Effects of Dietary Algal Docosahexaenoic Acid Oil Supplementation on Fatty Acid Deposition and Gene Expression in Laying Tsaiya Ducks *

The current study was designed to determine the effects of dietary docosahexaenoic acid (DHA) on fatty acid deposition in egg yolk and various tissues of laying Tsaiya ducks, and on the mRNA concentrations of hepatic lipogenesis-related transcription factors. Thirty laying ducks were randomly assigned to three treatments with diets based on corn-soybean meal (ME: 2803 kcal/kg; CP: 17.1%; Ca: 3.4%) supplemented with 0% (control diet), 0.5% or 2% algal DHA oil. The DHA content in egg yolks of the ducks was elevated significantly (p<0.01) with the supplementation of dietary DHA. The DHA percentage of the total fatty acids in the egg yolk of laying ducks was 0.5%, 1.3% and 3.4% for 0%, 0.5% and 2% algal DHA oil treatments, respectively, for the 1 week, and 0.5%, 1.5% and 3.3% for the 2 week. Therefore, algal DHA oil can be utilized by laying Tsaiya ducks to enhance the egg-yolk DHA content. The concentrations of triacylglycerol (TG) and cholesterol in plasma of laying Tsaiya ducks were not affected by dietary DHA treatments (p>0.05). The DHA concentration in plasma, liver, and skeletal muscle was increased with the addition of dietary algal DHA oil (p<0.05). The mRNA abundance of sterol regulatory element binding protein 1 (SREBP1) and SREBP2 in the livers of laying Tsaiya ducks was not affected by dietary DHA, suggesting that the expression of these transcription factors is tightly controlled and not sensitive to DHA treatments. (


INTRODUCTION
Dietary fatty acids (FA) are essential to maintain normal physiological functions and can be incorporated into different tissues of animals.In pigs, changes in FA composition of liver, muscle and adipose tissue are achieved by feeding different dietary FA sources (Innis et al., 1996;Smith et al., 1996;Ding et al., 2003).In avian species, dietary FA can be deposited into egg yolks and into other tissues (Cruickshank, 1934;Donaldson, 1967;Ding and Lilburn, 1997).Cruickshank (1934) showed that the unsaturated, but not the saturated FA composition of egg yolks is modified by dietary FA.Feeding hens with fish oil increases n-3 polyunsaturated fatty acid (PUFA) levels in the egg yolk lipids, indicating that the FA composition of egg yolks can be changed by dietary fat sources (Navarro et al., 1972;Oh et al., 1988;van Elswyk et al., 1992).
In the current study, we enriched egg-yolk, plasma, liver, and skeletal muscle DHA through dietary supplementation with algal DHA oil.Dietary DHA did not change the expression of liver lipogenesis-related transcription factors (SREBP1 and SREBP2), suggesting fatty acid and cholesterol synthesis, important for egg-yolk production are not regulated by dietary DHA in laying ducks.

Animals and diets
Thirty laying Tsaiya ducks were placed in individual cages and distributed randomly to three treatments.Cornsoybean meal based diets (ME: 2,803 kcal/kg; CP: 17.1%; Ca: 3.4%; Table 1) were supplemented with 0% (control diet), 0.5% or 2% algal DHA oil (Martek Bio).Vitamin E concentration was raised to 37.5 IU/kg of diet instead of the NRC recommendation level of 5 IU/kg (National Research Council, 1994) because the additional dietary DHA may compromise the antioxidant system of the laying Tsaiya ducks.The dietary FA compositions are listed in Table 2.After feeding the ducks with the control diet (2% butter) for one week, the birds were fed the experimental diets for 14 days.Egg production rate was determined for the first and second weeks.Egg weight and yolk weight were measured on the 7 th and 14 th day.Individual blood samples were obtained from the brachial vein (V. ulnaris) using EDTA as an anticoagulant.Plasma, dried egg yolk and diet samples were frozen at -80°C until analysis.Ducks were killed by cervical dislocation on the 14 th day, and tissue samples (liver and skeletal muscle) were removed, and immediately frozen in liquid nitrogen to be stored at -80°C.The animal protocol used in the present experiment was approved by the Animal Care and Use Committee of the National Taiwan University.

Fatty acid analysis
Total lipids of diet, tissue, and yolk were extracted according to the procedure of Folch et al. (1957).Before the extraction, tissue and yolk samples were freeze-dried.Onetenth g of yolk, 1 g of diet, or 0.5 g of tissue sample powders was used to extract total lipids.Heptadecanoic acid (17:0; 1,000 nmol), as di-17:0 L-α-phosphatidylcholine (Sigma, St. Louis, MO) was added to the sample as an internal standard before extraction.Total plasma lipids were extracted with methanol: benzene (4:1 v/v) as modified by Kates (1986).
Total lipids were then converted to fatty acid methyl esters (FAME) and separated by gas chromatography on a 30 m long by 0.25 mm ID with 0.20 um film thickness (SP-2380 capillary column; Supelco Inc., USA) with a Varian Star 3400cx gas chromatograph equipped with a hydrogen flame-ionization detector.Individual FA were identified by comparison to the retention times of standards (Nu Check Prep, Inc., Elysian, MN)

Plasma TG and total cholesterol analysis
The plasma TG and total cholesterol concentrations were determined spectrophotometrically using a commercial test kit (1.14856.0001for TG; 1.14830.0001for total cholesterol; Merck, Taipei, Taiwan).

RNA analysis
Total RNA was extracted from the frozen liver and muscle using the guanidinium-phenol -chloroform extraction method (Chomczynski and Sacchi, 1987); modifications were described previously (Hsu and Ding, 2003;Ying et al., 2003;Yang et al., 2004).The RNAs were separated by denatured electrophoresis, blotted to nylon membranes, and hybridized with radiolabeled cDNA probes in Ultrahyb (Ambion Inc., Austin, TX).Probes for Northern analysis were produced by PCR procedures described by Wang et al. (2004).The SREBP1 primer pair was generated from the chicken sequence whereas the SREBP2 primer  pair was from the human sequence.The primer sequences are 5'-GCGCTACCGCTATCCATCA-3' and 5'-GGTCGGC ATCTCCATCACCT-3' for SREBP1 and 5'-TAATACG ACTCACTATAGCG-3' and 5'-TCAAGTCCTTCAGCCTC AAG-3' for SREBP2.These lipogenesis-related transcriptional factors were cloned from the liver of a Tsaiya duck (Yen et al., 2005), and the cDNA fragments were used to generate probes for Northern analysis.The SREBP1 cDNA fragment is 283 bp with 93% homology when compared with the chicken SREBP1 (GenBank, AJ310768).The SREBP2 cDNA fragment is 386 bp with 83% homology when compared with the chicken SREBP2 (GenBank, AJ310769).The 18S ribosomal RNA primer sequences and PCR conditions are indicated in Liu et al. (2005).Hybridization results were quantified by phosphorimage analysis.The densitometric value for an individual transcript in a sample lane was normalized to the densitometric value for the 18S ribosomal RNA in the same lane.Duplicate RNA samples for each tissue from each bird were analyzed.

Statistical analysis
All data were analyzed by analysis of variance using the general linear model procedures of the SAS Institute (SAS, 2001).The differences between means were detected with the Duncan's New Multiple Range Test.A P value≤0.05 was considered significant.

Production parameters
Egg production, egg weight, and yolk weight of the laying ducks were not affected by the algal DHA oil supplement in the diet (p>0.05).The egg production ranged from 87±18 to 98±5%.The egg weights ranged from 63.9±4.5 to 67.0±6.4 g.The average weight of a yolk was between 19.4±1.9 and 20.0±1.2 g.

Plasma TG and total cholesterol concentrations
Neither the plasma TG nor total cholesterol concentrations of laying ducks were affected by dietary algal DHA oil (p>0.05).The plasma TG concentration ranged from 94±22 to 116±27 mg/dl whereas the plasma cholesterol ranged from 919±499 to 1,342±395 mg/dl in the laying ducks.

Plasma DHA concentration
The plasma DHA concentration was significantly increased by one or two week dietary supplementation with algal DHA oil (p<0.01;Table 3).

Tissue DHA concentrations
The liver DHA concentration data were previously reported by Ko et al. (2004).It was significantly increased by the dietary algal DHA oil supplement after two weeks (p<0.01;Table 4).The muscle DHA concentration of the laying Tsaiya ducks was increased by dietary DHA (p<0.01;Table 4).The increase was smaller than that observed in the liver.

The mRNA concentrations of SREBP1 and SREBP2
Neither the SREBP1 mRNA concentration nor the SREBP2 mRNA concentration in liver and muscle were affected by the dietary DHA treatment for two weeks (p>0.05;Figure1A; 1B).

DISCUSSION
Unsaturated FA concentrations in egg yolks are altered by dietary unsaturated FA in avian species (Cruickshank, 1934;Chen et al., 1965;Navarro et al., 1972).Fish meal, fish oil, linseed, flaxseed, and algae products have been incorporated into diets to increase the n-3 PUFA concentrations in chicken eggs and tissues.However, fishy odor is a concern when high amounts of fish oil are added to the diet (Nash et al., 1995).In the current study, we found that DHA concentrations in eggs, livers and muscles were increased whereas the production parameters of the laying ducks were not affected by dietary supplementation with algal DHA oil.The results on the production parameters in the current study are different from those of others that indicate the egg production, egg weight, and yolk size is reduced by dietary PUFA supplementation (Whitehead et al., 1993;Scheideler et al., 1994;van Elswyk et al., 1994).However, similar to the current study, other reports show no effect (Hargis et al., 1991;Ferrier et al., 1995;Meluzzi et al., 2000;Chen and Hsu, 2003;Cheng et al., 2004).We observed that changes in egg-yolk fatty acid composition had already taken place after on week of dietary DHA supplementation with no further change at two weeks (Table 5).Furthermore, several groups indicate that incorporation of dietary PUFA, including DHA, into eggyolk lipids at 14 to 18 days of treatment (Meluzzi et al., 2000;Chen and Hsu, 2003;Cheng et al., 2004).Thus, two weeks was a sufficient time to feed the algal DHA.A longer time (12 weeks) of dietary PUFA supplementation also increases egg-yolk DHA (Watkins et al., 2003).Nitsan et al. (1999) indicate that laying chickens fed with diets containing 1% algal meal had increased plasma DHA (30%).We found a greater increase (about three-to seven-fold) with the dietary supplementations of 0.5% or 2% algal DHA oil in laying ducks (Table 3) as well as in our previous study with laying hens (Cheng et al., 2004).Lopez-Ferrer et al. (2001) demonstrate that PUFA composition in the muscles of broilers was affected through dietary linoleic acid, linolenic acid or fish oil supplementation.The DHA concentration in the skeletal muscle and liver is increased by elevated dietary DHA in the current study, confirming data in the literature (Huang et al., 1990;Leskanich and Noble, 1997;Lopez-Ferrer et al., 2001;Schiavone et al., 2004).Other FA were either not affected or only slightly changed.
Dietary fish oil decreases plasma TG and cholesterol concentrations in mammals (Sanders and Hochland, 1983;Kromhout et al., 1985;Daviglus et al., 1997).Feeding the high DHA algae oil to laying ducks did not affect plasma Table 6.Effect of 14-d dietary algal DHA treatment on fatty acid compositions in the egg yolk of laying Tsaiya ducks* Fatty acid profile Control 0.5% DHA oil 2% DHA oil -------------% of total fatty acids ---------------C16:0 25.4±5.927.2±0.927.5±1.TG or cholesterol concentrations; similar to what is observed in laying chickens (Cheng et al., 2004).Physiologically, the de novo lipogenesis in the liver of laying birds is high in order to produce lipids for yolk deposition.Such function may be related to high estrogen concentrations in laying birds (Harms et al., 1972;Polin and Wolford, 1977;Dashti et al., 1983).The lipids generated in liver are transported into egg yolks by very low density lipoprotein (VLDL) and vitellogenin, both of which contain high TG and cholesterol (Walzem, 1996).If the efficiency of fat deposition in yolks was reduced, the egg production would be negatively affected.These observations suggest that in order to maintain egg production, the plasma TG and cholesterol concentrations in laying hens are not readily changed by dietary lipid composition as observed in mammals.
Cholesterol and FA synthesis in birds is primarily in the liver (Leveille et al., 1968;1975).Fatty acid synthase (FAS) and 3-hydroxyl-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) are the two key enzymes involved in lipid synthesis (Back et al., 1986;Semenkovich, 1997;Horton et al., 1998;Gondret et al., 2001).These enzymes are regulated by two lipogenic transcription factors-SREBP1 and SREBP2, respectively.Recent data show that the dietary long-chain PUFA (from fish oil or high DHA algae) decrease the SREBP1 mRNA abundance in rodent and pig livers (Xu et al., 1999;Yahagi et al., 1999;Hsu et al., 2004).Several days of feeding fish oil causes a reduction in mouse hepatic SREBP1 mRNA (Nakatani et al., 2003).In contrast to observations in mammals, there was no effect of dietary DHA on the expression of these transcription factors in the liver and skeletal muscle of laying ducks after two weeks feeding with the DHA oil (Figure 1).This observation is similar to that in laying hens (Cheng et al., 2004).A longer time of feeding might decrease the SREBPs, but the evidence from mammals suggests these transcription factors rapidly respond to PUFA.In the laying duck dietary DHA is incorporated into liver and muscle, but the SREBP transcription factors are not responsive.The necessity to produce egg-yolk lipids appears to negate the regulation of the SREBPs by dietary fatty acids in laying birds.
Taken together, although dietary algal DHA oil did not change the hepatic SREBP1 and SREBP2 mRNA in the laying ducks, the enrichment of DHA in the egg yolk will increase the value of the egg products for human consumption.

Figure 1 .
Figure1.The effect of dietary algal DHA supplementation on the abundance of SREBP1 (A) and SREBP2 mRNA (B) in laying Tsaiya ducks.Tsaiya ducks were fed diets supplemented with 0%, 0.5% or 2% algal docosahexaenoic acid (DHA) oil for 14 days (10 ducks per group).At the day of sampling, ducks were killed 2 h after feeding.The SREBP1 and 18S rRNA were determined by Northern analysis.The SREBP1 mRNA abundances were depicted relative to the control.The mRNA values were normalized to 18S rRNA content with n = 10 replicates/treatment.There was no treatment effect detected (p>0.05).

Table 2 .
Fatty acid composition 1 of experimental diets

Table 3 .
Effect of dietary algal DHA on DHA levels in the plasma of laying Tsaiya ducks*

Table 4 .
Effect of dietary algal DHA on DHA levels in the livers and skeletal muscles of laying Tsaiya ducks

Table 5 .
Effect of 7-d dietary algal DHA treatment on fatty acid compositions in the egg yolk of laying Tsaiya ducks*