a Values are means and their standard error of 3 analysis per sample.
C14:0	1.65 ± 0.01	N.D.
C16:0	17.59 ± 0.21	16.39 ± 0.03
C16:1	0.78 ± 0.05	N.D.
C17:0	0.23 ± 0.00	N.D.
C18:0	9.49 ± 0.11	2.08 ± 0.21
C18:1	20.28 ± 0.01	24.30 ± 0.51
C18:2 (LA, ω-6)	5.74 ± 0.05	53.84 ± 0.69
C18:3 (GLA, ω-6)	3.58 ± 0.10	1.27 ± 0.35
C18:3 (ALA, ω-3)	0.65 ± 0.05	N.D.
C18:4 (STA, ω-3)	0.60 ± 0.04	N.D.
C20:0	0.72 ± 0.01	0.34 ± 0.04
C20:1	0.23 ± 0.01	N.D.
C20:2	0.29 ± 0.00	N.D.
C22:3	3.12 ± 0.03	N.D.
C20:4 (AA, ω-6)	24.43 ± 0.75	N.D.
C20:4 (ETA, ω-3)	0.32 ± 0.00	N.D.
C20:5 (EPA, ω-3)	6.09 ± 0.15	N.D.
C22:0	1.26 ± 0.04	N.D.
C22:5 (DPA, ω-6)	N.D.	N.D.
C22:5 (DPA, ω-3)	N.D.	N.D.
C22:6 (DHA, ω-3)	N.D.	N.D.
C24:0	0.96 ± 0.01	N.D.
C24:1	0.69 ± 0.01	N.D.
Total ω-6 PUFAs	33.75 ± 0.90	55.12 ± 0.34
Total ω-3 PUFAs	7.65 ± 0.07	1.27 ± 0.35

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The fatty acid analysis procedure was as follows: approximately 50 mg of dry mycelia and 100 mg (wet base) of standard diet was used for each lipid extraction. Fatty acids were extracted and methyl esterified as described previously.²² Fatty acid profiles were analyzed as their methyl esters by GC and GC/MS. GC analysis was performed with a gas chromatograph (GC-2010, Shimadzu Co., Kyoto, Japan) with a 30 m × 0.32 mm DB-Wax column (film thickness 0.25 μm). The following temperature program was used: 120 °C for 3 min, ramp to 190 °C at 5 °C per min, ramp to 220 °C at 4 °C per min, hold for 20 min. Nitrogen served as the carrier gas at a constant flow of 3 mL min⁻¹. FAMEs were identified by comparing with commercial FAME standards (GLC-463, Nu-Chek, MN). GC/MS was performed using a GC/MS-QP2010 (Shimadzu Co., Kyoto, Japan). Separation was accomplished using Rtx-Wax column (30 m × 0.25 mm i.d.; film thickness 0.25 μm) (Restek Co., US). The column temperature was kept at 40 °C for 5 min, raised to 120 °C at a rate of 20 °C min⁻¹, raised to 190 °C at a rate of 5 °C min⁻¹ and kept for 5 min, and then raised to 220 °C at a rate of 5 °C min⁻¹ and kept for 17 min. Helium was the carrier gas at constant linear velocity (0.94 mL min⁻¹). Injection volume and mode: 1.0 μL; split (10 : 1). The MS conditions were as follows: ionization voltage: 70 eV; ion source temperature: 220 °C; full scan mode in the m/z range 50–550.

Animals and diet formulation

Forty-five laying hens, 30 weeks of age at the start of the experiment, were individually housed. The hens received 14 h light per day throughout the trial. Feed and water were supplied ad libitum. The overall experimental phase lasted 43 days: adaptation period (14 days), supplementation period with M. alpina (15 days) and wash-out period (14 days). During the adaptation period, the hens were fed with the standard diet to adapt to the new environment. The chemical composition of the standard feed is shown in Table 1. After the adaptation period, the hens were randomly attributed to three groups and fed with one of three diets: (1) 100% standard diet, (2) 95% standard diet + 5% heated-air dried M. alpina, and (3) 90% standard diet + 10% heated-air dried M. alpina. All hens were fed with their respective experimental diet for 15 days (supplementation period). Samples were gathered daily. Finally, all hens again received the standard diet for 14 days (wash-out period). All experimental protocols were approved by the Animal Ethics Committee of Jiangnan University, China, and were performed according to the ethical guidelines of the European Community guidelines (Directive 2010/63/EU).

Performance parameters of laying hens. Feed intake, egg production, egg weight and feed conversion were recorded daily during the three periods. The average body weight was calculated by total weight divided by total number of hens, and measured at the start and at the end of the supplementation period (day 0 and day 15, respectively). The diets were provided regularly at 4:00 pm daily ad libitum. Average feed intake was calculated as the difference between the combined weights of offered feed at 4:00 pm and the remaining weight at 4:00 pm the next day.

Egg production. Egg production was calculated as the number of eggs produced per day divided by the number of live hens housed, and expressed as a percentage.

Feed conversion. Feed conversion was calculated as the weight of total feed consumed divided by the total weight of eggs per day.

Egg quality analysis. The following parameters were calculated at day 10 of the M. alpina supplementation period in five eggs per treatment. Shell thickness was measured at three different points with a micrometer.

Formulas for egg quality measurements. Egg shape index = transversal axis/longitudinal axis; yolk ratio (%) = yolk weight/egg weight; shell ratio (%) = shell weight/egg weight; Haugh units = 100 × log 10 (H − 1.7 × W^0.37 + 7.57), where: H = the height of thick albumen, W = the egg weight.

Egg fatty acids analysis. Yolk was separated using a yolk separator (S010, SSGP, Korea), then homogenized using an Ultrasonic cell crusher (JY92-IIDN, Scientz Co, Ningbo, China). Approximately 50 mg of yolk was used for each lipid extraction. Total lipids and fatty acid profiles for M. alpina were determined by the same method described as Wang et al.²² Three eggs per treatment were analyzed in triplicate.

Color determination. The color of egg yolks was determined by two different methods. First, the yolk color was scored visually by comparison with a Roche yolk color fan (NFN, Robotmation, Japan). Second, a colorimeter (UltraScan Pro, HunterLab, US) was used to determine the color of egg yolk. The L*, a*, b* values were measured after the general analysis of color (L*, brightness; a*, redness; b*, yellowness), thus constituted the CIELAB value (Table 4). Three eggs per treatment were determined in triplicate.

Total cholesterol analysis. Egg yolk cholesterol was extracted by the method of Zhang et al.²³ For determination of total cholesterol of egg yolks, the corresponding diagnostic kits (Great Wall Clinical Reagents Co. Ltd, Baoding, China) designed for measuring cholesterol in serum were used according to the instructions of the manufacturer. 80 μL of mixed solution was used for measurement of absorbance. Three eggs per treatment were determined in triplicate.

Sampling and storage of eggs. For each dietary treatment, eggs were collected daily and stored at 4 °C within 3 hours.

Statistical analysis. The differences among all groups were evaluated with SPSS 17.0. Differences among means of treatments were compared by Tukey's test with P = 0.05 (SigmaPlot 11, Systat Software Inc., Illinois, USA).

Results

Yolk lipid composition

The levels of the ω-3 PUFAs ALA, EPA and DHA in egg yolk at day 0, day 5, day 10, and day 15 of the M. alpina supplementation period and at the end of the wash-out period (day 30) are shown in Table 3. Palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) were detected and the proportion were approximately 25%, 2.5%, 10%, 34% and 18% respectively (expressed as percent of total fatty acids). There was no significant differences for the five kinds of fatty acid above among the eggs in the three groups. The percentage of total fatty acids in eggs in the three groups was 22.37 ± 0.54, 21.91 ± 0.83 and 23.51 ± 0.93 respectively, and showed no significant difference among groups.

Table 3 The composition of fatty acids in egg yolk at day 0, day 5, day 10, and day 15 of the M. alpina supplementation period, and at the end of the wash-out period (day 30)^a

Day	Group	C18:3 (ALA, ω-3)	C20:3 (DGLA, ω-6)	C20:4 (AA, ω-6)	C20:5 (EPA, ω-3)	C22:5 (DPA, ω-6)	C22:5 (DPA, ω-3)	C22:6 (DHA, ω-3)	Total ω-6 PUFAs	Total ω-3 PUFAs	Ratio (ω-6/ω-3)
a Values are means and their standard error of 3 eggs per treatment. All the fatty acid results are shown as means ± SEMs in g/100 g FAMEs. Values with the same letter in the same column are not significantly different (P < 0.05).
0		0.25 ± 0.01^a	0.19 ± 0.04^a	2.56 ± 0.11^a	N.D.	0.20 ± 0.02^a	0.91 ± 0.07^ab	0.42 ± 0.10^a	21.73 ± 0.71^a	1.85 ± 0.01^a	11.73 ± 0.40^b
5	0%	0.26 ± 0.02^a	0.22 ± 0.01^a	2.69 ± 0.14^a	N.D.	0.21 ± 0.03^a	0.65 ± 0.12^a	0.45 ± 0.11^a	22.00 ± 0.61^a	1.75 ± 0.11^a	12.64 ± 1.12^c
	5%	0.20 ± 0.04^a	0.15 ± 0.03^a	3.02 ± 0.37^a	N.D.	0.19 ± 0.01^a	0.68 ± 0.02^a	0.78 ± 0.07^ab	21.49 ± 0.69^a	1.73 ± 0.14^a	12.44 ± 0.58^c
	10%	0.23 ± 0.03^a	0.22 ± 0.01^a	4.29 ± 0.51^ab	N.D.	0.32 ± 0.03^b	0.88 ± 0.06^ab	1.09 ± 0.03^bc	22.88 ± 0.29^a	2.96 ± 0.15^b	7.75 ± 0.29^ab
10	0%	0.23 ± 0.03^a	0.22 ± 0.01^a	2.61 ± 0.14^a	N.D.	0.26 ± 0.09^a	0.74 ± 0.06^a	0.47 ± 0.11^a	21.97 ± 0.20^a	1.65 ± 0.08^a	13.33 ± 0.57^c
	5%	0.28 ± 0.08^a	0.17 ± 0.04^a	3.26 ± 0.08^a	N.D.	0.25 ± 0.02^a	0.74 ± 0.01^a	1.06 ± 0.05^bc	21.56 ± 3.04^a	2.08 ± 0.04^a	10.38 ± 1.28^ab
	10%	0.25 ± 0.04^a	0.21 ± 0.01^a	5.88 ± 0.49^b	0.07 ± 0.01^a	0.45 ± 0.04^b	1.21 ± 0.15^b	1.27 ± 0.11^c	23.93 ± 0.10^a	3.18 ± 0.32^b	7.58 ± 0.73^a
15	0%	0.24 ± 0.02^a	0.21 ± 0.02^a	2.76 ± 0.18^a	N.D.	0.21 ± 0.04^a	0.60 ± 0.04^a	0.42 ± 0.09^a	22.06 ± 0.24^a	1.70 ± 0.07^a	12.95 ± 0.40^c
	5%	0.25 ± 0.05^a	0.15 ± 0.01^a	3.30 ± 0.09^a	N.D.	0.24 ± 0.04^a	0.62 ± 0.03^a	1.09 ± 0.07^bc	21.96 ± 2.01^a	1.96 ± 0.07^a	11.23 ± 1.43^abc
	10%	0.24 ± 0.05^a	0.25 ± 0.03^a	5.71 ± 0.94^b	0.05 ± 0.02^a	0.37 ± 0.06^b	0.85 ± 0.13^ab	1.25 ± 0.06^c	22.45 ± 0.33^a	2.83 ± 0.10^a	7.91 ± 0.36^a
30	0%	0.23 ± 0.05^a	0.17 ± 0.04^a	2.49 ± 0.14^a	N.D.	0.19 ± 0.01^a	0.77 ± 0.06^a	0.41 ± 0.05^a	22.01 ± 0.47^a	1.67 ± 0.01^a	13.18 ± 0.07^c
	5%	0.25 ± 0.03^a	0.21 ± 0.00^a	2.39 ± 0.34^a	N.D.	0.30 ± 0.00^a	0.86 ± 0.02^ab	0.44 ± 0.09^a	22.02 ± 2.14^a	1.81 ± 0.04^a	12.20 ± 0.32^c
	10%	0.23 ± 0.08^a	0.22 ± 0.03^a	2.66 ± 0.28^a	N.D.	0.20 ± 0.06^a	0.72 ± 0.04^a	0.43 ± 0.13^a	22.10 ± 0.22^a	1.60 ± 0.04^a	13.81 ± 0.23^c

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No EPA was detected in eggs in the control groups during the three periods. When 5.0% M. alpina was added, the level of ALA and EPA did not change compared to control, whereas the level of DHA increased two fold after 10 days feeding. Furthermore, when hens were fed 10.0% M. alpina for 10 days, DHA levels increased three fold compared to control (Table 3). The percentage of AA in total fatty acids increased from 2.69% to 3.26% and 5.88%, respectively, 10 days after 5.0% and 10.0% M. alpina was added to hens' feed. Feeding hens longer than 10 days with M. alpina did not further increase AA and DHA levels. Hence, it seems that AA and DHA provided in feed and deposited in eggs reached a plateau around the 10th day, and further supplementation did not raise the levels of AA and DHA (Table 3). Compared to adding 5% M. alpina, adding 10% M. alpina mainly increased the concentration of AA and had less impact on DHA than AA. When hens were treated with the standard diet again for 14 days in the wash-out period, AA and DHA decreased to normal levels seen before the supplementation of 5% and 10.0% M. alpina.

In conclusion, EPA in M. alpina does not accumulate to a great extent in egg yolks and is largely converted to DHA and deposited in the yolk, as observed by Fredriksson et al.²⁴ According to these results (Table 3), M. alpina added to hens' feed raises the levels of AA and DHA in egg yolk, resulting in a more balanced proportion of DHA and AA.

Egg yolk color

Yolk color values at day 0, day 5, day 10, and at the end (day 15) of the M. alpina supplementation period, and at the end of the wash-out period (day 30) are shown in Table 4.

Table 4 Color values of egg yolk measured at day 0, day 5, day 10, and day 15 of the M. alpina supplementation period and at the end of the wash-out period (day 30) from hens fed graded levels of M. alpina^a

Day	Group	Roche value	CIELAB value
			L*	a*	b*
a Values are means and their standard error of 3 eggs per treatment. Values with the same letter in the same column are not significantly different (P < 0.05).
0		8.05 ± 1.34^a	79.92 ± 0.39^a	11.96 ± 0.67^a	61.59 ± 1.21^a
5	0%	9.10 ± 2.67^a	81.36 ± 0.91^a	12.38 ± 1.04^a	58.89 ± 2.31^a
	5%	9.25 ± 0.35^a	82.06 ± 0.81^a	11.67 ± 1.29^a	59.67 ± 2.04^a
	10%	8.65 ± 1.91^a	78.86 ± 0.35^a	13.24 ± 0.82^a	60.58 ± 1.26^a
10	0%	8.35 ± 0.92^a	80.64 ± 0.62^a	12.91 ± 1.21^a	61.81 ± 2.40^a
	5%	9.10 ± 1.27^a	81.97 ± 0.26^a	11.30 ± 0.48^a	59.45 ± 1.26^a
	10%	8.55 ± 0.78^a	80.76 ± 0.77^a	13.58 ± 1.33^a	61.87 ± 2.19^a
15	0%	8.50 ± 0.71^a	82.51 ± 0.43^a	11.27 ± 0.38^a	61.26 ± 0.95^a
	5%	8.15 ± 1.20^a	80.67 ± 0.54^a	12.62 ± 0.49^a	60.03 ± 1.28^a
	10%	10.1 ± 1.27^a	81.53 ± 0.89^a	13.08 ± 0.91^a	62.06 ± 1.56^a
30	0%	9.35 ± 0.92^a	78.82 ± 1.40^a	11.12 ± 1.36^a	59.68 ± 0.82^a
	5%	9.30 ± 0.42^a	78.68 ± 0.83^a	12.28 ± 1.67^a	62.34 ± 0.88^a
	10%	8.90 ± 1.27^a	80.95 ± 0.29^a	11.29 ± 0.89^a	59.39 ± 0.37^a

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After feeding hens with M. alpina, the Roche value of egg yolk was unaffected. Similarly, the L* value (measure for brightness), a* value (measure for redness) and the b* value (measure for yellowness) were unchanged in all treatment. Yolk color did not change significantly after feeding hens with 5% and 10% M. alpina.

Performance parameters of laying hens

The performance parameters during the M. alpina supplementation period of 15 days are summarized in Table 5.

Table 5 The mean feed intake (g per hen per day), egg production (egg per day, %), feed conversion (g feed per g eggs) and average body weight (g per hen) from hens fed graded levels of M. alpina during the supplementation period of 10 days^a

Group	0%	5%	10%
a Values with the same letter in the same row are not significantly different (P < 0.05). The hens' average body weight in the three groups all decreased.
Feed intake, g per hen per day	102.25 ± 11.39^a	121.44 ± 7.16^a	109.39 ± 3.83^a
Egg production, %	62.15 ± 0.28^a	62.85 ± 0.48^a	63.11 ± 1.36^a
Feed conversion, g feed per g eggs	3.09 ± 0.25^a	3.57 ± 0.41^a	3.38 ± 0.23^a
Initial average body weight, g per hen	1895.3	1806.7	1785.5
Final average body weight, g per hen	1873.7	1770.8	1768.1
Change in average body weight, g per hen	21.6	35.9	17.4

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The average feed intake, egg production, feed conversion, initial and final average body weight during the 15 day M. alpina supplementation were not significantly affected.

Egg quality criteria and total cholesterol level

The effects of supplementing M. alpina for 10 days on egg quality criteria and total cholesterol level are summarized in Table 6.

Table 6 The effects of supplementing M. alpina for 15 days on egg quality criteria and total cholesterol level^a

Group	Egg weight (g)	Yolk weight (g)	Yolk ratio (%)	Shell weight (g)	Shell ratio (%)	Shell thickness (mm)	Haugh units	Total cholesterol (mg per egg)
a Values are means and their standard error of 3 eggs per treatment. Values with the same letter in the same column are not significantly different (P < 0.05).
0%	53.62 ± 1.80^a	19.45 ± 0.58^a	36.18 ± 0.13^a	4.72 ± 0.73^a	10.19 ± 1.02^a	0.39 ± 0.02^a	61.93 ± 6.53^a	642.74 ± 112.35^a
5%	51.83 ± 2.61^a	17.91 ± 1.93^a	34.50 ± 1.99^a	4.48 ± 1.00^a	8.72 ± 1.50^a	0.35 ± 0.03^a	64.25 ± 4.29^a	805.95 ± 127.82^a
10%	50.11 ± 1.02^a	17.46 ± 2.84^a	34.71 ± 2.87^a	4.45 ± 0.63^a	8.76 ± 0.55^a	0.34 ± 0.01^a	61.28 ± 2.32^a	661.74 ± 3.56^a

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Haugh unit is considered the parameter indicating the fresh degree of eggs,²⁵ and M. alpina supplementation maintained the Haugh unit from 61 to 64. Also, egg weight, yolk weight and shell thickness did not change significantly. M. alpina also did not affect total cholesterol. Hence, parameters for egg quality criteria were not affected significantly during the M. alpina supplementation.

Discussion

Different ω-3 PUFAs sources, such as flaxseed, fishmeal and fish oil, are used to produce “ω-3 PUFAs enriched” eggs with fatty acids beneficial to health. To date, no research has been published reporting that M. alpina was used as an alternative source to enrich eggs with ω-3 PUFAs, since M. alpina is oleaginous microorganism producing high levels of ω-6 PUFAs. ω-3 PUFAs, mainly EPA, can be enriched in M. alpina modified by gene engineering technology.²¹ The M. alpina strain used in the present study (CCFM442, CGMCC No. 10259) contains approximately 11% total lipids (wet base) (Table 1) and has an interesting fatty acid profile (Table 2), containing ω-3 PUFAs, mainly EPA (8% of total fatty acids) and ω-6 PUFAs (34% of total fatty acids) (Table 2).

This study shows that feeding M. alpina did not affect the performance parameters of hens. Feed intake, egg weight and egg production remained unchanged compared with the control diet. Feeding M. alpina did not decrease egg quality criteria. Shell thickness, shell percentage, yolk percentage and yolk color were not significantly affected by M. alpina supplementation (odor and taste of eggs were not examined here). When flaxseed was added to hens' feed, performance parameters and egg quality criteria were variable, probably depending on the strain and age of the hens.^10,26

Not surprisingly, feeding hens with M. alpina rich in EPA affected the fatty acid profile of egg yolks (Table 3). EPA in M. alpina was barely deposited in egg yolks, but largely converted to DHA and accumulated predominantly in this form. Thus, DHA, rather than EPA, was preferentially incorporated into egg lipids, as was also observed by Fredriksson et al.²⁴ AA, abundant in M. alpina, was also deposited in yolk lipids (Table 3). The ratio of ω-6 to ω-3 PUFAs in the eggs decreased significantly (from 12.6 : 1 to 7.5 : 1), which is a desirable outcome due to the health benefits of ω-3 PUFAs.

Control eggs contained 107 mg AA and 18 mg DHA per egg. After feeding hens with 5.0% M. alpina for 15 days, eggs contained 131 mg AA and 42 mg DHA per egg, while doubling the concentration of M. alpina, i.e. supplementation with 10.0% M. alpina, resulted in 234 mg AA and 50 mg DHA per egg. After feeding 5% M. alpina, 36% of the total ω-3 PUFAs in feed was deposited into egg yolks, similar to the control group. However, the efficiency of deposition for total ω-3 PUFAs from the feed to egg yolks decreased to 29% after feeding 10% M. alpina. This decrease was mentioned in previous publication,¹⁰ which reported that the retention rate for ω-3 PUFAs is reduced with increasing dietary concentration. The efficiency of deposition of ω-3 PUFAs from M. alpina, mainly EPA, was higher than that from flaxseed, which is mainly ALA.¹⁶ This may be primarily due to the low activity of desaturase and enlongase involved in ALA conversion to EPA, and the high amounts of ω-6 PUFAs in the diet increasing the competition for the desaturase involved in conversion of ALA. In contrast, the higher activity of desaturase involved in conversion of EPA to DHA may have caused the increase in DHA deposition in eggs. One of the factors relevant to the efficiency of deposition may be the low digestibility of the cell wall. The efficiency of deposition of ω-3 PUFAs (mainly EPA) in microalgae was low (15–20%), which can presumably be explained by biopolymer formation, which prevented enzymatic degradation of the cell wall, similar to the results reported by Gelin et al.^18,27 Fish oil supplementation of feed worked well to produce eggs enriched with ω-3 PUFAs, for its extremely abundant EPA and DHA, even though the efficiency of deposition of ω-3 PUFAs for fish oil was rarely reported. However, unpleased flavors were produced with supplementation of as little as 1.5% fish oil, as it has a propensity to oxidize.⁷

Conclusion

The present study clearly shows that by adding M. alpina in the diet of laying hens, fatty acid profiles in the eggs changed and egg yolks were enriched with long-chain ω-3 PUFAs, mainly DHA. AA was also deposited in egg yolks following M. alpina supplementation, which may also be beneficial, as AA is an important additive in infant formula. Also, egg quality and performance parameters of laying hens were not decreased. Total cholesterol in egg yolk did not change significantly. This study shows that M. alpina can work as another supplement to produce “functional eggs”, which contain abundant DHA and AA. This is the first report supporting the feasibility of producing nutritious eggs by feeding laying hens with M. alpina. Generally, the eggs, which were produced by feeding hens with genetically modified organisms, were accepted. Humans, especially children, consuming this type of egg enriched with AA and DHA may gain beneficial health effects.

Conflict of interest

There are no conflicts of interest.

Acknowledgements

This study was supported by the Program for New Century Excellent Talents (NCET-13-0831), the National Natural Science Foundation of China (No. 21276108, 31471128), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249), and the Fundamental Research Funds for the Central Universities (No. JUSRP51320B). This study is also supported by the Jiangsu province “Collaborative Innovation Center for Food safety and quality control” industry development program.

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