consume about 1.5 to 3.0 g of CLA daily (Decker, 1995). This amount of CLA is ...... mary cancer prevention by conjugated dienoic derivative of linoleic acid.
Effects of Dietary Conjugated Linoleic Acid on the Productivity of Laying Hens and Egg Quality During Refrigerated Storage X. G. Shang, F. L. Wang, D. F. Li,1 J. D. Yin, and J. Y. Li Animal Science and Technology College, China Agricultural University, Beijing, China, 100094 ABSTRACT Five hundred and four 40-wk-old Brown Dwarf hens (1.51 ± 0.08 kg BW) were fed corn-soybean meal diets containing 0, 1, 2, 3, 4, 5, or 6% conjugated linoleic acid (CLA) for 56 d to measure the effects of dietary CLA on laying hen productivity and egg quality during refrigerated storage. Four hens were placed in 1 cage, and 3 cages were grouped as 1 replicate resulting in 6 replicates per treatment. After feeding the experimental diets for 11 d, eggs were collected to determine the fatty acid composition of egg yolks. From d 12 to 18, eggs from hens fed diets containing 0, 2, 4, and 6% CLA diets were stored at 4°C for up to 28 d. At designated times (1, 14, or 28 d), eggs were taken, broken, and shelled to evaluate water content, pH, and ion concentration. Firmness of hard-cooked egg yolk was also determined. With increased dietary CLA, feed intake, BW gain, rate of egg production, egg weight, and feed efficiency all decreased linearly (P < 0.01). The weight of the yolk, albumen, and shell decreased linearly (P < 0.01) with increasing dietary CLA. Concentration of CLA in the yolk lipids increased
quadratically (P < 0.01), with increasing dietary CLA. Concurrent increases (P < 0.01) in the concentration of myristic, palmitic, and stearic acids and decreases (P < 0.01) in oleic, linoleic, linolenic, and archidonic acids in egg yolk lipids were observed. Days of storage and CLA (P < 0.01) increased yolk firmness. Egg yolk water content and pH increased with storage and CLA content (P < 0.01). Corresponding decreases were observed in albumen pH. Regardless of dietary treatment, the concentrations of Na, K, and Mg in egg yolks increased with longer storage time. At 28 d of storage, there was a linear (P < 0.01) increase in Na, K, and Mg content in egg yolks as dietary CLA increased. In contrast to the egg yolk, the concentrations of Na, K, and Mg in egg albumen decreased with storage time. On d 28, there was a linear decrease (P < 0.01) in the Na content of albumen with increasing CLA. This study suggests that the greater firmness of CLA-fed eggs might be related to the change of pH, water content, and ion concentrations during refrigerated storage.
(Key words: conjugated linoleic acid, laying hen, yolk lipid, yolk firmness) 2004 Poultry Science 83:1688–1695
INTRODUCTION Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid (Ha et al., 1987). Consumption of CLA by humans has been shown to elicit many favorable health benefits such as modulating immune function, weight reduction, and providing protection against diseases such as cancer and arteriosclerosis (Chin et al., 1992; Lee et al., 1994; Nicolosi et al., 1997). However, to obtain this health benefit, it has been predicted that a 70-kg human would have to consume about 1.5 to 3.0 g of CLA daily (Decker, 1995). This amount of CLA is significantly greater than the estimated consumption of 0.5 to 1.0 g of CLA per person daily in the US and other countries (Ip et al., 1991). As a result, there is considerable interest in supplementing
2004 Poultry Science Association, Inc. Received for publication December 24, 2003. Accepted for publication May 27, 2004. 1 To whom correspondence should be addressed: defali@public2. bta.net.cn.
CLA in animal feed to improve its content in animal product for human consumption. Eggs from CLA-fed hens are a good source of CLA in the human diet (Chamruspollert and Sell, 1999; Du et al., 1999; Raes et al., 2002; Sun et al., 2003). However, Ahn et al. (1999) reported that dietary CLA increased the firmness of hard-cooked egg yolks, and that the yolks of hard-cooked CLA egg yolks were rubbery and elastic, which may reduce their acceptability to consumers. Watkins et al. (2003) reported that dietary CLA increased the concentration of C18:0 in the vitelline membrane. Such a change would probably increase membrane permeability (Abou-Ashour and Edward, 1970). Ahn et al. (1999) speculated that the textural changes in CLA eggs could be related to an increase of yolk water content, the movement of ions between the yolk and albumen membrane, or change of egg yolk pH during storage. Aydin et al. (2001) reported that dietary CLA caused abnormal pH changes
Abbreviation Key: CLA = conjugated linoleic acid; MUFA = monounsaturated fatty acid; SFA = saturated fatty acid.
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EFFECT OF CONJUGATED LINOLEIC ACID ON EGG PRODUCTION AND QUALITY TABLE 1. Ingredient composition of diets fed to laying hens (% as-fed basis) Ingredients and composition Ground corn Soybean meal Wheat bran Soybean oil Conjugated linoleic acid1 Limestone Dicalcium phosphate Salt Vitamin-mineral premix2 DL-Methionine Ethoxyquin
Level of dietary conjugated linoleic acid (%) 0
1
2
3
4
5
6
41.52 32.00 7.00 7.80 0.00 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 6.50 1.30 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 5.20 2.60 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 3.90 3.90 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 2.60 5.20 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 1.30 6.50 8.70 1.50 0.37 1.00 0.07 0.04
41.52 32.00 7.00 0.00 7.80 8.70 1.50 0.37 1.00 0.07 0.04
1 The commercial CLA source contained 77% CLA, which consisted of 43.2% cis-9, trans-11; 40.3% trans-10, cis-12; 5.2% cis-11, trans-13; 1.6% cis-9, cis-11; 2.0% cis-10, cis-12; and 7.6% trans-9, trans-11 positional isomers. 2 Premix provided per kilogram of diet: Fe, 70 mg; Cu, 7 mg; Zn, 70 mg; Mn, 70 mg; Se, 0.36 mg; retinyl acetate, 8,000 IU; cholecalciferol, 2,750 IU; α-tocopherol, 15 IU; thiamine, 1.5 mg; riboflavin, 4.0 mg; vitamin B6, 2.9 mg; vitamin B12, 10 µg; niacin, 25 mg; pantothenic acid, 10 mg; folic acid, 0.38 mg; biotin, 50 mg; choline, 400 mg.
of albumen and yolk when eggs were stored at 4°C. Furthermore, after refrigerated storage for 10 wk, eggs from CLA-fed hens had greater Mg and Na concentrations in yolk, and lower Mg and Na concentrations in albumen relative to those from hens fed corn oil, which is believed to change the vitelline membrane permeability during cold storage by feeding CLA. Therefore, it was hypothesized that during refrigerated storage, alkaline ions such as Na, K, and Mg might move from the albumen to the yolk. The migration of alkaline ions might permute with hydrogen ions in the yolk, and cause yolk pH to increase. This pH change could then induce denaturation of the protein in the yolk, increasing yolk firmness. To test this hypothesis, the experiments were designed to determine the effects of dietary CLA concentration on yolk firmness, pH, water content, and ions during refrigerated storage. The influence of dietary CLA on productive performance of laying hens and its transfer to egg yolk was also determined.
or slightly exceeded the nutrient requirements recommended by the Ministry of Agriculture (2002).
Bird Management Four hens were placed in one cage, and 3 cages were grouped as a replicate, resulting in 6 replicates per treatment. The birds were housed in wire-floored cages measuring 45 cm × 40 cm, with a height of 45 cm. Feed and water were provided ad libitum. The photoperiod was set at 17L:7D throughout the 56-d experiment. Room temperature was maintained at 25 ± 5°C. Egg productions and feed consumption for each replicate were recorded daily or weekly. Body weight of hens was determined at d 0, 28, and 56 of the experiment, and rates of egg production and feed efficiency (kilograms of feed needed to produce a kilogram of eggs) were calculated at the end of the experiment. On d 28 and 56, eggs were weighed, broken open, and the yolks and albumens were separated. The weight of egg, albumen, yolk, and shell was recorded.
MATERIALS AND METHODS Feed Analysis Dietary Treatments Five hundred and four 40-wk-old Brown Dwarf laying hens (1.51 ± 0.08 kg BW) were fed corn-soybean meal based diets containing 0, 1, 2, 3, 4, 5, or 6% CLA (Table 1). The Aohai Company, Qingdao, Shandong Province (PR China), supplied the CLA in the form of free fatty acids. The CLA source (providing 77% CLA) was included at 0, 1.3, 2.6, 3.9, 5.2, 6.5, and 7.8% of the diet. To equalize the concentration of total fat, the CLA source was substituted for soybean oil on an equal weight basis. To avoid fat oxidation, fresh diets were prepared each week and ethoxyquin (0.04%) was added to all diets. The experimental diets were formulated to provide 2,855 kcal of ME/kg, 0.94% lysine, 0.45% methionine, 3.78% Ca, and 0.41% available P. Sufficient vitamins and minerals were added so that all dietary treatments met
Feed samples from each diet were taken after mixing, and stored at 4°C until analysis. The diets were analyzed for CP by Kjeldahl methodology (N × 6.25; AOAC, 1998). Total lipids in diets were determined by the method of Folch et al. (1957), and the fatty acid composition of lipids was analyzed as described below. The CP, total lipid, and fatty acid composition of the diets are presented in Table 2.
Sampling and Storage of Eggs According to Chamruspollert and Sell (1999), the maximum level of CLA in egg yolk lipids was reached 10 to 11 d after the initiation of feeding CLA. Therefore, on d 11, eggs were collected, broken open, and the yolk and albumen were separated to determine fatty acid composi-
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SHANG ET AL. TABLE 2. The crude protein, total lipid, and fatty acid composition1 of diets (% as fed) containing different concentrations of conjugated linoleic acid2 (CLA) Levels of dietary CLA (%)
Chemical analysis Crude protein Total lipid Fatty acid profile Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Conjugated linoleic acid
0
1
2
3
4
5
6
19.78 10.22
19.47 10.11
19.90 10.18
19.51 10.12
19.80 10.15
19.98 10.24
19.99 10.36
1.30 0.38 1.95 5.26 0.82 0.06
1.14 0.34 1.78 4.60 0.69 0.92
1.07 0.32 1.66 3.90 0.57 1.91
1.02 0.31 1.59 3.33 0.47 2.90
0.91 0.27 1.45 2.66 0.33 3.91
0.85 0.26 1.37 2.03 0.21 4.91
0.80 0.24 1.27 1.43 0.10 6.01
1
Shown as the percentage of methyl ester of each fatty acid. The commercial CLA source contained 77% CLA, which consisted of 43.2% cis-9, trans-11; 40.3% trans-10, cis-12; 5.2% cis-11, trans-13; 1.6% cis-9, cis-11; 2.0% cis-10, cis-12; and 7.6% trans-9, trans-11 positional isomers. 2
tion of egg yolks. On d 12 to 18, all eggs from hens fed 0, 2, 4, or 6% CLA were collected and stored to determine the effect of dietary CLA on egg keeping quality. Eggs were stored according to the methods described by Ahn et al. (1999). All eggs were labeled according to date of production and treatment, weighed, and then stored at 4°C for 1, 14, and 28 d. At the designated times, eggs representing each dietary treatment were processed to evaluate yolk firmness and water content, yolk and albumen pH, as well as yolk and albumen ion concentrations.
Fatty Acid Analysis Fatty acid analysis was carried out according to the method described by Raes et al. (2002). Heptadecanoic acid (C17:0) was added as an internal standard. The fatty acid methyl esters were analyzed by gas chromatography using a Hewlett-Packard HP6890 GC System2 installed with a Chrompack Capillary Column (CP–Sil 88 column;3 100 m × 250 µm × 0.25 µm). The chromatography conditions were as follows: 250°C injector temperature; 250°C detector temperature; Helium carrier gas, 1/40 split ratio; temperature program set for 180°C for 45 min, followed by an increase of 10°C/min to 215°C, and then maintained for 17 min. Peaks were identified by comparison of retention times with those of the corresponding standards from Sigma4 and Matreya Biochemicals.5 Identification of the peaks included fatty acids between C14:0 and C24:1, and 6 different CLA isomers. Trans-8, cis-10 CLA isomer was probably present in the CLA source, but its peak overlapped with that of cis-9, trans-11 CLA and could not be detected.
2
Hewlett-Packard Co., Wilmington, DE. Varian Co., Palo Alto, CA. 4 Sigma Chemical Co., St. Louis, MO. 5 Matreya Biochemicals, State College, PA. 6 Hitachi Co., Tokyo, Japan. 7 Rheotech Co., Tokyo, Japan. 3
Water Content, pH, and Ion Concentration Measurements The water content of egg yolks was determined by freeze-drying. Yolks were placed into drying pans and set in the freeze drier (4 yolks/pan). The freeze-drying program was carried out as follows: −40°C for 60 min, followed by −30°C for 60 min, −25°C for 60 min, −15°C for 60 min, −10°C for 30 min, 5°C for 2 h, 10°C for 15 h, and then samples were stored at 15°C for 2 h. The prefreeze temperature was −50°C. The difference in weight between the wet and dried yolk was expressed as percentage moisture. The pH of the egg yolk or albumen was measured on an Accumet pH meter 910 after diluting samples with 5 volumes of deionized distilled water and stirring with a glass rod during pH measurements. Ion concentrations were analyzed as described by Aydin et al. (2001). The digest solutions were diluted to the 100-mL calibration mark and analyzed by Z-500 Polarized Zeeman Atomic Absorption Spectrophometer6 with the exception of chloride, which was determined as described by AOAC (1998).
Firmness The firmness of egg yolks was determined according to the method described by Ahn et al. (1999) using a Fudoh Rheo Meter.7 The crosshead speed of the Instron was 6 cm/min.
Statistical Analysis All data were analyzed using the statistics software of SPSS 9.0 to determine the effects of dietary CLA and refrigerated storage. The effects of dietary CLA concentrations on layer performance, egg composition, yolk profile, and CLA concentration in yolk lipids was determined by regression analysis. The effects of increasing CLA were partitioned into linear and quadratic components using orthogonal polynomial contrasts. Yolk quality data were
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EFFECT OF CONJUGATED LINOLEIC ACID ON EGG PRODUCTION AND QUALITY 1
TABLE 3. Influence of dietary conjugated linoleic acid (CLA) on performance, change in BW, and egg composition of 40-wk-old Brown Dwarf hens Levels of dietary CLA (%)
Feed consumption (g/hen per d) BW gain (g/hen) Rate of egg production (%) Feed efficiency (kg:kg) Egg weight (g/egg) Yolk weight (g/egg) Albumen weight (g/egg) Shell weight (g/egg) Percentage of egg, % Yolk Albumen Shell
P value
0
1
2
3
4
6
7
SEM
Linear
Quadratic
92.6 23.2 81.4 1.96 58.1 16.3 35.3 5.6
85.1 38.2 70.4 2.11 57.2 16.4 35.0 5.6
84.5 40.7 68.7 2.15 57.3 15.9 35.9 5.8
85.4 26.5 72.8 2.13 55.1 15.0 34.0 5.5
84.0 22.7 71.4 2.19 53.7 14.5 33.3 5.3
79.1 −7.3 67.1 2.23 52.8 13.7 32.5 5.3
78.4 −52.7 69.0 2.20 51.6 13.1 33.1 5.3
1.8 14.9 2.8 0.07 0.6 0.3 0.8 0.1
