Egg yolk omega-6 and omega-3 fatty acids modify tissue lipid components, antioxidant status, and ex vivo eicosanoid production in chick cardiac tissue

IMMUNOLOGY, HEALTH, AND DISEASE
Egg yolk omega-6 and omega-3 fatty acids modify tissue lipid components,
antioxidant status, and ex vivo eicosanoid production in chick cardiac tissue
J. Bautista-Ortega , D. E. Goeger , and G. Cherian 1 Department of Animal Sciences, Oregon State University, Corvallis 97331 ABSTRACT The effects of maternal n-6 and n-3 fatty higher in the tissues of medium and high n-3 chicks
acid (FA) supplementation on hatched chick tissue FA than in the tissue of low n-3 chicks (P < 0.05). There profile, antioxidant status, and ex vivo eicosanoid pro- was no effect of egg FA on docosahexaenoic acid (22:6n- duction by the cardiac tissue were investigated. Eggs 3) in the heart of low, medium, and high n-3 chicks (P with low, medium, and high levels of n-3 FA were ob- > 0.05). There were no differences in total glutathione, tained by feeding Cobb breeder hens were fed a corn- glutathione peroxidase, glutathione reductase, or super- soybean meal-based diet containing 3.5% sunflower oil oxide dismutase activities in the tissues of chicks from (low n-3), 1.75% sunflower oil plus 1.75% fish oil (me- low n-3, medium n-3, and high n-3 eggs (P > 0.05). dium n-3), or 3.5% fish oil (high n-3). Total n-3 FA The medium n-3 and high n-3 chicks had lower catalase in the yolk ranged from 1.8, 10.3, and 13.3% for low, activity in the heart than did the low n-3 chicks (P = medium, and high n-3 eggs, respectively (P < 0.001). 0.013). The TBA reactive substances were significantly Total long-chain (>20 C) n-6 FA in the egg yolk were lower in the liver of high n-3 chicks than in that of low 7.4, 2.1, and 1.3 for low n-3, medium n-3, and high n-3 and medium n-3 chicks (P < 0.05). Heart tissue pros- eggs, respectively (P < 0.001). No differences were ob- taglandin E2 concentration was higher in low n-3 chicks served in total fat content of the eggs, which was 33.3, than in those hatched from medium or high n-3 eggs (P 31.6, and 31.9% for low n-3, medium n-3, and high n-3 < 0.05). Heart tissue thromboxane A3 was lowest in low eggs, respectively (P > 0.05). Hatchability for the low, n-3 chicks (P < 0.05). There was no effect of yolk FA on medium, and high n-3 eggs was 89, 85, and 83%, respec- ex vivo prostaglandin E3 or thromboxane A2 production tively (P > 0.05). The total lipid content of chick liver, in cardiac tissue (P > 0.05). These results indicate that heart, brain, and lungs can be placed in the following modulating egg yolk n-3 FA enhances tissue n-3 FA and descending order: liver > brain > heart > lung and was reduces proinflammatory cardiac eicosanoid production not affected by egg FA (P > 0.05). Total n-3 FA were without affecting hatchability. Key words: egg , omega-3 fatty acid , antioxidant enzyme , thiobarbituric acid reactive substance , prostaglandin
2009 Poultry Science 88 :1167–1175 doi: 10.3382/ps.2009-00027 INTRODUCTION
C) n-6 and n-3 PUFA are the precursors of eicosanoids. Eicosanoids derived from n-6 FA are proinflammatory Egg yolk fatty acids (FA) are the major modifiable
factor known to influence the lipid and FA composition 2 (PGE2)] and prothrombotic (throm-
of the developing chick (Cherian and Sim, 1991; Cheri- 2) and those derived from n-3 FA are anti or less proinflammatory and less prothrombotic [pros- an et al., 1997). Among the different FA in egg, much work has been reported on yolk linoleic (18:2n-6) and 3 (PGE3) and thromboxane A3 (TXA3);
(Calder, 2006)]. Convincing evidence has been pub- α-linolenic (18:3n-3) due to their role in polyunsaturat- lished that shows that alterations in egg yolk n-6 and ed FA (PUFA) synthesis during embryogenesis. Arachi-
n-3 FA composition brought about by maternal dietary donic acid (20:4n-6) is the major n-6 PUFA derived from lipid source result in significant changes in tissue n-6 linoleic acid and eicosapentaenoic acid (EPA, 20:5n-3)
and n-3 PUFA content, liver desaturase enzyme activ- and docosapentaenoic acid (DPA, 22:5n-3) and doco-
ity, immune responses, and PUFA-derived eicosanoid sahexaenoic acid (DHA, 22:6n-3) are the major n-3
synthesis in progeny chicks (Cherian and Sim, 2001; PUFA derived from α-linolenic acid. Long-chain (>20 Liu and Denbow, 2001; Ajuyah et al., 2003a,b; Wang et al., 2004; Hall et al., 2007). These results suggest a 2009 Poultry Science Association Inc.
unique role of maternal (egg yolk) n-6 and n-3 FA in Received January 15, 2009.
modulating lipid and eicosanoid metabolism and im- Accepted February 18, 2009.
1 Corresponding author: [email protected] mune and inflammatory responses in the progeny. BAUTISTA-ORTEGA ET AL.
Altering the n-6 and n-3 FA content in egg yolk also the hatchability of fertile eggs and antioxidant status increases the degree of unsaturation leading to lipid of hatchlings.
peroxidation in tissue and it may compromise the an- tioxidant status of progeny. The bird's antioxidant MATERIALS AND METHODS
system includes enzymes (e.g., superoxide dismutase, glutathione peroxidase, glutathione reductase, and Egg Enrichment of n-6 and n-3 FA,
catalase) and molecules (e.g., glutathione, vitamin A Incubation, and Chick Tissue Collection
and E, and carotenoids) (Surai, 1999). Hatching time is considered to be a period of high oxidative stress due to A total of 270 eggs were collected from Cobb breeder long-chain PUFA accretion in tissues (Noble and Coc- hens (n = 24, 36 wk old) fed a corn-soybean diet con- chi, 1990; Cherian and Sim, 1992; Cherian et al., 1997), taining either 3.5% fish oil (high n-3), a mixture of exposure to atmospheric oxygen, onset of pulmonary 1.75% sunflower oil and 1.75% fish oil (medium n-3), respiration, and sudden increase in rate of oxidative or 3.5% sunflower oil (low n-3). The hens were fed the metabolism (Speake et al., 1998) and the hatchlings experimental diets for 8 wk before egg collection. The are expected to react with a compensatory induction breeder hen diets were isonitrogenous (16% CP) and of endogenous antioxidants. Tissue-specific differences isocaloric (2,866 kcal of ME). The FA composition of in the antioxidant system during avian embryogenesis the breeder hen diet is shown in Table 1. The eggs were and the degree of lipid peroxidation have been reported collected during a period of 7 d and kept in a cooler previously (Surai et al., 1996, 1999). However, there at 65°F (18.3°C). Eighteen eggs from each treatment is limited information regarding the effect of maternal were collected randomly and the yolks were separated. (egg yolk) n-3 and n-6 FA enrichment on antioxidant Three egg yolks were pooled to get a sample size of status and lipid peroxidation in progeny.
6 per treatment. An aliquot of each yolk sample pool Modern-day meat-type broiler chickens have fast was taken for total lipid and FA analysis. The remain- growth rates and high feed conversion ratios and meta- ing 252 eggs were incubated at a dry bulb and wet bolic rates. These features promote an increased work- bulb temperature of 37.5 and 29.4°C, respectively. At load on the cardiovascular system, predisposing birds 18 d of incubation, the eggs were candled and infertile to metabolic disorders such as right ventricular failure, eggs were removed and counted. The eggs were trans- ascites syndrome, cardiac arrhythmias, cardiopulmo- ferred to hatch baskets and the hatch was pulled at nary disorders, and sudden death (Olkowski and Clas- 21.5 d. Hatched chicks from all treatments were count- sen, 1998; Julian, 2005). A growing body of information ed and those eggs that did not hatch were removed supports the concept that inflammation is an important from the hatcher and were also counted. Eighteen predisposing risk factor contributing to cardiovascular newly hatched chicks were randomly chosen from each diseases such as heart failure and hypertension (Ghosh treatment group, blood was collected from the jugular et al., 2007). The role of dietary FA in modulating car- vein, and the chicks were killed. Chick tissue samples diac health through alteration in eicosanoid synthesis (heart, lung, brain, and liver) were quickly removed, in the heart has been reported in humans (Rana et snap-frozen with liquid nitrogen, and stored at −80°C al., 2007). Previously, we reported that thrombocytes until analysis. Tissue and blood samples (n = 3) from from chicks hatched to hens fed a high n-3 FA diet pro- each treatment were pooled to obtain a sample size of duced higher leukotriene B5 (less inflammatory) than 6 (n = 6) per treatment for analytical purposes. All did those from chicks hatched from hens fed a low n-3 protocols were approved by Oregon State University's FA diet (Hall et al., 2007). However, no information is Animal Care and Use Committee to ensure adherence available on the role of maternal (yolk) FA composition to the Animal Care Guidelines.
on eicosanoid production in the heart tissue of newly hatched chicks. In view of the roles of yolk FA in mod- Total Lipid and FA Analysis
ulating lipid metabolism in the progeny, the present study was designed to determine the effects of yolk n-6 Total lipids were extracted from feed, egg yolk, and and n-3 FA on antioxidant status, lipid peroxidation, chick tissues and plasma by the method of Folch et tissue PUFA profile (heart, lungs, liver, and brain), al. (1957). The mass of total lipid content was deter- and ex vivo eicosanoid generation in the cardiac tissue mined gravimetrically. Fatty acid methyl esters were of newly hatched chicks. These tissues were selected prepared as reported earlier (Cherian et al., 2002). due to their respective roles in lipid assimilation (liver), Fatty acid analysis was performed with an HP 6890 oxidation (heart), functional long-chain PUFA incor- gas chromatograph (Hewlett-Packard Co., Wilmington, poration (brain), and oxidative metabolism (lung). It DE) equipped with an autosampler, flame ionization is hypothesized that: 1) as n-3 PUFA in the maternal detector, and SP-2330 fused silica capillary column (30 source (yolk) is increased, n-6 PUFA accretion in prog- mm × 0.25 mm i.d.). Samples (1 μL) were injected with eny tissue is decreased with a concomitant reduction helium as a carrier gas onto the column programmed in n-6 PUFA-derived eicosanoids and 2) the increase for ramped oven temperatures (initial temperature was in n-3 PUFA in maternal source (yolk) will not affect 110°C, held for 1 min, then ramped at 15°C/min to YOLK OMEGA-3 FATTY ACIDS AND CARDIAC EICOSANOIDS Table 1. Fatty acid composition of the breeder hen diets
Fatty acid (% of total fatty acids) Linoleic (18:2n-6) α-Linolenic (18:3n-3) Arachidonic (20:4n-6) Total monounsaturated 1Low n-3, medium n-3, and high n-3 represent a corn-soybean meal-based maternal (breeder hen) diet supple- mented with 3.5% sunflower oil, a mixture of 1.75% sunflower oil and 1.75% fish oil, or 3.5% fish oil, respectively. In addition, the maternal diet contained corn (55.1%), wheat middling (5.8%), soybean meal (21%), alfalfa (5.0%), limestone (6.7%), calcium phosphate (1.9%), salt (0.5%), and premix (0.5%). The premix composition included (per kg of feed): vitamin A, 12,500 IU; vitamin D3, 4,000 IU; vitamin E, 25 IU; vitamin B12, 0.014 mg; riboflavin, 8 mg; pantothenic acid, 12 mg; niacin, 40 mg; menadione, 2.5 mg; choline, 500 mg; thiamine, 1.75 mg; folic acid, 0.75 mg; pyridoxine, 2 mg; d-biotin, 0.15 mg; and ethoxyquin, 2.5 mg.
190°C and held for 55 min, then ramped at 5°C/min to 230°C and held for 5 min). Inlet and detector tem- peratures were both 220°C. Fatty acid methyl esters Blood samples were homogenized in 1% picric acid were identified by comparison with retention times of and then centrifuged at 12,600 × g for 2 min. Total glu- authentic standards (Nuchek Prep, Elysian, MN). Peak tathione was determined according to Griffith (1985). areas and percentages were calculated using Hewlett- Briefly, 0.7 mL of 0.3 mM NAD phosphate (NADPH)
Packard ChemStation software (Agilent Technologies buffer, 0.1 mL of 6 M 5,5′-dithio-bis (2-nitrobenzoic Inc., Wilmington, DE). Fatty acid values were reported acid) solution, and 0.15 mL of supernatant (diluted as percentages.
1:20 with distilled water) were mixed and the absor- bance was measured at 412 nm. A standard curve was made by substituting the supernatant for 1, 2, 3, and 4 TBA Reactive Substances
nmol of glutathione, and appropriate amounts of water and total glutathione concentration in the sample were Lipid peroxidation in tissues was measured as TBA calculated and expressed in micromoles per gram of reactive substances (TBARS) expressed in malondial-
dehyde equivalents as described by Cherian et al. (2007). Briefly, 2 g of tissues were minced and weighed into 50-mL test tubes, and 18 mL of 3.86% perchloric acid and butylated hydroxytoluene (50 μL in 4.5% ethanol) Glutathione peroxidase activity was assayed using the was added, after which the samples were homogenized. method of Paglia and Valentine (1967). Briefly, 1.2 mL The homogenate was filtered and the filtrate was mixed of a cofactor solution (0.25 mM NADPH, 0.5 U/mL of with 20 mM TBA in distilled water and incubated in glutathione reductase, and 1.25 mM glutathione) and the dark at room temperature for 17 h. Absorbance was 0.20 mL of collected supernatant were mixed. The reac- determined at 531 nm. The TBARS were expressed as tion was started by the addition of 0.1 mL of cumene milligrams of malondialdehyde per gram of sample.
hydroxide. The change in absorbance at 340 nm was measured. The supernatant of brain tissue was used undiluted, whereas that of heart, liver, and lung was diluted 1:10 with PBS. Glutathione peroxidase activity Antioxidant concentration (total glutathione) and was expressed as units per gram of protein. One unit activities of glutathione peroxidase, glutathione re- of glutathione peroxidase was defined as micromoles of ductase, superoxide dismutase, and catalase were mea- NADPH oxidized per minute.
sured in liver, heart, lung, and brain tissue. About 0.5 g of tissues was homogenized in 5 volumes of ice-cold PBS for 30 s. After centrifugation at 10, 000 × g for 30 min at 4°C, the supernatant was collected and stored at Glutathione reductase activity was determined using −80°C until assay. In the case of blood, only total glu- the method of Goldberg and Spooner (1983) as follows. tathione was determined and was done on fresh blood Briefly, 1 mL of phosphate-EDTA buffer (0.11 M po- samples collected.
tassium and 0.55mM EDTA buffer, pH 7.2), 0.04 mL BAUTISTA-ORTEGA ET AL.
of oxidized glutathione, and 0.04 mL of undiluted su- added and the mixture was centrifuged at 1,500 × g for pernatant were mixed and the reaction was started by 10 min and the supernatant was applied to a 3-mL C18 adding 0.02 mL of a cofactor solution (9.3 mM NADPH solid phase extraction (SPE) column previously acti-
in 1% sodium bicarbonate). The change in absorbance vated with 3 mL of methanol and equilibrated with 3 at 340 nm was documented. Glutathione reductase ac- mL of 15% methanol in 20 mM acetic acid. The column tivity values were expressed as units of activity (1 unit was washed with 3 mL of 15% methanol in 20 mM ace- of activity = 1 μmol of NADPH oxidized per minute) tic acid followed by another wash with 3 mL of water. per gram of protein.
The sample was eluted with 2 mL of methanol and evaporated under a stream of nitrogen, and the residue was dissolved in 1 mL of methanol.
Superoxide dismutase was assayed using the method of Paoletti and Mocali (1990). In short, 0.8 mL of tri- ethanolamine-diethanolamine-HCl buffer (0.1 M each), The eicosanoids were separated by HPLC (Shimadzu 0.04 mL of NADH (7.5 mM), 0.025 EDTA-MnCl2 (0.1 LC-2010AHT, Shimadzu Corp., Kyoto, Japan) using a M EDTA and 50 mM MnCl2), and 0.1 mL of super- 25 cm × 4.6 mm 5 μM C18 SPE column at 40°C and natant were mixed and the reaction was started with a 1 mL/min isocratic mobile phase of 70% A (78.6% addition of 0.1 mL of mercaptoethanol (10 mM). The 1 mM acetic acid and 21.4% methanol) and 30% B linear change in absorbance at 340 nm was measured. (acetonitrile). An appropriate aliquot of the SPE elute Superoxide dismutase activity was expressed as units was dried, dissolved in mobile phase A, filtered, and per gram of protein.
injected on the HPLC. Fractions containing the desired eicosanoids, determined previously by elution times of authentic standards, were collected and extracted with 2 mL of ethyl acetate. The extract was centrifuged at Catalase activity was determined using the method of 1,500 × g for 10 min, after which the organic phase was Aebi (1990). About 0.1 mL of supernatant was placed collected and dried under nitrogen, and the residue was in a 15 × 75 mm test tube held on ice and the reaction dissolved in 1 mL of ethanol. An appropriate volume of was started sequentially at 15-s intervals by adding 1 ethanol fraction was dried and dissolved in PBS con- mL of ice-cold 6 mM H2O2. After 3 min, the reaction taining 1% BSA and quantitated by ELISA.
was stopped in the same order in which it was started, by adding 0.2 mL of 6 N H2SO4. After adding 1.4 mL of 0.1 M KMnO Eicosanoid Quantification by ELISA
4 to each tube, the change in absorbance at 480 nm was recorded. A standard was prepared by The eicosanoids were quantitated by ELISA using adding 1.4 mL of 0.1 KMnO4, 1.1 mL of 0.01 M potas- a procedure by Krämer et al. (1993). In short, high- sium phosphate buffer (pH 7), and 0.2 mL of 6 N H2SO4 binding 96-well enzyme immunoassay-RIA plates were to a test tube. Catalase activity was expressed in units coated with either PGE2 or thromboxane B2 (TXB2)
per milligram of protein.
that had been conjugated with BSA by the carbodiim- ide method of Dray et al. (1982). The eicosanoid-BSA Tissue Protein Determination
conjugates diluted in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6), 1.25 and 0.125 μL/mL Total protein was determined as described by Lowry for PGE2 and TXB2 conjugate, respectively, were added et al. (1951) using Folin reagent. The protein content at 0.2 mL/well to 96-well plates and incubated at 37°C was expressed as milligrams of protein per milliliter of for 2 h. The plates were washed 3 times with 0.3 mL/ well of PBS containing 0.1% Tween 20 (PBS-T) and
then blocked with 0.25 mL of PBS-BSA/well by incu- Cardiac Tissue Preparation
bating for 2 h at room temperature. After the plates for Eicosanoid Extraction
were washed 3 times with PBS-T, PGE2 and PGE3 samples in PBS-BSA were added at 0.1 mL/well to Eicosanoids were extracted from heart tissue using PGE2-BSA-coated plates and TXB2 and TXB3 sam- a modification of the method by Powell (1980) and ples to TXB2-BSA-coated plates. Anti-PGE2 (20 μL/ Murphy et al. (1999). About 0.5 g of heart tissue was mL) and anti-TXB2 (50 μL/mL) antibodies (Cayman minced and homogenized in 2 mL of PBS diluted 1:10 Chemical Co.) in PBS-BSA were added at 0.1 mL/ containing 1 mM EDTA and 10 μM indomethacin and well to the prostaglandin and thromboxane plates, was incubated at 37°C for 45 min on a shaking water respectively, followed by overnight incubation at 4°C. bath, after which the tubes were cooled on ice for 5 The plates were then washed as before and 0.2 mL of min. Three milliliters of methanol was added to the PBS-BSA containing 0.5 μL/mL of either anti-mouse homogenate and mixed. After incubation at room tem- or anti-rabbit IgG biotin conjugates was added to the perature for 5 min, 15 mL of 20 mM acetic acid was prostaglandin or thromboxane plates, respectively, fol- YOLK OMEGA-3 FATTY ACIDS AND CARDIAC EICOSANOIDS lowed by incubation for 2 h at room temperature on a There was no association between dietary treatment rotary shaker. After washing, 0.2 mL of 1 μL of extra- and hatchability (P = 0.l4).
vidin peroxidase conjugate/mL of PBS was added to the wells of both plates followed by incubation for 2 h Chick Tissue Total Lipids and FA
at room temperature on a rotary shaker. After washing with 0.3 mL of PBS-T/well 4 times, the plates were The total lipid and PUFA content in the tissues of stained by incubating with 0.2 mL/well of 2,2′-azino- newly hatched chicks is shown in Table 3. Yolk PUFA bis(3-ethylbenzthiazoline-6-sulphonic acid) (0.66 mg/ composition had no significant effect on the content of mL) and H2O2 (0.33 μL/mL) in 42 mM citric acid and total lipids in the tissues of chicks hatched from low, 58 mM Na2HPO4, pH 4.2, overnight at 4°C. The plates medium, or high n-3 eggs. The total lipid content of were read at 405 nm on a plate reader and unknowns tissues can be placed in the following descending or- quantitated against authentic standards.
der: liver > brain > heart > lung. However, the PUFA composition of tissues was significantly altered by the maternal (yolk) lipid source. The yolk content of n-3 PUFA (EPA, DPA, DHA) was associated with a sig- The effect of high, medium, and low n-3 maternal nificant increase in total n-3 PUFA (EPA+ DPA + lipid source (yolk) on chick tissue total lipids, PUFA DHA) in the heart, lung, liver, and brain. It is impor- content, antioxidant status, TBARS, and ex vivo ei- tant to emphasize that the amounts of DHA (22:6n-3) cosanoid production were analyzed by 1-way ANOVA were similar in the hearts of low, medium, and high using S-PLUS (MathSoft, 1988). Results were present- n-3 chicks. The increase in n-3 PUFA in the tissues ed as mean ± SE. Percentage data underwent angular of medium and high n-3 chicks was associated with transformation (arc sine square root percentage trans- a concomitant decrease in arachidonic acid (20:4n-6) formation) before analysis. Means were compared us- and other long-chain n-6 FA such as 22:4n-6 and 22:5n- ing the Tukey method for multiple comparisons. Values 6. Arachidonic acid (20:4n-6) constituted the major were considered significant at P < 0.05.
PUFA in the heart, lung, and liver. Docosahexaenoic acid (22:6n-3) was the major PUFA in the chick brain and was significantly higher (P < 0.05) in the brain tissue of high n-3 and medium n-3 chicks than that of Egg Yolk FA and Hatchability
the low n-3 chicks.
The FA composition of maternal diet is shown in Effect of Maternal Diet on Antioxidant
Table 1. Egg PUFA composition reflected the dietary Status of Hatched Chicks
source (Table 2). Docosahexaenoic acid (22:6n-3) was the major long-chain n-3 FA in the yolk and ranged Total Glutathione. There were no significant differ-
from 1.5, 8.8, and 10.7% for low n-3, medium n-3, and ences in total glutathione concentration in blood and high n-3 eggs, respectively (P < 0.001). The predomi- tissues (P > 0.05; data not shown). In terms of total nant n-6 long-chain PUFA in egg yolk was arachidonic glutathione concentration, chick tissues can be placed acid (20:4n-6), constituting 4.6, 2.1, and 1.3 for low in the following descending order: blood > brain > liver n-3, medium n-3, and high n-3 eggs, respectively. Oth- > heart > lung.
er long-chain PUFA such as EPA (20:5n-3) and DPA Glutathione Peroxidase, Glutathione Reductase,
(22:5n-3) were found in medium and high n-3 egg yolks. Superoxide Dismutase, and Catalase. There was
No differences were observed among the 3 diets in total no difference in glutathione peroxidase, glutathione fat content of eggs, which was 33.3, 31.6, and 31.9% for reductase, and superoxide dismutase activities in the low n-3, medium n-3, and high n-3 eggs, respectively tissues of chicks from low n-3, medium n-3, and high (P > 0.05). Hatchability for the low n-3, medium n-3, n-3 eggs. However, there was a significant difference in and high n-3 groups was 89, 85, and 83%, respectively. heart catalase activity between treatments. The cata- Table 2. Fatty acid composition of eggs from broiler breeder hens fed low, medium, or high n-3 oils
a–cFor each fatty acid, values with different superscripts are significantly different (P < 0.05).
1Low n-3, medium n-3, and high n-3 represent a corn-soybean meal-based maternal (breeder hen) diet supplemented with 3.5% sunflower oil, a mixture of 1.75% sunflower oil and 1.75% fish oil, or 3.5% fish oil, respectively. Values are means of 6 observations (n = 6).
BAUTISTA-ORTEGA ET AL.
lase activity in the heart was 0.38, 0.16, and 0.16 U/ Heart Tissue Ex Vivo Eicosanoid Production
mg of protein for low, medium, and high n-3 chicks (P = 0.013). Catalase activity was not detectable in brain Heart tissue PGE2 concentration was significantly higher in low n-3 chicks than in those hatched from medium or high n-3 eggs (P < 0.05; Figure 1). Heart tissue TXA3 was lowest in low n-3 chicks (P < 0.05). Lipid Peroxidation in Chick Tissues
There was no effect of yolk PUFA composition on ex vivo PGE3 or thromboxane A2 production in cardiac Thiobarbituric acid reactive substances were signifi- tissue (P > 0.05).
cantly lower in the liver tissue of high n-3 chicks than in the liver of those hatched from hens fed the medium n-3 diet (P < 0.05), but not significantly different from hens fed the low n-3 diet. The liver TBARS were 0.64, The effect of maternal (yolk) n-6 and n-3 FA levels on 0.85, and 0.57 mg of malondialdehyde/g for low, me- the tissue PUFA profile, antioxidant status, lipid per- dium, and high n-3, respectively (P < 0.05). There were oxidation, and ex vivo eicosanoid production by cardiac no significant differences in heart or lung TBARS levels tissue in progeny is investigated. As reported earlier, among treatments (data not shown). Overall, lung tis- egg n-6 and n-3 FA composition reflected the dietary sue was more prone to lipid peroxidation followed by source (Cherian and Sim, 1991; Cherian et al., 1997). heart and liver tissue.
No effect of yolk n-6 and n-3 FA on hatchability was no- Table 3. Total lipids and polyunsaturated fatty acid (PUFA) content in tissues of chicks hatched
from eggs containing low, medium, or high n-3 fatty acids Lipids/fatty acid (%) a,bFor each fatty acid, values with different superscripts are significantly different (P < 0.05).
1Low n-3, medium n-3, and high n-3 represent a corn-soybean meal-based maternal (breeder hen) diet supple- mented with 3.5% sunflower oil, a mixture of 1.75% sunflower oil and 1.75% fish oil, or 3.5% fish oil, respectively. Values are means ± SE (n = 6).
YOLK OMEGA-3 FATTY ACIDS AND CARDIAC EICOSANOIDS ticed in the current study, which corroborates previous research (Cherian, 2008). However, Pappas et al. (2006) reported increased embryonic mortality in eggs laid by hens fed a 5.5% fish oil diet compared with hens fed diets with the same amount of soybean oil. In the cur- rent study, a 3.5% inclusion of fish oil was used, which may explain discrepancies with the work by Pappas et al. (2006). The egg yolk n-6 and n-3 FA composition did not affect total lipid content of heart, lung, liver, and brain of newly hatched chicks. However, alterations in n-3 and n-6 FA in the maternal reserves (yolk) led to significant changes in the tissue n-6 and n-3 PUFA status of chick liver, brain, and heart. This is not sur- prising because, from a nutritional standpoint, yolk FA are the major source of long-chain C20 and C22 PUFA to the developing chick and are incorporated into the tissue phospholipids serving as structural components. Changes in the tissue n-6 and n-3 PUFA composition brought about by maternal source as reported in the current study are in accordance with previous studies on the tissue FA composition of broiler chicks hatched from eggs varying in n-3 and n-6 FA (Cherian and Sim, 1991; Ajuyah et al., 2003a,b). The availability of yolk EPA (20:5n-3) and DPA (22:5n-3) is evidenced by the significant increase in EPA (20:5n-3) and DPA in the Figure 1. Ex vivo prostaglandin E2 (PGE2) and thromboxane A3
heart of medium and high n-3 chicks. However, the in- (TXA3) production in the cardiac homogenate of day-old chicks from broiler breeder hens fed low n-3, medium n-3, and high n-3 diets. Low crease in EPA (20:5n-3) and DPA (22:5n-3) did not n-3, medium n-3, and high n-3 represent a basal corn-soybean breeder lead to any change in the DHA (22:6n-3), suggesting hen diet supplemented with 3.5% sunflower oil, a mixture of 1.75% limited Δ4-desaturase in chick heart. Although there sunflower oil and 1.75% fish oil, or 3.5% fish oil, respectively. Values are means of 6 observations (n = 6). a,bIndicate significant differences was no effect of yolk n-3 FA on DHA content of chick (P < 0.05).
heart, as a percentage of total n-3 long-chain PUFA, DHA (22:6n-3) was the major FA in the cardiac tissue of day-old chicks in all the treatments, constituting 100, donic acid (20:4n-6) was for PGE2 production. Arachi- 74.1, and 67% in low, medium, and high n-3 chicks, donic acid (20:4n-6) and EPA (20:5n-3) are esterified at respectively, suggesting the importance of DHA in car- the sn-2 position of phospholipids and after release by diac cell membrane lipids.
phospholipase A2 are further metabolized by cyclooxy- The amount and type of long-chain PUFA released in genases and lipooxygenase to different eicosanoids. In response to inflammatory stimuli depend on cell mem- the cardiovascular system, arachidonic acid-derived ei- brane phospholipid PUFA content. The availability of cosanoids are responsible for smooth muscle constric- arachidonic acid (20:4n-6) and EPA (20:5n-3) in cardi- tion, platelet aggregation, and decreasing thrombus for- ac tissue of the chick is evidenced by changes in the ex mation. The alterations of n-6 and n-3 PUFA-derived vivo eicosanoid production observed. The higher levels eicosanoids observed in the current study demonstrate of unesterified arachidonic acid in the low n-3 chicks that the newly hatched chick has already developed a led to a significant increase in ex vivo PGE2 production highly active phospholipase A2 system capable of cata- by the cardiac homogenate. The substantial decrease lyzing eicosanoid formation. The role of EPA (20:5n- in PGE2 biosynthesis in the cardiac homogenate of me- 3) and DHA (22:6n-3) in reducing the beating rate of dium and high n-3 chicks was consistent with the con- cardiac myocytes exposed to arrhythmogenic agents in centrations of arachidonic acid (20:4n-6), the precursor vitro (Kang and Leaf, 1996) and in providing protection of PGE2, which was over 22 and 36% less in the cardiac against ischemia-induced arrhythmia in pigs has been tissues of medium and high n-3 chicks when compared reported (Nair et al., 1997). Thus, the high cardiac con- with that of low n-3 chicks. Similarly, the higher levels centration of arachidonic acid (20:4n-6) and absence of EPA in the heart lipids of medium and high n-3 of EPA (20:5n-3) in low n-3 chicks could contribute chicks may explain the increase in ex vivo TXA3 pro- to a proinflammatory, proarrhythmic, and prothrom- duction observed in the current study. Although EPA botic state. Previous studies have shown that addition (20:5n-3) was higher in the cardiac tissue of medium of fish oil (rich n-3 FA source) to the maternal diet led and high n-3 chicks, no difference was observed in ex to a significant reduction in ex vivo PGE2 production vivo PGE3 production among the 3 treatments. Raisz by tibiae in Japanese quail (Liu and Denbow, 2001) et al. (1989) have reported that EPA (20:5n-3) was only and in leukotriene B4 by thrombocytes in chickens (Hall one-tenth as effective for PGE3 formation as arachi- et al., 2007). However, to our knowledge, the effect of BAUTISTA-ORTEGA ET AL.
maternal diet on cardiac eicosanoid production in the chicks. Considering the role of n-6 PUFA-derived eico- progeny of avians has not been reported. Considering sanoids in the pathobiology of various disease condi- the role of inflammation as an important risk factor tions, results from the present study and those reported contributing to cardiovascular diseases (Ghosh et al., earlier (Hall et al., 2007) on the role of maternal diet in 2007; Rana et al., 2007), the role of maternal diet in modulating eicosanoid production in the hatched chick modulating eicosanoid formation and progeny cardiac may have practical applications. Under commercial health needs to be investigated.
conditions, mortality during the first 2 wk of growth is In the present study, total glutathione and the anti- around 5% and this remains a problem for the broiler oxidant activities of enzymes such as glutathione perox- industry. In addition, metabolic disorders and heart- idase, glutathione reductase, and superoxide dismutase related conditions are the major cause of mortalities in the newly hatched chick were not affected by yolk and morbidities in fast-growing broiler birds and have PUFA reserves. At hatching, tissue phospholipids con- been reported in birds as early as 3 d of age (Gardiner tain large amounts of PUFA (Cherian and Sim, 1993; et al., 1988). Therefore, the amounts and balance of n-6 Noble and Speake, 1997). In the present study, tissue and n-3 PUFA in the maternal diet and yolk reserves total PUFA was either similar or lower in medium n-3 will affect the eicosanoid precursor pool and thereby and high n-3 as compared with low n-3 chicks. In the modulate eicosanoid-controlled functions, with effects lung tissue, there were no differences in percentage of on cardiovascular disease and metabolic disorders in total PUFA, but in the brain, heart, and liver tissues, broiler birds.
total n-6 + n-3 PUFA was significantly lower in the high n-3 as opposed to low n-3 chicks. Therefore, it is possible that the high levels of n-3 PUFA in medium and high n-3 chicks were not capable of producing dif- ferences in the antioxidant enzymatic activities in the This study was supported in part by the National tissues studied. However, maternal n-3 PUFA led to Research Initiative of the USDA Cooperative State Re- a decreased catalase activity in the heart of high n-3 search, Education and Extension Service, grant number and medium n-3 chicks compared with low n-3 chicks. 2004-35204-14654, and Oregon State University Exper- Normally, catalase does the same job as glutathione iment Station Hatch Project. The generous donation peroxidase in destroying H of menhaden oil from Omega Protein Inc. (Reedville, 2O2 produced during cell metabolism, once superoxide dismutase has converted VA) is appreciated. The assistance of Irene Pilgrim and Mare Goeger of the Department of Animal Sciences, 2-derived toxic molecules into H2O2. Catalase activ- ity was moderately correlated to the long-chain n-6:n-3 Oregon State University, for care and management of FA ratio (r = 0.61) in the heart. It may be that as the breeder hens, analytical aspects, and egg quality mea- long-chain n-6:n-3 FA ratio increases, catalase activity surements, is acknowledged.
decreases in the heart, suggesting an increased need for antioxidants. Inclusion of vitamin E in the hen diet has been reported to enhance catalase activity in the liver of chicks (Lin et al., 2005). These authors suggested Aebi, H. 1990. Catalase in vitro. Pages 121–127 in Methods of En- that elevation of catalase activity in chicks could be zymatic Analysis. H. U. Bergmeyer, J. Bermeyer, and M. Bral, an indication of increased antioxidant protection of the ed. Vol. III. Enzymes 1: Oxidoreductases, Transferases. Verlag Chemie, Weinheim, Germany.
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