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Long-Term Feeding of Conjugated Linoleic Acid and Fish Oil to Laying Hens: Effects on Hepatic Histopathology, Egg Quality, and Lipid Components
©2007 Poultry Science Association, Inc.
Long-Term Feeding of Conjugated Linoleic Acid and Fish Oil to Laying Hens: Effects on Hepatic Histopathology, Egg Quality, and Lipid Components G. Cherian,1 D. Gonzalez, K. S. Ryu,2 and M. P. Goeger Department of Animal Sciences, Oregon State University, Corvallis 97331
SUMMARY The present study was conducted to investigate the effect of long-term feeding of conjugated linoleic acid (CLA) and fish oil on egg quality characteristics, production performance, liver pathology, and egg fatty acid content of laying hens. Single Comb White Leghorn laying hens (n = 112), 21 wk old, were placed in cages and randomly assigned to 4 diets (28 hens/diet, 4 replicates of 7 hens) containing 3.0% yellow grease (control), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), and 2.75% yellow grease + 0.25% fish oil (YG-FO). The experimental diets were fed for 12 mo. Eggs were collected daily for 12 mo. Feed consumption, hen-day egg production, and feed efficiency were monitored. At the end of the trial, hepatic tissue was collected for histopathology. No effect of diet was found on feed consumption, hen-day egg production, feed efficiency, egg weight, yolk weight, shell weight, or Haugh unit. The YG-CLA and YG-CLA-FO diets produced an increase in CLA and saturated fatty acids in the egg and liver tissue with a concomitant reduction in monounsaturated fatty acids (P < 0.05). Feeding YG-CLA-FO and YG-FO increased the n-3 fatty acids in egg yolk and liver of hens (P < 0.05). No difference was observed in the number of fat vacuoles in the liver tissue. The total fat content of hepatic and abdominal fat pads did not differ among treatments (P > 0.05). Regardless of the diet, as the hens aged, egg weight, yolk weight, and egg total fat increased, and shell weight decreased (P < 0.05). These data demonstrate that eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications without affecting the production performance or health of birds. Key words: egg, conjugated linoleic acid, n-3 fatty acid 2007 J. Appl. Poult. Res. 16:420–428
DESCRIPTION OF PROBLEM Conjugated linoleic acids (CLA) and longchain n-3 fatty acids (n-3 FA, >20-carbon) have received considerable attention during the past decade for their beneficial health effects. These 1
include the following: reduction of body fat, inhibition of carcinogenesis, modulation of immune system and eicosanoid synthesis, prevention of atherosclerosis, and minimizing the progression of cardiovascular diseases in animal models [1, 2, 3, 4]. Human intake of CLA is
Corresponding author: [email protected] Present address: Department of Animal Resources and Biotechnology, College of Agriculture and Life Science, Chonbuk National University, Chonju, Korea, 561-756.
2
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Primary Audience: Nutritionists, Researchers, Feed Formulators, Egg Producers
CHERIAN ET AL.: CONJUGATED LINOLEIC ACID AND FISH OIL
[14]. Similarly, a higher incidence of hepatic hemorrhage and lipidosis has been reported in hens fed flax or fish oil [15, 16]. Consequently, the objective of this study was to determine bird performance during the peak production period, egg quality characteristics, yolk lipid content, fatty acid incorporation, and metabolism over an entire laying cycle (12 mo) in hens fed diets containing low levels of CLA and n-3 PUFA along with yellow grease. In addition, hepatic histopathology and total fat content of hepatic tissue and abdominal fat pads of the birds fed the different lipid sources were assessed. Feeding high-fat (5 to 10%) diets to layer birds has been shown to cause an increase in egg mass output and feed efficiency [17]. In the current study, fat sources constituted 3% of the experimental diet. However, the information on hen performance as measured by feed intake, egg mass, egg production, and feed conversion was measured only during a short period of laying cycle (27 to 31 wk). The information on egg quality characteristics (egg weight, yolk weight, shell weight, yolk color, Haugh unit) was measured throughout the laying cycle. Low levels of CLA were used in the current study to minimize the adverse textural effects on egg yolk [18] and hepatic lipidosis [14] associated with feeding a high dietary concentration of CLA to hens.
MATERIALS AND METHODS These experiments were reviewed by the Oregon State University Animal Care Committee to ensure adherence to Animal Care Guidelines. Birds, Diets, and Housing One hundred twenty Hy-Line strain, Single Comb White Leghorn pullets [19] were reared in floor pens to 18 wk of age. The diets were provided as follows: commercial chick starter from 0 to 6 wk, grower from 16 to 21 wk, and the experimental layer from 21 wk until the end of the experiment (73 wk). At 18 wk, 112 hens were moved to individual laying cages in a room maintained at 20°C, and a step-up lighting schedule was used. Hens received 15L:9D from 18 until 21 wk of age and 16L:8D beginning at 21 wk of age and maintained until the end of the trial. Hens were randomly assigned to 4 diets
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predominantly from ruminant-derived foods, and long-chain n-3 FA is provided by marinederived foods. Current intake of CLA is estimated to be several hundred milligrams per day [5], and long-chain n-3 FA are estimated to be 0.2 g/d [6]. Based on animal data, it is estimated that approximately 3 g/d of CLA would be required to produce beneficial effects in humans [7]. Although no official dietary recommendation has been made in the United States, nutritional scientists suggest inclusion of 0.65 g/d of long-chain n-3 FA. Therefore, an additional 4to 5-fold increase in the supply of CLA and n3 FA is needed in the current US diet. Ruminantderived foods and marine products are the primary sources of CLA and long-chain n-3 FA. However, consumer preference for lean beef cuts, low-fat dairy foods, seasonal availability, and cost of marine foods limits the intake of CLA and n-3 FA in the western diet. Therefore, manipulating CLA and n-3 FA in other animal food lipids offers potential for developing health-enhancing value-added animal products. During the past decade, several different feeding strategies have been adopted by poultry scientists to modify the n-3 FA and CLA content of chicken eggs [8, 9]. Feeding synthetic CLA oil, flax, or marine oil has been the common method to increase the content of CLA or n-3 polyunsaturated FA (PUFA) in eggs [9, 10]. However, success of egg lipid modification strategies will depend upon maintaining egg quality characteristics, production performance, and health of hens during the entire laying cycle. Most of the reported studies on feeding CLA to laying hens were short-term feeding trials (under 6 wk) with small bird numbers varying in age and production cycle except for those reported by Jones et al. [11], Scha¨fer et al. [12], and Suksombat et al. [13]. However, the results reported by these authors were not consistent due to the level of CLA fed, strain of birds used, and the duration of the study. Little or no emphasis was given to bird health in these long-term trials. A combined incorporation of CLA along with n-3 PUFA into egg yolk and its effect on egg quality aspects and bird health have not been addressed. We have reported previously that feeding CLA (0.5% of diet) to hens for 6 wk can lead to an enhancement of hepatic lipid infiltration
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422 Table 1. Composition of experimental diets Ingredients
%
Corn Wheat middling Wheat grain Soybean meal Limestone Dicalcium phosphate Salt Oil1 Vitamin-mineral premix2
38.5 29.5 10.0 9.0 6.5 2.5 0.5 3.0 0.5 2,900 16.0 3.8 0.45 0.74 0.89 0.20
1
Oil sources include yellow grease, conjugated linoleic acid, and fish oil. Supplied the following per kilogram 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 g.
Sample Collection
(28 hens/diet), and the treatments were replicated 4 times with 7 birds per replicate. Water and feed were provided ad libitum. The diets were corn-soybean meal-based and contained 3.0% yellow grease (YG; control), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), and 2.75% yellow grease + 0.25% fish oil (YG-FO). The composition of the basal diet is shown in Table 1, and the fatty acid composition of the diets is shown in Table 2. Menhaden fish oil was used as a source of n-3 fatty acid. The CLA oil was made up of equal amounts of cis-9,trans-11 and cis-12,trans-10 CLA isomers. Yellow grease (restaurant grease) is commonly used in commercial poultry feeding and was used for the basal diet. The diets were mixed weekly and stored in a cold room (4°C) in air-tight containers. The experimental diets were fed for a period of 12 mo.
Hepatic Histopathology and Criteria for Fat Scoring
2
Production Parameters and Egg Quality Characteristics Production performance (egg production, feed intake, feed conversion, egg mass) was
After 352 d of feeding the experimental diets, 8 birds per treatment (n = 8, two per replicate) were randomly selected and euthanized by exsanguination. Liver and abdominal fat pads were collected.
A slice of liver about 1 × 1 × 0.5 cm thick was taken from the right lobe of each hen, fixed in 10% neutral buffered formalin for 48 h, embedded in paraffin, sectioned (8 m), and stained with hematoxylin and eosin stain before microscopic examination by a veterinary pathologist at the Oregon State University Veterinary Hospital. For each section of liver, 3 randomly located areas were graded at 40× magnification using a grid within a 10× ocular piece. Fat content of each grid was assessed in 2 patterns: a) total number of fat vacuoles in the grid and b) number of cells within the grid having >75% lipid vacuolation of cytoplasm. For each method, the sum total from each bird was divided by 3 to give an average value per grid [14]. The 2 methods were used, because it is common for lipid-laden cells to be largely occupied by only 1 or 2 vacuoles in severe fatty metamorphosis. A single section from each bird was assessed in this manner. A final average for each group was then calculated. The remaining liver tissue was kept frozen (−20°C) for further lipid analysis.
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Calculated composition ME (kcal/kg) CP (%) Ca Available P TSAA Lys Na
measured during the peak production period (27 to 31 wk of hen age). Egg production was expressed as average hen-day production, calculated from the total eggs divided by the number of days. For egg quality characteristics, 8 eggs from each treatment (n = 8, two per replicate) were collected every 4 wk until the end of the trial (12 mo). The eggs were weighed, and yolks were separated using an egg separator and weighed. Albumen weight was calculated by subtracting yolk and shell weight from total egg weight. Albumen height was also documented, and Haugh unit was calculated. Yolk color was determined by comparing yolk color to the Roche color fan. Egg total lipid and fatty acid composition were determined at every 8-wk interval.
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Table 2. Major fatty acid composition of experimental diets1 Dietary treatments2 Fatty acids3 (%)
YG-CLA
YG-CLA-FO
YG-FO
18.20 0.62 4.92 34.46 37.74 3.09 0.31 0.20 0.00 0.00 0.00 23.67 35.50 3.09 0.00
18.65 0.61 4.93 32.14 37.80 3.09 0.39 0.18 0.99 0.72 0.00 24.47 32.93 3.09 1.71
18.46 0.99 4.94 32.71 36.13 2.91 0.41 0.47 0.77 0.68 0.37 23.81 34.81 3.79 1.46
19.94 0.90 4.97 31.35 36.38 3.37 0.40 0.00 0.00 0.00 0.37 26.83 33.05 3.74 0.00
1
n = 6. YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 conjugated linoleic acid (CLA), 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 3 SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; total n-3 PUFA = n-3 polyunsaturated fatty acids. 2
Total Lipid and Fatty Acid Analysis Total lipids were extracted from feed, liver, and abdominal fat pads and egg yolk by the method of Folch et al. [20]. One gram of sample was weighed into a screw-capped test tube with 18 mL of chloroform:methanol (2:1, vol/vol) and homogenized with a polytron [21] for 10 to 15 s at high speed. After an overnight incubation at 4°C, the homogenate was filtered through Whatman number 1 filter paper into a 100-mL graduated cylinder, and 4 mL of 0.88% sodium chloride solution was added and mixed. After phase separation, the volume of lipid layer was recorded, and the top layer was completely siphoned off. Total lipids were determined gravimetrically. Analysis of fatty acid composition was performed with an Agilent 6890 gas chromatograph [22] equipped with an autosampler, flame ionization detector, and fused silica capillary column, 100 m × 0.25 mm × 0.2 m film thickness [23]. Sample (1 L) was injected with He as a carrier gas onto the column programmed for ramped oven temperatures (initial temperature was 110°C, held for 1 min, then ramped at 150°C/ min to 190°C and held for 55 min, then ramped at 5°C/min to 230°C and held for 5 min). Inlet and detector temperatures were both 220°C.
Peak areas and fatty acid percentages were calculated using Agilent ChemStation software. Fatty acid methyl esters were identified by comparison with retention times of authentic standards [24]. Fatty acid values and total lipids are expressed as milligrams per 100 g of fatty acids. Statistical Analysis A 2-way ANOVA was used to compare different egg quality aspects and egg lipid and fatty acid composition between dietary treatment and months. The diet and months were the fixed effects. Significant differences among treatment means were analyzed by Student-NewmanKeuls multiple range test and significance was set at P < 0.05 [25]. Means of interaction were analyzed by comparing the feeding period (mo) separately for each diet. The effect of diet on tissue total lipids, hepatic fatty acids, and histopathology was analyzed by 1-way ANOVA using SAS [26]. Computations were done using the GLM procedure of the SAS [26]. Mean values and SEM are reported.
RESULTS The laying hen diets were isocaloric and isonitrogenous and provided 2,900 kcal/kg of ME and 16% CP, respectively (Table 1). The
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:0 C20:1 C18:2 cis-9,trans-11 C18:2 cis-12,trans-10 C22:6 n-3 Total SFA Total MUFA Total n-3 PUFA Total CLA
YG
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Table 3. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on hen performance during peak production period (27 to 31 wk) Diet1 YG YG-CLA YG-CLA-FO YG-FO P-value PSE
Egg production (%)
Egg weight (g)
Egg mass (g)
Feed intake (g/hen per d)
Feed conversion (g of feed/g of egg mass)
93.36 ± 2.02 93.35 ± 0.97 93.97 ± 1.85 91.63 ± 1.85 0.80 0.81
55.42 ± 0.62 55.76 ± 0.33 55.45 ± 0.43 55.34 ± 0.35 0.91 0.20
51.74 ± 1.39 52.05 ± 0.56 52.09 ± 1.02 50.78 ± 1.34 0.81 0.52
120.9 ± 3.18 120.3 ± 2.24 116.5 ± 2.49 119.1 ± 2.33 0.63 1.23
2.338 ± 0.04 2.311 ± 0.05 2.236 ± 0.04 2.350 ± 0.03 0.23 0.02
YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. PSE = pooled SE.
1
Hen Performance and Egg Characteristics Egg production, egg mass (daily individual egg weight), feed intake, and feed conversion during the peak production period (27 to 31 wk of production) were unaffected by incorporating CLA or fish oil (Table 3). However, when egg quality characteristics were assessed over the entire experimental period (12 mo), a significant increase in egg weight, yolk weight, and shell weight was observed for YG-CLA eggs when compared with eggs from hens fed YG or YGCLA-FO diets (Table 4). The shell weight was higher for YG-CLA eggs (P < 0.05). Duration of feeding time (mo) was significantly different for all the egg characteristics assessed. No difference due to dietary treatment was observed for Haugh unit, albumen weight, or height. The weights of yolk and shell when expressed as percentages of egg weight were also found not to be different among treatments (P > 0.05). However, a significant effect of hen age on percentages of shell weight and yolk weight and Haugh unit was observed. As the hens aged, egg
quality, assessed by Haugh unit, and shell weight percentage deteriorated significantly in all treatments (P < 0.05). Yolk weight as a percentage of egg weight increased as the hen aged (P < 0.05). Yolk color was higher for YG-CLA, YGCLA-FO, and YG-FO when compared with YG eggs (P < 0.05). There was no difference in the BW of the birds. No mortality was noticed among the birds during the experimental period. Egg Total Fat and Fatty Acids No difference was noted in egg total fat content due to dietary treatments (Table 5). However, a significant difference due to hen age on yolk fat was observed. Eggs from 22-wk-old hens contained significantly lower levels of fat compared with all other age groups (P < 0.05). Yolk FA profile clearly reflected the dietary FA composition (Table 5). The CLA was present only in eggs from YG-CLA and YG-CLA-FO (P < 0.05). The cis-9,trans-11 CLA was the only CLA isomer detected in egg yolk. Addition of 0.25% CLA resulted in a significant increase in saturated FA (SFA) with a concomitant reduction in monounsaturated FA (MUFA). The effects of CLA supplementation on n-6 FA varied for different n-6 FA. Arachidonic acid (20:4 n6) was lowest in YG-CLA-FO eggs when compared with the other 3 treatments (P < 0.05). However, it was the reverse for linoleic acid (18:2 n-6), with YG-FO incorporating the lowest concentration (P < 0.05). Similarly, docosahexaenoic acid (22:6 n-3) was lower in YG-CLAFO eggs than YG-FO eggs, although both diets contained equal levels of fish oil (P < 0.05).
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fatty acid composition of the diet is shown in (Table 2). The CLA was present only in the CLA-supplemented diet (YG-CLA and YGCLA-FO) and consisted of cis-9,trans-11 and cis-12,trans-10 isomers at 1.0 and 0.7%, respectively. Inclusion of fish oil resulted in the incorporation of long-chain n-3 FA such as docosahexaenoic acid (22:6 n-3) in YG-CLA-FO and YG-FO diets. α-Linolenic acid (18:3 n-3) was the only source of n-3 FA in the YG and YGCLA diets. The content of other fatty acids such as 16:0, 18:0, 18:1, and 18:2 n-6 was similar among the 4 diets.
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Table 4. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on egg characteristics during the entire 12-mo production period Dietary treatments1 Egg components
YG
YGCLA
YG-CLAFO
YG-FO
Pooled SEM
Diet
Month
Diet × month
57.06b 15.75b 6.10b
58.67a 16.48a 6.31a
57.06b 15.94b 6.11b
57.43ab 16.16ab 6.12b
0.45 0.18 0.05
0.043 0.034 0.029
<0.0001 <0.0001 <0.0001
0.95 0.61 0.63
27.49 10.73 5.34b 94.93 35.2 8.98
28.15 10.78 5.87a 93.41 35.87 8.78
27.80 10.74 5.68a 94.49 35.14 8.89
28.06 10.70 5.90a 94.55 35.01 8.82
0.22 0.08 0.08 0.26 0.32 0.11
0.169 0.950 <0.0001 0.180 0.25 0.59
<0.0001 <0.0001 <0.0001 <0.0001 0.0034 <0.0001
0.59 0.59 0.002 0.70 0.98 0.85
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO.
a,b 1
Hepatic Histopathology, Total Lipids, and Fatty Acids No significant gross abnormalities were noted in the hepatic tissue of any of the birds examined. No significant changes in number of fat vacuoles per grid or number of cells with >75% fat vacuoles were noted. The pathology report indicated no other significant abnormalities associated with fatty liver such as dilated sinusoids, intrasinusoidal fibrin deposition, telangiectasias, and lymphocyte aggregates. Liver fatty acid concentrations were significantly affected by dietary lipids (Table 6). The increase in CLA isomers was higher (P < 0.05) in birds fed YG-CLA and YG-CLA-FO than YG or YG-FO. The increase in CLA was at the expense of MUFA (16:1, 18:1). The 16:0 and 18:0 increased significantly in liver from birds fed CLA. A significant increase in 22:6 n-3 was observed in the hepatic tissue of hens fed diets containing fish oil. As observed with eggs, arachidonic acid was lowest in the hepatic tissue of hens fed YG-CLA-FO when compared with the other 3 treatments (P < 0.05). No difference was observed in the total fat content of hepatic tissue. There was no difference in the total fat content of the abdominal fat pads, which constituted 75.6, 74.0, 74.7, and 74.3% for YG, YG-CLA, YG-CLA-FO, and YG-FO diets, respectively (P > 0.05).
DISCUSSION Incorporating n-3 FA and CLA into eggs has been widely documented [8, 9, 27]. However, long-term feeding trials, investigating the incorporation of CLA along with long-chain n-3 FA in laying hen diets and its effect on production performance or egg fatty acids, are lacking. In general, feeding CLA or CLA along with longchain n-3 FA did not alter the performance of hens. Other researchers have reported a 7% reduction in egg production and no effect on egg or yolk weight in a 68-wk-long feeding trial when CLA was included at 0.5 and 1.0 g/kg of diet [11]. However, Scha¨efer et al. [12] reported no effect on egg production or egg weight when hens were fed diets containing 29 g/kg of CLA for 80 wk. In the current study, when compared with YG diets, we observed an increase in egg weight, yolk weight, and shell weight when CLA was included in the diet. The difference between our study and those reported by others [11, 12] may be due to the level of CLA used in the current study. Although incorporating fish oil and CLA did not affect other egg quality parameters, shell weight when expressed in percentage of egg weight deteriorated significantly as the hens aged. However, egg size increased due to age, and this would mean that economic loss associated with shell breakage will be higher in larger eggs compared with medium eggs or small-sized eggs.
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Egg weight (g) Yolk weight (g) Shell weight (g) Yolk weight (% egg weight) Shell weight (%) Yolk color Haugh unit Albumen weight (g) Albumen height (mm)
P-value
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Table 5. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on egg total fat and fatty acid composition during the entire 12-mo production period Dietary treatments1,2 Egg lipids3
YG-CLA c
26.00 3.19a 9.4d 42.94a 15.30ab 0.35a 1.71a 0.00b 0.65c 35.86c 46.13a 17.01a 1.00b 30.27
a
30.03 1.99d 14.41a 34.22c 15.22ab 0.24ab 1.78a 0.61a 0.60c 45.35a 36.20c 17.00a 0.84b 30.03
YG-CLA-FO a
29.74 2.37c 13.50b 34.23c 15.58a 0.27ab 1.50b 0.61a 1.48b 43.64b 36.61c 17.12a 2.02a 29.68
YG-FO b
26.85 2.96b 10.65c 40.60b 14.86b 0.23b 1.66a 0.00b 1.60a 37.82c 43.57b 16.57b 2.04a 29.77
Diet
Month
Diet × month
0.19 0.06 0.28 0.03 0.03 0.04 0.04 0.001 0.03 0.91 0.30 0.18 0.08 0.21
<0.0001 <0.0001 <0.0001 <0.0001 0.03 0.13 0.002 <0.0001 <0.0001 <0.0001 <0.0001 0.062 <0.0001 0.167
<0.0001 <0.0001 0.002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.1113 <0.0001 <0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001 <0.0001 0.0009 0.096 0.17 <0.0001 <0.0001 0.0018 <0.0001 0.0002 0.3057 0.1906
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 2 Mean of 8 observations for each period. 3 Σ SFA = total saturated fatty acids; Σ MUFA = total monounsaturated fatty acids; Σ n-6 = total n-6 polyunsaturated fatty acids; Σ n-3 = total n-3 polyunsaturated fatty acids. a–c 1
The results obtained on egg CLA and n-3 FA enrichment in YG-CLA, YG-CLA-FO, and YG-FO eggs are in agreement with our previous reported results and also of others [14, 28]. In the eggs from hens fed YG-CLA and YG-CLAFO, a significant increase in SFA was observed at the expense of MUFA. This is in agreement with our previous results and those of others [14, 29]. Inclusion of fish oil along with CLA did not rescue the MUFA-suppressing effect of CLA in YG-CLA-FO eggs. The ∆9-desaturase enzyme is responsible for the conversion of stearic acid (18:0) to oleic acid (18:1). Dietary CLA may have an inhibitory action on ∆9-desaturases, thereby leading to the reduction of MUFA. A recent study also reported a decrease in mRNA expression of stearoyl coenzyme A in CLA-fed rats affecting the synthesis of MUFA and accumulation of SFA [30]. Higher levels of SFA in yolk lipids due to CLA supplementation may be of concern, because increased consumption of SFA, especially 14:0 and 16:0, is associated with increasing total and low-density lipoprotein cholesterol concentrations in blood plasma and changes related to cardiovascular diseases [31]. However, if the antiatherogenic activity of CLA found in rabbits [32], hamsters [33], and mice [34] could be extrapolated to humans, the ad-
verse consequences of increased SFA levels could be counteracted by CLA. The effects of CLA supplementation on PUFA varied for 18and 20-carbon n-6 FA. The CLA, when fed along with fish oil, reduced the concentration of arachidonic acid (20:4 n-6) and increased linoleic acid (18:2 n-6) in YG-CLA-FO eggs. The reason for the lower incorporation of long-chain (>20-carbon) FA is not clear. ∆6-Desaturase enzyme is involved in the formation of arachidonic acid (20:4 n-6) from linoleic acid (18:2 n-6). The structure of CLA is more comparable to linoleic acid, and it is therefore possible that CLA is able to make a complex with ∆6- and ∆4-desaturase in the same way as linoleic (18:2 n-6) does, thus preventing the incorporation of longchain 20-carbon FA in the YG-CLA-FO eggs. Maintaining egg yolk textural property is of utmost importance in developing CLA-modified eggs, because most eggs are used after cooking or after further processing. Several investigators reported hard and rubbery yolks [18], altered yolk shape [12] and changes in yolk color [9] upon storage of eggs from hens fed CLA over 0.5%. We did not find any change in color or shape of CLA-rich eggs in the current study when stored up to 2 mo (data not shown). Overall, incorporating CLA and fish oil in the diet
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:4 n-6 cis-9,trans-11 CLA C22:6 n-3 Σ SFA Σ MUFA Σ n-6 Σ n-3 Σ Lipids (%)
YG
P-value
Pooled SEM
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Table 6. Total lipid and fatty acid composition of the hepatic tissue Dietary treatments1,2 Fatty acids3 (%)
YG-CLA b
25.81 4.36a 10.75c 44.06a 11.24a 0.11a 2.25a 0.14 0.30a 0.82b 36.77b 48.40a 13.90 0.93b 0.00b 154.3
a
29.87 3.56b 15.45b 37.00b 10.60a 0.18a 1.95a 0.00 0.14ab 0.54b 45.32a 38.20b 15.30 0.81b 0.37a 156.97
YG-CLA-FO a
30.59 3.26b 16.51a 35.37b 10.63a 0.18a 1.74a 0.00 0.00 1.33a 47.1a 38.6b 12.4 1.51a 0.40a 158.60
YG-FO 24.58b 3.99ab 10.78c 43.37a 11.19a 0.17a 2.11a 0.00 0.00 1.48a 36.55b 48.5a 13.3 1.65a 0.00b 125.20
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25% conjugated linoleic acid (CLA); 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 2 Mean of 8 observations. 3 Σ SFA = total saturated fatty acids; Σ MUFA = total monounsaturated fatty acids; Σ n-6 = total n-6 polyunsaturated fatty acids; Σ n-3 = total n-3 polyunsaturated fatty acids; TCLA = total conjugated linoleic acids. a–c 1
of the hen at 0.25% produced no detrimental long-term effects on egg quality or liver pathology. The hens performed equally when CLA and fish oil were added to the diets and comparable amounts of CLA and n-3 FA were incorporated into their eggs. Egg size and yolk size increased with hen age. For example, the egg yolk from a 22-wk-old hen weighed 14 g compared with 17.5 g from an older hen and contained signifi-
cantly higher levels of fat than the egg from a younger hen. Consequently, eggs from an older hen could provide a greater proportion of the daily requirement of n-3 PUFA and CLA for a person. Taking into account the current per capita consumption of eggs at 254 in the United States [35], this implies that a minor hen diet modification could provide value-added healthful egg or egg products to the human diet.
CONCLUSIONS AND APPLICATIONS 1. In addition to its role in enriching eggs with CLA and n-3 fatty acids, feeding CLA and fish oil at low levels did not lead to any changes in hepatic histopathology or production performance of birds. However, the current study used only 120 birds, and a small window of production performance was investigated in detail. Feeding trials with a larger number of birds for a longer period would be ideal to investigate the effects of fish oil and CLA in layer diets. 2. Incorporation of CLA in eggs (4.2 mg/g of fat or 21 mg/egg) was higher than that reported in ruminant-derived foods (3 mg of CLA/g of fat). 3. Consuming 2 large eggs from hens fed CLA and the fish oil diet could provide over 70 mg of CLA and 225 mg of n-3 FA to the human diet. This represents over 2 and 35% requirements of CLA and long-chain n-3 FA to the human diet. 4. Development of egg-based products such as pasta, baked goods, salad dressings, mayonnaise with CLA and n-3 FA-modified eggs may help in increasing the CLA and n-3 FA content in the human diet.
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:4 n-6 C22:4 n-6 C22:5 n-6 C22:6 n-3 Σ SFA Σ MUFA Σ n-6 Σ n-3 TCLA Σ Lipids (mg/g)
YG
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REFERENCES AND NOTES 1. Pariza, M. W., Y. Park, and M. E. Cook. 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 4:283–298. 2. Belury, M. A. 2002. Dietary conjugated linoleic acid in health: Physiological effects and mechanisms of action. Annu. Rev. Nutr. 22:505–531. 3. Kris-Etherton, P. M., W. S. Harris, and L. J. Appel. 2003. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 23:20–30.
5. Fritsche, J., R. Rickert, H. Steinhart, M. P. Yurawecz, M. M. Mossaba, N. K. Sehat, K. Roach, J. K. G. Kramer, and Y. Ku. 1999. Conjugated linoleic acid (CLA) isomers: Formation, analysis, amounts in foods and dietary intake. Fett Lipid 101:272–276. 6. Kris-Etherton, P. M., D. S. Taylor, S. Yu-Poth, P. Huth, K. Moriarty, V. Fishell, and R. L. Hargrove. 2000. The polyunsaturated fatty acids in the food chain in the United States. Am. J. Clin. Nutr. 71:179S–188S. 7. Ip, C., S. Banni, E. Angioni, G. Carta, J. McGinley, H. J. Thompson, D. Barbano, and D. E. Bauman. 1999. Conjugated linoleic acid-enriched butterfat alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr. 129:2135–2142.
18. Ahn, D. U., J. L. Sell, C. Jo, M. Chamruspollert, and M. Jeffrey. 1999. Effect of dietary conjugated linoleic acid on the quality characteristics of chicken eggs during refrigerated storage. Poult. Sci. 78:922–928. 19. Hy-Line International, West Des Moines, IA. 20. Folch, J., M. Lees, and G. H. Sloane-Stanely. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–507. 21. Type PT10/35, Brinkman Instruments, Westbury, NY. 22. Agilent Technologies Inc. Palo Alto, CA. 23. Sp-2560, Supelco, Bellefonte, PA. 24. Matreya, Pleasant Gap, PA. 25. Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., Toronto, Canada. 26. SAS Institute. 2001. SAS User’s Guide: Statistics. Release 8.2. SAS Inst. Inc, Cary, NC. 27. Leskanich, C. O., and R. C. Noble. 1997. Manipulation of the n-3 polyunsaturated fatty acid composition of avian eggs and meat. World’s Poult. Sci. J. 53:155–183.
8. Gonzalez-Esquerra, R., and S. Leeson. 2001. Alternatives for enrichment of eggs and chicken meat with omega-3 fatty acids. Can. J. Anim. Sci. 81:295–305.
28. Du, M., D. U. Ahn, and J. L. Sell. 1999. Effect of dietary conjugated linoleic acid on the composition of egg yolk lipids. Poult. Sci. 78:1639–1645.
9. Cherian, G. 2005. Conjugated linoleic acid (CLA) for health: Chicken eggs as alternative sources of CLA. Pages 49–64 in The Amazing Egg: Nature’s Perfect Functional Food for Health Promotion. J. S. Sim and H. H. Sunwoo, ed. Univ. Alberta, Edmonton, Alberta, Canada.
29. Raes, K., G. Huyghebaert, S. D. Smet, L. Nollet, S. Arnouts, and D. Demeyer. 2002. The deposition of conjugated linoleic acids in eggs of laying hens fed diets varying in fat level and fatty acid profile. J. Nutr. 132:182–189.
10. Cherian, G. 2002. Lipid modification strategies and nutritionally functional poultry foods. Food science and product technology. Pages 77–82 in Food Science and Product Technology. T. Nakano and L. Ozimek, ed. Res. Sign Post, Trivandrum, India. 11. Jones, S., D. W. L. Ma, F. E. Robinson, C. J. Field, and M. T. Clandinin. 2000. Isomers of conjugated linoleic acid (CLA) are incorporated into egg yolk lipids by CLA-fed laying hens. J. Nutr. 130:2002–2005. 12. Scha¨fer, K., K. Ma¨nner, A. Sagredos, K. Eder, and O. Simon. 2001. Incorporation of dietary linoleic and conjugated linoleic acids and related effects on eggs of laying hens. Lipids 36:1217–1222. 13. Suksombat, W., S. Samitayotin, and P. Lounglawan. 2006. Effects of conjugated linoleic acid supplementation in layer diet on fatty acid compositions of egg yolk and layer performance. Poult. Sci. 85:1603–1609. 14. Cherian, G., T. B. Holsonbake, M. P. Goeger, and R. Bildfell. 2002. Dietary conjugated linoleic acid alters yolk and tissue fatty acid composition and hepatic histopathology of laying hens. Lipids 37:751–757. 15. Bean, L. D., and S. Leeson. 2003. Long-term effects of feeding flaxseed on performance and egg fatty acid composition of brown and white hens. Poult. Sci. 82:388–394. 16. Van Elswyk, M. E., B. M. Hargis, J. D. Williams, and P. S. Hargis. 1994. Dietary menhaden oil contributes to hepatic lipidosis in laying hens. Poult. Sci. 73:653–662. 17. Grobas, S., J. Mendez, R. Lazaro, C. de Blas, and G. G. Mateo. 2001. Influence of source and percentage of fat added to diet
30. Choi, Y., Y. Kim, Y. Han, Y. Park, M. W. Pariza, and J. M. Ntambi. 2000. The trans-10, cis-2 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3L1 adipocytes. J. Nutr. 13:1920–1924. 31. Bonanome, A., and S. M. Grundy. 1998. Effect of dietary stearic acid on plasma cholesterol and lipoprotein. N. Engl. J. Med. 318:1244–1248. 32. Lee, K. N., D. Kritchevevsky, and M. W. Pariza. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 10:19–25. 33. Nicolosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277. 34. Munday, J. S., K. G. Thompson, and K. A. C. James. 1999. Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model. Br. J. Nutr. 81:251–255. 35. USDA Economic Research Service. Food consumption and nutrient intake tables. http://www.ers.usda.gov Accessed Jul. 20, 2006.
Acknowledgments The Ott Professorship awarded to G. Cherian is acknowledged. The CLA used in this study was kindly supplied by Pharmanutrients, Lake Bluff, Illinois. The generous donation of menhaden oil from Omega Protein Inc, Reedville, Virginia, is appreciated. The assistance of Irene Pilgrim and the Oregon State University poultry farm staff is acknowledged.
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4. Terpstra, A. H. 2004. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: An overview of the literature. Am. J. Clin. Nutr. 79:352–361.
on performance and fatty acid composition of egg yolks of two strains of laying hens. Poult. Sci. 80:1171–1179.
Long-Term Feeding of Conjugated Linoleic Acid and Fish Oil to Laying Hens: Effects on Hepatic Histopathology, Egg Quality, and Lipid Components G. Cherian,1 D. Gonzalez, K. S. Ryu,2 and M. P. Goeger Department of Animal Sciences, Oregon State University, Corvallis 97331
SUMMARY The present study was conducted to investigate the effect of long-term feeding of conjugated linoleic acid (CLA) and fish oil on egg quality characteristics, production performance, liver pathology, and egg fatty acid content of laying hens. Single Comb White Leghorn laying hens (n = 112), 21 wk old, were placed in cages and randomly assigned to 4 diets (28 hens/diet, 4 replicates of 7 hens) containing 3.0% yellow grease (control), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), and 2.75% yellow grease + 0.25% fish oil (YG-FO). The experimental diets were fed for 12 mo. Eggs were collected daily for 12 mo. Feed consumption, hen-day egg production, and feed efficiency were monitored. At the end of the trial, hepatic tissue was collected for histopathology. No effect of diet was found on feed consumption, hen-day egg production, feed efficiency, egg weight, yolk weight, shell weight, or Haugh unit. The YG-CLA and YG-CLA-FO diets produced an increase in CLA and saturated fatty acids in the egg and liver tissue with a concomitant reduction in monounsaturated fatty acids (P < 0.05). Feeding YG-CLA-FO and YG-FO increased the n-3 fatty acids in egg yolk and liver of hens (P < 0.05). No difference was observed in the number of fat vacuoles in the liver tissue. The total fat content of hepatic and abdominal fat pads did not differ among treatments (P > 0.05). Regardless of the diet, as the hens aged, egg weight, yolk weight, and egg total fat increased, and shell weight decreased (P < 0.05). These data demonstrate that eggs with increased n-3 fatty acids and CLA can be generated by minor diet modifications without affecting the production performance or health of birds. Key words: egg, conjugated linoleic acid, n-3 fatty acid 2007 J. Appl. Poult. Res. 16:420–428
DESCRIPTION OF PROBLEM Conjugated linoleic acids (CLA) and longchain n-3 fatty acids (n-3 FA, >20-carbon) have received considerable attention during the past decade for their beneficial health effects. These 1
include the following: reduction of body fat, inhibition of carcinogenesis, modulation of immune system and eicosanoid synthesis, prevention of atherosclerosis, and minimizing the progression of cardiovascular diseases in animal models [1, 2, 3, 4]. Human intake of CLA is
Corresponding author: [email protected] Present address: Department of Animal Resources and Biotechnology, College of Agriculture and Life Science, Chonbuk National University, Chonju, Korea, 561-756.
2
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Primary Audience: Nutritionists, Researchers, Feed Formulators, Egg Producers
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[14]. Similarly, a higher incidence of hepatic hemorrhage and lipidosis has been reported in hens fed flax or fish oil [15, 16]. Consequently, the objective of this study was to determine bird performance during the peak production period, egg quality characteristics, yolk lipid content, fatty acid incorporation, and metabolism over an entire laying cycle (12 mo) in hens fed diets containing low levels of CLA and n-3 PUFA along with yellow grease. In addition, hepatic histopathology and total fat content of hepatic tissue and abdominal fat pads of the birds fed the different lipid sources were assessed. Feeding high-fat (5 to 10%) diets to layer birds has been shown to cause an increase in egg mass output and feed efficiency [17]. In the current study, fat sources constituted 3% of the experimental diet. However, the information on hen performance as measured by feed intake, egg mass, egg production, and feed conversion was measured only during a short period of laying cycle (27 to 31 wk). The information on egg quality characteristics (egg weight, yolk weight, shell weight, yolk color, Haugh unit) was measured throughout the laying cycle. Low levels of CLA were used in the current study to minimize the adverse textural effects on egg yolk [18] and hepatic lipidosis [14] associated with feeding a high dietary concentration of CLA to hens.
MATERIALS AND METHODS These experiments were reviewed by the Oregon State University Animal Care Committee to ensure adherence to Animal Care Guidelines. Birds, Diets, and Housing One hundred twenty Hy-Line strain, Single Comb White Leghorn pullets [19] were reared in floor pens to 18 wk of age. The diets were provided as follows: commercial chick starter from 0 to 6 wk, grower from 16 to 21 wk, and the experimental layer from 21 wk until the end of the experiment (73 wk). At 18 wk, 112 hens were moved to individual laying cages in a room maintained at 20°C, and a step-up lighting schedule was used. Hens received 15L:9D from 18 until 21 wk of age and 16L:8D beginning at 21 wk of age and maintained until the end of the trial. Hens were randomly assigned to 4 diets
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predominantly from ruminant-derived foods, and long-chain n-3 FA is provided by marinederived foods. Current intake of CLA is estimated to be several hundred milligrams per day [5], and long-chain n-3 FA are estimated to be 0.2 g/d [6]. Based on animal data, it is estimated that approximately 3 g/d of CLA would be required to produce beneficial effects in humans [7]. Although no official dietary recommendation has been made in the United States, nutritional scientists suggest inclusion of 0.65 g/d of long-chain n-3 FA. Therefore, an additional 4to 5-fold increase in the supply of CLA and n3 FA is needed in the current US diet. Ruminantderived foods and marine products are the primary sources of CLA and long-chain n-3 FA. However, consumer preference for lean beef cuts, low-fat dairy foods, seasonal availability, and cost of marine foods limits the intake of CLA and n-3 FA in the western diet. Therefore, manipulating CLA and n-3 FA in other animal food lipids offers potential for developing health-enhancing value-added animal products. During the past decade, several different feeding strategies have been adopted by poultry scientists to modify the n-3 FA and CLA content of chicken eggs [8, 9]. Feeding synthetic CLA oil, flax, or marine oil has been the common method to increase the content of CLA or n-3 polyunsaturated FA (PUFA) in eggs [9, 10]. However, success of egg lipid modification strategies will depend upon maintaining egg quality characteristics, production performance, and health of hens during the entire laying cycle. Most of the reported studies on feeding CLA to laying hens were short-term feeding trials (under 6 wk) with small bird numbers varying in age and production cycle except for those reported by Jones et al. [11], Scha¨fer et al. [12], and Suksombat et al. [13]. However, the results reported by these authors were not consistent due to the level of CLA fed, strain of birds used, and the duration of the study. Little or no emphasis was given to bird health in these long-term trials. A combined incorporation of CLA along with n-3 PUFA into egg yolk and its effect on egg quality aspects and bird health have not been addressed. We have reported previously that feeding CLA (0.5% of diet) to hens for 6 wk can lead to an enhancement of hepatic lipid infiltration
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%
Corn Wheat middling Wheat grain Soybean meal Limestone Dicalcium phosphate Salt Oil1 Vitamin-mineral premix2
38.5 29.5 10.0 9.0 6.5 2.5 0.5 3.0 0.5 2,900 16.0 3.8 0.45 0.74 0.89 0.20
1
Oil sources include yellow grease, conjugated linoleic acid, and fish oil. Supplied the following per kilogram 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 g.
Sample Collection
(28 hens/diet), and the treatments were replicated 4 times with 7 birds per replicate. Water and feed were provided ad libitum. The diets were corn-soybean meal-based and contained 3.0% yellow grease (YG; control), 2.75% yellow grease + 0.25% CLA (YG-CLA), 2.5% yellow grease + 0.25% CLA + 0.25% fish oil (YG-CLA-FO), and 2.75% yellow grease + 0.25% fish oil (YG-FO). The composition of the basal diet is shown in Table 1, and the fatty acid composition of the diets is shown in Table 2. Menhaden fish oil was used as a source of n-3 fatty acid. The CLA oil was made up of equal amounts of cis-9,trans-11 and cis-12,trans-10 CLA isomers. Yellow grease (restaurant grease) is commonly used in commercial poultry feeding and was used for the basal diet. The diets were mixed weekly and stored in a cold room (4°C) in air-tight containers. The experimental diets were fed for a period of 12 mo.
Hepatic Histopathology and Criteria for Fat Scoring
2
Production Parameters and Egg Quality Characteristics Production performance (egg production, feed intake, feed conversion, egg mass) was
After 352 d of feeding the experimental diets, 8 birds per treatment (n = 8, two per replicate) were randomly selected and euthanized by exsanguination. Liver and abdominal fat pads were collected.
A slice of liver about 1 × 1 × 0.5 cm thick was taken from the right lobe of each hen, fixed in 10% neutral buffered formalin for 48 h, embedded in paraffin, sectioned (8 m), and stained with hematoxylin and eosin stain before microscopic examination by a veterinary pathologist at the Oregon State University Veterinary Hospital. For each section of liver, 3 randomly located areas were graded at 40× magnification using a grid within a 10× ocular piece. Fat content of each grid was assessed in 2 patterns: a) total number of fat vacuoles in the grid and b) number of cells within the grid having >75% lipid vacuolation of cytoplasm. For each method, the sum total from each bird was divided by 3 to give an average value per grid [14]. The 2 methods were used, because it is common for lipid-laden cells to be largely occupied by only 1 or 2 vacuoles in severe fatty metamorphosis. A single section from each bird was assessed in this manner. A final average for each group was then calculated. The remaining liver tissue was kept frozen (−20°C) for further lipid analysis.
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Calculated composition ME (kcal/kg) CP (%) Ca Available P TSAA Lys Na
measured during the peak production period (27 to 31 wk of hen age). Egg production was expressed as average hen-day production, calculated from the total eggs divided by the number of days. For egg quality characteristics, 8 eggs from each treatment (n = 8, two per replicate) were collected every 4 wk until the end of the trial (12 mo). The eggs were weighed, and yolks were separated using an egg separator and weighed. Albumen weight was calculated by subtracting yolk and shell weight from total egg weight. Albumen height was also documented, and Haugh unit was calculated. Yolk color was determined by comparing yolk color to the Roche color fan. Egg total lipid and fatty acid composition were determined at every 8-wk interval.
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Table 2. Major fatty acid composition of experimental diets1 Dietary treatments2 Fatty acids3 (%)
YG-CLA
YG-CLA-FO
YG-FO
18.20 0.62 4.92 34.46 37.74 3.09 0.31 0.20 0.00 0.00 0.00 23.67 35.50 3.09 0.00
18.65 0.61 4.93 32.14 37.80 3.09 0.39 0.18 0.99 0.72 0.00 24.47 32.93 3.09 1.71
18.46 0.99 4.94 32.71 36.13 2.91 0.41 0.47 0.77 0.68 0.37 23.81 34.81 3.79 1.46
19.94 0.90 4.97 31.35 36.38 3.37 0.40 0.00 0.00 0.00 0.37 26.83 33.05 3.74 0.00
1
n = 6. YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 conjugated linoleic acid (CLA), 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 3 SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; total n-3 PUFA = n-3 polyunsaturated fatty acids. 2
Total Lipid and Fatty Acid Analysis Total lipids were extracted from feed, liver, and abdominal fat pads and egg yolk by the method of Folch et al. [20]. One gram of sample was weighed into a screw-capped test tube with 18 mL of chloroform:methanol (2:1, vol/vol) and homogenized with a polytron [21] for 10 to 15 s at high speed. After an overnight incubation at 4°C, the homogenate was filtered through Whatman number 1 filter paper into a 100-mL graduated cylinder, and 4 mL of 0.88% sodium chloride solution was added and mixed. After phase separation, the volume of lipid layer was recorded, and the top layer was completely siphoned off. Total lipids were determined gravimetrically. Analysis of fatty acid composition was performed with an Agilent 6890 gas chromatograph [22] equipped with an autosampler, flame ionization detector, and fused silica capillary column, 100 m × 0.25 mm × 0.2 m film thickness [23]. Sample (1 L) was injected with He as a carrier gas onto the column programmed for ramped oven temperatures (initial temperature was 110°C, held for 1 min, then ramped at 150°C/ min to 190°C and held for 55 min, then ramped at 5°C/min to 230°C and held for 5 min). Inlet and detector temperatures were both 220°C.
Peak areas and fatty acid percentages were calculated using Agilent ChemStation software. Fatty acid methyl esters were identified by comparison with retention times of authentic standards [24]. Fatty acid values and total lipids are expressed as milligrams per 100 g of fatty acids. Statistical Analysis A 2-way ANOVA was used to compare different egg quality aspects and egg lipid and fatty acid composition between dietary treatment and months. The diet and months were the fixed effects. Significant differences among treatment means were analyzed by Student-NewmanKeuls multiple range test and significance was set at P < 0.05 [25]. Means of interaction were analyzed by comparing the feeding period (mo) separately for each diet. The effect of diet on tissue total lipids, hepatic fatty acids, and histopathology was analyzed by 1-way ANOVA using SAS [26]. Computations were done using the GLM procedure of the SAS [26]. Mean values and SEM are reported.
RESULTS The laying hen diets were isocaloric and isonitrogenous and provided 2,900 kcal/kg of ME and 16% CP, respectively (Table 1). The
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:0 C20:1 C18:2 cis-9,trans-11 C18:2 cis-12,trans-10 C22:6 n-3 Total SFA Total MUFA Total n-3 PUFA Total CLA
YG
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Table 3. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on hen performance during peak production period (27 to 31 wk) Diet1 YG YG-CLA YG-CLA-FO YG-FO P-value PSE
Egg production (%)
Egg weight (g)
Egg mass (g)
Feed intake (g/hen per d)
Feed conversion (g of feed/g of egg mass)
93.36 ± 2.02 93.35 ± 0.97 93.97 ± 1.85 91.63 ± 1.85 0.80 0.81
55.42 ± 0.62 55.76 ± 0.33 55.45 ± 0.43 55.34 ± 0.35 0.91 0.20
51.74 ± 1.39 52.05 ± 0.56 52.09 ± 1.02 50.78 ± 1.34 0.81 0.52
120.9 ± 3.18 120.3 ± 2.24 116.5 ± 2.49 119.1 ± 2.33 0.63 1.23
2.338 ± 0.04 2.311 ± 0.05 2.236 ± 0.04 2.350 ± 0.03 0.23 0.02
YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. PSE = pooled SE.
1
Hen Performance and Egg Characteristics Egg production, egg mass (daily individual egg weight), feed intake, and feed conversion during the peak production period (27 to 31 wk of production) were unaffected by incorporating CLA or fish oil (Table 3). However, when egg quality characteristics were assessed over the entire experimental period (12 mo), a significant increase in egg weight, yolk weight, and shell weight was observed for YG-CLA eggs when compared with eggs from hens fed YG or YGCLA-FO diets (Table 4). The shell weight was higher for YG-CLA eggs (P < 0.05). Duration of feeding time (mo) was significantly different for all the egg characteristics assessed. No difference due to dietary treatment was observed for Haugh unit, albumen weight, or height. The weights of yolk and shell when expressed as percentages of egg weight were also found not to be different among treatments (P > 0.05). However, a significant effect of hen age on percentages of shell weight and yolk weight and Haugh unit was observed. As the hens aged, egg
quality, assessed by Haugh unit, and shell weight percentage deteriorated significantly in all treatments (P < 0.05). Yolk weight as a percentage of egg weight increased as the hen aged (P < 0.05). Yolk color was higher for YG-CLA, YGCLA-FO, and YG-FO when compared with YG eggs (P < 0.05). There was no difference in the BW of the birds. No mortality was noticed among the birds during the experimental period. Egg Total Fat and Fatty Acids No difference was noted in egg total fat content due to dietary treatments (Table 5). However, a significant difference due to hen age on yolk fat was observed. Eggs from 22-wk-old hens contained significantly lower levels of fat compared with all other age groups (P < 0.05). Yolk FA profile clearly reflected the dietary FA composition (Table 5). The CLA was present only in eggs from YG-CLA and YG-CLA-FO (P < 0.05). The cis-9,trans-11 CLA was the only CLA isomer detected in egg yolk. Addition of 0.25% CLA resulted in a significant increase in saturated FA (SFA) with a concomitant reduction in monounsaturated FA (MUFA). The effects of CLA supplementation on n-6 FA varied for different n-6 FA. Arachidonic acid (20:4 n6) was lowest in YG-CLA-FO eggs when compared with the other 3 treatments (P < 0.05). However, it was the reverse for linoleic acid (18:2 n-6), with YG-FO incorporating the lowest concentration (P < 0.05). Similarly, docosahexaenoic acid (22:6 n-3) was lower in YG-CLAFO eggs than YG-FO eggs, although both diets contained equal levels of fish oil (P < 0.05).
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fatty acid composition of the diet is shown in (Table 2). The CLA was present only in the CLA-supplemented diet (YG-CLA and YGCLA-FO) and consisted of cis-9,trans-11 and cis-12,trans-10 isomers at 1.0 and 0.7%, respectively. Inclusion of fish oil resulted in the incorporation of long-chain n-3 FA such as docosahexaenoic acid (22:6 n-3) in YG-CLA-FO and YG-FO diets. α-Linolenic acid (18:3 n-3) was the only source of n-3 FA in the YG and YGCLA diets. The content of other fatty acids such as 16:0, 18:0, 18:1, and 18:2 n-6 was similar among the 4 diets.
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Table 4. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on egg characteristics during the entire 12-mo production period Dietary treatments1 Egg components
YG
YGCLA
YG-CLAFO
YG-FO
Pooled SEM
Diet
Month
Diet × month
57.06b 15.75b 6.10b
58.67a 16.48a 6.31a
57.06b 15.94b 6.11b
57.43ab 16.16ab 6.12b
0.45 0.18 0.05
0.043 0.034 0.029
<0.0001 <0.0001 <0.0001
0.95 0.61 0.63
27.49 10.73 5.34b 94.93 35.2 8.98
28.15 10.78 5.87a 93.41 35.87 8.78
27.80 10.74 5.68a 94.49 35.14 8.89
28.06 10.70 5.90a 94.55 35.01 8.82
0.22 0.08 0.08 0.26 0.32 0.11
0.169 0.950 <0.0001 0.180 0.25 0.59
<0.0001 <0.0001 <0.0001 <0.0001 0.0034 <0.0001
0.59 0.59 0.002 0.70 0.98 0.85
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO.
a,b 1
Hepatic Histopathology, Total Lipids, and Fatty Acids No significant gross abnormalities were noted in the hepatic tissue of any of the birds examined. No significant changes in number of fat vacuoles per grid or number of cells with >75% fat vacuoles were noted. The pathology report indicated no other significant abnormalities associated with fatty liver such as dilated sinusoids, intrasinusoidal fibrin deposition, telangiectasias, and lymphocyte aggregates. Liver fatty acid concentrations were significantly affected by dietary lipids (Table 6). The increase in CLA isomers was higher (P < 0.05) in birds fed YG-CLA and YG-CLA-FO than YG or YG-FO. The increase in CLA was at the expense of MUFA (16:1, 18:1). The 16:0 and 18:0 increased significantly in liver from birds fed CLA. A significant increase in 22:6 n-3 was observed in the hepatic tissue of hens fed diets containing fish oil. As observed with eggs, arachidonic acid was lowest in the hepatic tissue of hens fed YG-CLA-FO when compared with the other 3 treatments (P < 0.05). No difference was observed in the total fat content of hepatic tissue. There was no difference in the total fat content of the abdominal fat pads, which constituted 75.6, 74.0, 74.7, and 74.3% for YG, YG-CLA, YG-CLA-FO, and YG-FO diets, respectively (P > 0.05).
DISCUSSION Incorporating n-3 FA and CLA into eggs has been widely documented [8, 9, 27]. However, long-term feeding trials, investigating the incorporation of CLA along with long-chain n-3 FA in laying hen diets and its effect on production performance or egg fatty acids, are lacking. In general, feeding CLA or CLA along with longchain n-3 FA did not alter the performance of hens. Other researchers have reported a 7% reduction in egg production and no effect on egg or yolk weight in a 68-wk-long feeding trial when CLA was included at 0.5 and 1.0 g/kg of diet [11]. However, Scha¨efer et al. [12] reported no effect on egg production or egg weight when hens were fed diets containing 29 g/kg of CLA for 80 wk. In the current study, when compared with YG diets, we observed an increase in egg weight, yolk weight, and shell weight when CLA was included in the diet. The difference between our study and those reported by others [11, 12] may be due to the level of CLA used in the current study. Although incorporating fish oil and CLA did not affect other egg quality parameters, shell weight when expressed in percentage of egg weight deteriorated significantly as the hens aged. However, egg size increased due to age, and this would mean that economic loss associated with shell breakage will be higher in larger eggs compared with medium eggs or small-sized eggs.
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Egg weight (g) Yolk weight (g) Shell weight (g) Yolk weight (% egg weight) Shell weight (%) Yolk color Haugh unit Albumen weight (g) Albumen height (mm)
P-value
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Table 5. Effect of conjugated linoleic acid (CLA) and fish oil supplementation in the diet of layer birds on egg total fat and fatty acid composition during the entire 12-mo production period Dietary treatments1,2 Egg lipids3
YG-CLA c
26.00 3.19a 9.4d 42.94a 15.30ab 0.35a 1.71a 0.00b 0.65c 35.86c 46.13a 17.01a 1.00b 30.27
a
30.03 1.99d 14.41a 34.22c 15.22ab 0.24ab 1.78a 0.61a 0.60c 45.35a 36.20c 17.00a 0.84b 30.03
YG-CLA-FO a
29.74 2.37c 13.50b 34.23c 15.58a 0.27ab 1.50b 0.61a 1.48b 43.64b 36.61c 17.12a 2.02a 29.68
YG-FO b
26.85 2.96b 10.65c 40.60b 14.86b 0.23b 1.66a 0.00b 1.60a 37.82c 43.57b 16.57b 2.04a 29.77
Diet
Month
Diet × month
0.19 0.06 0.28 0.03 0.03 0.04 0.04 0.001 0.03 0.91 0.30 0.18 0.08 0.21
<0.0001 <0.0001 <0.0001 <0.0001 0.03 0.13 0.002 <0.0001 <0.0001 <0.0001 <0.0001 0.062 <0.0001 0.167
<0.0001 <0.0001 0.002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.1113 <0.0001 <0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001 <0.0001 0.0009 0.096 0.17 <0.0001 <0.0001 0.0018 <0.0001 0.0002 0.3057 0.1906
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25 CLA, 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 2 Mean of 8 observations for each period. 3 Σ SFA = total saturated fatty acids; Σ MUFA = total monounsaturated fatty acids; Σ n-6 = total n-6 polyunsaturated fatty acids; Σ n-3 = total n-3 polyunsaturated fatty acids. a–c 1
The results obtained on egg CLA and n-3 FA enrichment in YG-CLA, YG-CLA-FO, and YG-FO eggs are in agreement with our previous reported results and also of others [14, 28]. In the eggs from hens fed YG-CLA and YG-CLAFO, a significant increase in SFA was observed at the expense of MUFA. This is in agreement with our previous results and those of others [14, 29]. Inclusion of fish oil along with CLA did not rescue the MUFA-suppressing effect of CLA in YG-CLA-FO eggs. The ∆9-desaturase enzyme is responsible for the conversion of stearic acid (18:0) to oleic acid (18:1). Dietary CLA may have an inhibitory action on ∆9-desaturases, thereby leading to the reduction of MUFA. A recent study also reported a decrease in mRNA expression of stearoyl coenzyme A in CLA-fed rats affecting the synthesis of MUFA and accumulation of SFA [30]. Higher levels of SFA in yolk lipids due to CLA supplementation may be of concern, because increased consumption of SFA, especially 14:0 and 16:0, is associated with increasing total and low-density lipoprotein cholesterol concentrations in blood plasma and changes related to cardiovascular diseases [31]. However, if the antiatherogenic activity of CLA found in rabbits [32], hamsters [33], and mice [34] could be extrapolated to humans, the ad-
verse consequences of increased SFA levels could be counteracted by CLA. The effects of CLA supplementation on PUFA varied for 18and 20-carbon n-6 FA. The CLA, when fed along with fish oil, reduced the concentration of arachidonic acid (20:4 n-6) and increased linoleic acid (18:2 n-6) in YG-CLA-FO eggs. The reason for the lower incorporation of long-chain (>20-carbon) FA is not clear. ∆6-Desaturase enzyme is involved in the formation of arachidonic acid (20:4 n-6) from linoleic acid (18:2 n-6). The structure of CLA is more comparable to linoleic acid, and it is therefore possible that CLA is able to make a complex with ∆6- and ∆4-desaturase in the same way as linoleic (18:2 n-6) does, thus preventing the incorporation of longchain 20-carbon FA in the YG-CLA-FO eggs. Maintaining egg yolk textural property is of utmost importance in developing CLA-modified eggs, because most eggs are used after cooking or after further processing. Several investigators reported hard and rubbery yolks [18], altered yolk shape [12] and changes in yolk color [9] upon storage of eggs from hens fed CLA over 0.5%. We did not find any change in color or shape of CLA-rich eggs in the current study when stored up to 2 mo (data not shown). Overall, incorporating CLA and fish oil in the diet
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:4 n-6 cis-9,trans-11 CLA C22:6 n-3 Σ SFA Σ MUFA Σ n-6 Σ n-3 Σ Lipids (%)
YG
P-value
Pooled SEM
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Table 6. Total lipid and fatty acid composition of the hepatic tissue Dietary treatments1,2 Fatty acids3 (%)
YG-CLA b
25.81 4.36a 10.75c 44.06a 11.24a 0.11a 2.25a 0.14 0.30a 0.82b 36.77b 48.40a 13.90 0.93b 0.00b 154.3
a
29.87 3.56b 15.45b 37.00b 10.60a 0.18a 1.95a 0.00 0.14ab 0.54b 45.32a 38.20b 15.30 0.81b 0.37a 156.97
YG-CLA-FO a
30.59 3.26b 16.51a 35.37b 10.63a 0.18a 1.74a 0.00 0.00 1.33a 47.1a 38.6b 12.4 1.51a 0.40a 158.60
YG-FO 24.58b 3.99ab 10.78c 43.37a 11.19a 0.17a 2.11a 0.00 0.00 1.48a 36.55b 48.5a 13.3 1.65a 0.00b 125.20
Means within a row with no common superscript differ (P < 0.05). YG, YG-CLA, YG-CLA-FO, and YG-FO represent diets containing 3.0% yellow grease (YG), 2.75% YG + 0.25% conjugated linoleic acid (CLA); 2.5% YG + 0.25% CLA + 0.25% fish oil (FO), and 2.75% YG + 0.25% FO. 2 Mean of 8 observations. 3 Σ SFA = total saturated fatty acids; Σ MUFA = total monounsaturated fatty acids; Σ n-6 = total n-6 polyunsaturated fatty acids; Σ n-3 = total n-3 polyunsaturated fatty acids; TCLA = total conjugated linoleic acids. a–c 1
of the hen at 0.25% produced no detrimental long-term effects on egg quality or liver pathology. The hens performed equally when CLA and fish oil were added to the diets and comparable amounts of CLA and n-3 FA were incorporated into their eggs. Egg size and yolk size increased with hen age. For example, the egg yolk from a 22-wk-old hen weighed 14 g compared with 17.5 g from an older hen and contained signifi-
cantly higher levels of fat than the egg from a younger hen. Consequently, eggs from an older hen could provide a greater proportion of the daily requirement of n-3 PUFA and CLA for a person. Taking into account the current per capita consumption of eggs at 254 in the United States [35], this implies that a minor hen diet modification could provide value-added healthful egg or egg products to the human diet.
CONCLUSIONS AND APPLICATIONS 1. In addition to its role in enriching eggs with CLA and n-3 fatty acids, feeding CLA and fish oil at low levels did not lead to any changes in hepatic histopathology or production performance of birds. However, the current study used only 120 birds, and a small window of production performance was investigated in detail. Feeding trials with a larger number of birds for a longer period would be ideal to investigate the effects of fish oil and CLA in layer diets. 2. Incorporation of CLA in eggs (4.2 mg/g of fat or 21 mg/egg) was higher than that reported in ruminant-derived foods (3 mg of CLA/g of fat). 3. Consuming 2 large eggs from hens fed CLA and the fish oil diet could provide over 70 mg of CLA and 225 mg of n-3 FA to the human diet. This represents over 2 and 35% requirements of CLA and long-chain n-3 FA to the human diet. 4. Development of egg-based products such as pasta, baked goods, salad dressings, mayonnaise with CLA and n-3 FA-modified eggs may help in increasing the CLA and n-3 FA content in the human diet.
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C16:0 C16:1 C18:0 C18:1 C18:2 n-6 C18:3 n-3 C20:4 n-6 C22:4 n-6 C22:5 n-6 C22:6 n-3 Σ SFA Σ MUFA Σ n-6 Σ n-3 TCLA Σ Lipids (mg/g)
YG
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Acknowledgments The Ott Professorship awarded to G. Cherian is acknowledged. The CLA used in this study was kindly supplied by Pharmanutrients, Lake Bluff, Illinois. The generous donation of menhaden oil from Omega Protein Inc, Reedville, Virginia, is appreciated. The assistance of Irene Pilgrim and the Oregon State University poultry farm staff is acknowledged.
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