Effect of dietary conjugated linoleic acid on laying hen performance, egg yolk fatty acid composition and egg quality during refrigerated storage

Sixty-four Lohman LSL white layers, 75 weeks of age, kept in individual cages were assigned randomly to four treatments of 16 hens per group. The hens received one of four diets with 0, 0.5, 1 or 2% CLA for 4 weeks. Eggs were collected daily and stored at 4oC for 1 or 42 days. The performance of hens and fatty acid composition, thiobarbituric acid reactive substances (TBARS) levels, pH of yolk and albumen, and yolk colour in eggs were measured. Feed intake, feed conversion and egg weight were influenced by the dietary CLA level (P<0.05). The concentration of CLA in the yolk lipids linearly increased as the dietary CLA increased. The addition of CLA to feed significantly (P<0.01) increased the saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) and decreased (P<0.01) the MUFA content in the egg yolk. The duration of refrigeration increased the proportion of egg yolk but decreased the albumen content. It also increased egg yolk pH but decreased albumen pH. Yolk colour was influenced by storage time but it was not influenced by the CLA level. TBARS values were significantly influenced by the dietary CLA and storage time (P<0.01).


INTRODUCTION
Conjugated linoleic acid (CLA) is a mixture of several geometrical and positional conjugated isomers of linoleic acid (C 18:2 cis-9, cis-12).CLA is currently recognized as having a beneficial effect on human health, atherosclerosis, hypercholesterolaemia and cancer (Pariza and Hargraves, 1985;Chin et al., 1992;Ip et al., 1994).Ha et al. (1990) reported that CLA is an antioxidant almost as effective as butylated hydroxytolune (BHT).
Ruminant products, such as milk, meat, and cheese, are major CLA sources in human diets (Chin et al., 1992).Foods obtained from non-ruminants, however, contain much less CLA.Poultry products contain relatively low CLA levels.Chicken meat and egg yolks contain approximately 0.9 and 0.6 mg of CLA/g of fat, respectively (Chin et al., 1992).Feeding animals synthetic CLA sources should be a good way to enrich foods in biologically effective CLAs.Ahn et al. (1999) reported that enriching eggs with CLA likely alters egg characteristics.Chamruspollert and Sell (1999) demonstrated that chicken eggs could be enriched in CLA to as high as 11% by feeding hens 5% CLA in the diet.
CLA is incorporated into all lipid classes of egg yolk in amounts proportional to the dietary level of CLA (Chamruspollert and Sell, 1999).Dietary CLA increased the proportion of saturated fatty acids and decreased monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) of yolk (Ahn et al., 1999).
The experiment was designed to determine the effect of dietary CLA levels on the concentration of CLA, other fatty acids and thiobarbituric acid reactive substances (TBARS) of egg yolk lipids at 4 o C. The influence of dietary CLA on the productive performance of laying hens and selected egg characteristics of commercial importance also was determined.
Beginning on day 21, all eggs were labeled according to date of laying and hen number, weighed and stored at 4 o C for 1 or 42 days.Eggs used for evaluation of yolks and albumen were broken and the weights of yolks, albumen, and shell, recorded.Yolk colour was determined using a 14-point Improved Roche Colour Fan.The pH of the egg yolk and white was measured after diluting with deionized water.

Chemical analysis
From three eggs in each group, 2 g of yolk was taken and lipids were extracted by the method of Folch et al. (1957).Fatty acids of the lipids were methylated by adding 10 mL of anhydrous 3N HCL-methanol to 180 to 200 μg of lipid and heating this mixture for 40 min at 60 o C (Chin et al., 1992).Analysis of fatty acid methyl esters (FAME) was performed by gas chromatography.
Thiobarbituric acid reactive substances were determined on egg samples as described by Salih et al. (1987) with some modifications (Cherian et al., 1995).Egg samples (2 g) were weighed into 50 mL test tubes, and 18 mL of 3.86% perchloric acid were added.The samples were homogenized.BHT (butylated hydroxiyanisole) was added to each sample.The homogenate was filtered through Whatman 1 filter paper.Two mL of filtrate were mixed with 2 mL of 20 mM TBA in distilled water, and incubated.Absorbance was determined at 531 nm.The TBA numbers were expressed as milligrams of malonaldehyde (MA) per kilogram of yolk.
Differences between groups were analysed with one-way and two-way analysis of variance (ANOVA) by using the statistical package SPSS for Windows (1999), version 10.0.Significant means were subjected to a multiple comparison test (Duncan) at α = 0.01 and 0.05 level.

RESULTS AND DISCUSSION
Hens fed the diet containing 1 or 2% CLA consumed less feed (P<0.01)than hens fed 0 or 0.5% CLA (Table 2).Feed efficiency and egg weight were also affected by feeding 0.5, 1 or 2% CLA.However, no effects of dietary CLA concentration were found on the rate of egg production.No mortality of hens occurred during the experiment.Ahn et al. (1999) and Shang et al. (2004) reported that with increasing dietary CLA supply, feed intake and the rate of egg production decreased linearly.Although initially no significant differences were observed between groups with respect to percentage of egg yolk at one day of storage, there were significant differences between percentage increases of egg yolks at the end of the storage period (Table 3).The greatest changes during storage occurred in eggs from hens fed 0.5 or 1% CLA, the dietary CLA by storage time interaction was not significant.Ahn et al. (1999) reported that the yolk percentage increased as dietary CLA increased (P<0.001).The percentage of albumen decreased (P<0.001) with time of refrigerator storage, but it was not affected by the dietary CLA level.The egg shell proportion was not influenced by CLA or storage time.
Egg yolk pH increased during refrigerated storage in all dietary treatments, but the increase was greatest in eggs from hens fed the 1 or 2% CLA diets (Table 4).Ahn et al. (1999) reported that ion movement between the yolks and albumen through the yolk membrane in CLA eggs was greater than that in the control eggs.It is speculated that incorporation of CLA, most of which was in the trans form, could have increased the permeability of the yolk membrane due to the cis-trans arrangements of CLA, which would have increased membrane fluidity and the size of the space between the fatty acid chains of phospholipids that constitute the membrane bilayer.Albumen pH of eggs from hens fed 0, 0.5 or 1% of CLA was decreased significantly by storage time, but it increased in eggs from hens fed 2% CLA.There was no effect of dietary CLA on yolk colour (Table 4).Yolk colour was significantly affected by refrigerated storage (P<0.01).
The fatty acid composition of yolk lipids was significantly altered by increasing concentrations of dietary CLA, but it was not changed by storage time (Table 5).The proportions of myristic, palmitic, stearic and CLA (9-cis, 11-trans and 10-trans, 12-cis) in egg yolk lipids was increased by dietary CLA, but those of palmitioleic, oleic, linoleic, linolenic and arachidonic acid were decreased.These results were similar to those reported by Chamruspollert (1998) and Ahn et al. (1999).Du et al. (1999) reported that n-3 and n-6 precursors have been used for the synthesis of long-chain-PUFA.It has been reported that dietary supplementation of the cis-9, trans 11-CLA isomer inhibited the activity of Δ 6-desaturase in rat liver (Bretillon et al.,1999).They concluded that decreases in unsaturated fatty acids could be caused by the competitive inhibition of Δ6desaturase by CLA.In this research, we increased the dietary supplementation of CLA on the basis of preceding reports and found that CLA in yolk lipids increased linearly.This also confirms earlier findings (e.g., Ahn et al., 1999;Chamruspollert and Sell, 1999;Cherian et al., 2002;Shang et al., 2004).The greatest concentration of CLA, cis-9, trans-11 plus trans-10, cis-12 isomers, in yolk lipids (6.04%) was obtained when the diet contained 2% CLA.The most accumulated CLA isomers were 9-cis, 11-trans observed from the group with 2% CLA (Table 5).Moreover, accumulation of cis-9, trans-11 was higher than that of trans-10, cis-12.Ahn et al. (1999) reported that the decrease in the concentration of linoleic and linolenic acids in yolk lipids of hens fed CLA probably reflects the relatively low concentration of these fatty acids in the CLA source as compared with soyabean oil.The content of arachidonic acid in yolk from hens fed diets enriched in CLA was lower than from the control diet, probably due to competition of metabolites of dietary CLA isomers with metabolites of linoleic acid, particularly in reaction with Δ6-, Δ5-desaturases and elongase (i.e.mechanism of anticancer activity of cis-9, trans-11CLA).Li and Watkins (1998) speculated that CLA reduced the concentration of oleic acid by inhibiting liver Δ9-desaturase activity and found that dietary CLA decreased the concentrations of palmitoleic and oleic acids, which agrees with the present study.The effects of dietary supplementation of CLA on total saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acid (PUFA) composition of yolk lipids are shown in Table 5. SFA and PUFA was increased (P<0.01) by dietary CLA compared with the control group (0% CLA), whereas the concentration of MUFA decreased (P<0.01).These results are in accordance with the findings of Chamruspollert and Sell (1999) and Szymczyk et al. (2003).Inclusion of CLA resulted in a significant (P<0.01)reduction in the TBARS values in eggs.TBARS values were increased during refrigerated storage in all dietary treatments.Supplementation of laying hen's diets with CLA is a novel way of increasing the intrinsic concentration of CLA in eggs.A higher concentration of CLA ameliorated lipid oxidation in eggs, resulting in a dietary CLA by storage time interaction (P<0.01).

TABLE 3
Influence of dietary conjugated linoleic acid (CLA) and days of refrigeration on percentage of yolks, albumens and shells of egg

TABLE 5
Influence of dietary conjugated linoleic acid (CLA) and days of refrigeration on fatty acid composition of egg yolk lipids Fatty acids, % Effect of dietary conjugated linoleic acid (CLA) and days of refrigerated storage on the thiobarbituric acid reactive substances (TBARS) values of egg yolk