Effect of conjugated linoleic acid , high-oleic sunflower oil and fish oil dietary supplementation on laying hen egg quality

An experiment was performed to determine the effect of supplementing the diet of laying hens with conjugated linoleic acid (CLA), fish oil (FO) and high-oleic sunflower oil (HOSO) on hen performance, egg yolk fatty acid (FA) concentrations, and egg sensorial quality and firmness. Four treatments were factorially designed involving two levels of supplementation of FO (0 and 17 g kg) and HOSO (30 and 35 g kg) for diets containing 3 g kg of CLA. A control diet was also designed, with 30 g kg HOSO but with no CLA or FO. Twenty five 38 week-old Warren laying hens were randomly assigned to 28 d dietary treatments with the above diets, following a pre-experimental period of 21 d. The type of diet did not affect egg production characteristics. The addition of CLA increased the yolk fat concentrations of CLA, saturated FA and C22:5 n-3, but reduced those of monounsaturated FA and C20:4 n-6. Supplementation with FO increased long-chain n-3 FA concentrations but reduced those of long-chain n-6 FAs, t10,c12 and tt-CLA. Supplementation with HOSO had little influence on yolk fat composition. Supplementation with CLA and FO impaired egg sensorial quality in an additive fashion, although yolk firmness was not affected. Eggs from hens fed diets with 3 g CLA kg and 30 g HOSO kg —but no added FO— contained 9 g CLA kg yolk fat and were of acceptable sensorial quality to trained panellists. However, no eggs double-enriched in CLA and n-3 FA were obtained that were also sensorially acceptable. Additional key words: CLA, egg quality, layers, n-3 fatty acids.


Introduction
Previous work has shown that supplementing the diet of laying hens with conjugated linoleic acid (CLA) increases the yolk fat CLA concentration of their eggs, but also increases the degree of yolk fat saturation.Even supplementation with as little as 5 g kg -1 can lead to a 33-46% increase in total yolk saturated fatty acids and reduce the level of total monounsaturated fatty acids to below 300 g kg -1 (Aydin et al., 2001;Cherian et al., 2002;Szymczyck and Pisulewski, 2003;Álvarez et al., 2004b).As a consequence, the hardness of the yolk of cooked eggs increases and the sensorial quality (for consumers) is reduced (Ahn et al., 1999;Aydin et al., 2001Aydin et al., , Álvarez et al., 2004a,b;,b;2005).The combined incorporation of high-oleic sunflower oil (HOSO, 30 g kg -1 ) and moderate levels of CLA (up to 2 g kg -1 ) can restore oleic acid yolk fat concentration to above 410 g kg -1 , and help produce eggs with an appreciable level of CLA (6.82 g kg -1 yolk fat) and acceptable sensorial quality (Álvarez et al., 2005).
Other studies indicate that CLA supplementation of fish oil-(FO) or flaxseed-added diets does not reduce and can even increase total n-3 fatty acid deposition in the yolk fat (Du et al., 2000;Cherian et al., 2002;Raes et al., 2002;Álvarez et al., 2004b).Further, CLA is an effective antioxidant (Ha et al., 1990), and might reduce the off-flavours associated with the feeding of f ish oil (Herber-McNeill and Van Elswyk, 1998;Leeson et al. 1998).However, the addition of FO to the diet does not reduce the firmness of hardboiled egg yolks from hens receiving CLA-supplemented diets (Álvarez et al., 2004b), and has been associated with reduced sensorial quality caused by the presence of off-flavours [for a review see González-Esquerra and Leeson (2001)].
This study determined the effects of incorporating different levels of FO (0 or 17 g kg -1 ) and high-oleic sunflower oil (30 or 35 g kg -1 ) (in combination) to diets containing 3 g of CLA kg -1 in an attempt to produce eggs enriched in CLA and n-3 fatty acids with an acceptable sensorial quality for consumers.

Material and Methods
Twenty five 38 week-old Warren laying hens were individually housed in cages (33 × 41 cm).Each hen was considered as one experimental unit.The dietary trial lasted for 28 d and started after a pre-experimental period of 21 d, during which the birds were allowed to adapt to the experimental diets.The length of the preexperimental period was considered sufficient since previous research (Chamruspollert and Sell, 1999) has shown that the maximum effects of dietary CLA on yolk fat occurs 10-11 days after feeding a CLA source.Feed was restricted to 115 g d -1 ; water was supplied ad libitum.All hens received 15 h light d -1 over the entire experimental period.Room temperature was controlled at close to 24ºC.
The hens were randomly assigned to receive one of five dietary treatments (five hens per treatment).Four of these were designed by factorially combining two levels of FO (0 and 17 g kg -1 ) and HOSO (30 and 35 g kg -1 ) to a base diet containing a fixed supplement of CLA (3 g kg -1 ).The fifth was a control diet containing 30 g HOSO kg -1 , but no CLA or FO.The CLA source used in this study was obtained from BASF Española, S. A. (Tarragona, Spain).This contained 560 g kg -1 CLA; therefore the actual amount of CLA source added was 5.4 g kg -1 .The FO used contained 68 g kg -1 of eicosapentanoic acid (EPA) and 184 g kg -1 of docosahexanoic acid (DHA), whereas the HOSO contained 757 g kg -1 of oleic and 144 g kg -1 of linoleic acid.Table 1 shows the ingredients and chemical composition of the base diet; Table 2 shows the fatty acid (FA) profiles of the experimental diets.Yolk and dietary lipids were extracted following the methods of Folch et al. (1957) and OJ (1998) respectively.Fatty acid profiles for the experimental fats, diets and the egg yolks obtained were determined according to Cherian and Sim (1992).The fat extracted from each sample was methylated (Metcalfe et al., 1961) and the FAs separated and identified using a Hewlett-Packard 5890 gas chromatograph (Varian Star 3400 CX, Walnut Creek, CA, USA ) equipped with a Supelco SP-2330 (30 m × 0.25 mm inside diameter) silica capillary column.The apparatus was programmed to provide an initial temperature of 150ºC for 4 min, with steps of 1.3ºC min -1 until a final temperature of 210ºC was reached.The temperature of the injector and detector was 250ºC.Hydrogen, at 11.5 psi, was used as the carrier gas.Calibration was performed and the different FA peaks identified by comparing retention times with that of a standard (Qualimix Fich S. Ref: 89-5550, LARODAN, Malmö, Sweden) of known composition.Yolk and albumen acidity (973.41) and moisture (920.116) were determined according to AOAC methods (2000).
Hen-day egg production and feed consumption were measured daily throughout the trial.Three eggs produced by each hen at the end of the 28 d experimental period were used to determine egg, albumen and yolk weight and yolk fat content, and to analyse the FA composition of the yolk fat.Pooled data were used for statistical analyses.In addition, six eggs per replicate were collected at the end of the experimental period.Three were used to determine their commercial value on the basis of shell thickness, albumen height and yolk colour, as measured by the Roche yolk colour fan (Vuilleumier, 1969), the remaining three were used to measure albumen and yolk pH and moisture.
Twelve additional eggs per treatment were randomly selected for sensorial evaluation.All were kept at 5ºC for 14 d.They were then boiled for 15 min and kept in warm water until tasted (Caston and Leeson, 1990) the consistency and reproducibility of scores awarded in accordance with the intensity of sensations perceived (UNE 87.020.93).The results obtained from the preliminary tasting sessions led to twelve panellists being selected.
These were asked to evaluate the eggs (warm, hardboiled) produced by the experimental hens after peeling them and cutting them in half.Eggs from the control and treatment hens were randomly placed in covered plastic dishes and labelled using random numbers.The questionnaires given the panellists were constructed following the instructions proposed by Gonzalez-Esquerra and Leeson (2000), and used the same attributes and definitions: aroma, taste, aftertaste, flavour, presence of off-flavours and overall-acceptability.The panellists were asked to score their dislikes or likes (0 to 10 on a 10 point scale) for aroma, taste, aftertaste, flavour and acceptability, as well as the intensity of off-flavours (absent = 0, very strong = 10).When this scale is used in the evaluation of n-3 enriched commercial eggs, scores below 4 are deemed as «not acceptable», 5 to 7 is «acceptable» and above 8 is «normal».The eggs were presented to the panellists under red lighting to avoid any bias associated with yolk colour.Unsalted crackers and water were offered to cleanse the palate between samples.
The firmness of the hardboiled yolks of six eggs from each treatment was evaluated using a Texture Analyser TA-XT2 and Expert software (v.2.61a) (Texture Technologies Corp., Scarsdale, NY).The tested eggs were stored for 21 days at 5ºC.They were then allowed to attain room temperature and were cooked in boiling water for 15 min.The cooked eggs were cooled to room temperature and the yolks separated from the shell and albumen.To determine the hardness of the yolks, the Texture Analyser was equipped with a TA-18 mm compression plate, and the resistance measured as the force (N) needed to compress the yolks to a distance of 6 mm between parallel planes.This distance was selected since it represented 20% of the original yolk height (Ahn et al., 1999).
Data were analysed as a completely randomised experimental design using the SAS software GLM procedure (SAS Institute Inc., 1990).The effects of the dietary supplementation level of FO and HOSO and their interaction were the main factors studied.Non-orthogonal contrasts were made to test the effect of incorporating 3 g CLA kg -1 to the control diet without added FO.Total dietary fat supplementation was included in the model as a covariate for yolk CLA concentrations.Panellist scores were included as a block for sensorial evaluation traits.Regression procedures (SAS Institute Inc., 1990) were used to predict the retention of CLA isomers in yolk fat as well as the overall acceptability of the eggs.
Supplementation with 17 g FO kg -1 of diets containing 3 g CLA kg -1 decreased (P < 0.05) the yolk content of t 10 ,c 12 and tt CLA by 0.15 and 0.2 g kg -1 respectively, but not the c 9 ,t 11 or total CLA contents.Fish oil supplementation had little influence on SFA and MUFA concentrations, although it slightly reduced C 18:0 and total saturated FAs by 7% and 5% respectively.The inclusion of FO in the diet did not affect C 18:2 n-6 yolk fat concentration, greatly reduced (P < 0.001) those of C 20:4 n-6 and C 22:4 n-6 , and increased (P < 0.001) those of C 20:5 n-3 , C 22:5 n-3, C 22:6 n-3 and total n-3 FA from 0.18, 0.88, 3.73 and 7.07 to 1.11, 3.46, 37.0 and 44.1 g kg -1 respectively.
Increasing the HOSO supplement from 30 to 35 g kg -1 in diets containing 3 g CLA kg -1 did not signif icantly affect yolk CLA, SFA or MUFA concentrations (including C 18:1 ), and had little effect on the n-6 and n-3 FA concentrations.Yolk contents of long chain polyunsaturated FA (PUFA) C 22:4 n-6 and C 22:6 n-3 tended to decrease by about 10% with increasing HOSO supplementation.
The interaction between FO and HOSO had a signif icant (P < 0.07) effect on the C 20:4 n-6 , C 20.5 n-3 , C 22:6 n-3 and total n-3 FA yolk contents; the effect of including 17 g kg -1 of FO on these traits was smaller when the amount of HOSO in the diet was increased from 30 to 35 g kg -1 .
Table 4 shows the effects of the different treatments on egg sensorial quality.The addition of 3 g CLA kg -1 to diets containing 30 g HOSO kg -1 but with no added FO, impaired (P < 0.05) the aroma, taste, aftertaste, flavour and acceptability of hardboiled eggs.Neither the presence of off-flavours nor yolk firmness were modified by the addition of CLA.
Supplementation with 17 g kg -1 of FO of diets containing 3g CLA kg -1 plus 30-35 g HOSO kg -1 did not influence yolk firmness but had a negative effect (P < 0.001) on all the egg sensorial traits evaluated.The eggs produced were considered unacceptable for human consumption by the evaluation panel.
Neither increasing HOSO from 30 to 35 g kg -1 nor the interaction between FO and HOSO significantly affected any of the sensorial traits studied.

Discussion
The type of diet had little effect on hen performance.However, the length of experimental period was short  (seven weeks) and the number of replicates small (five per treatment); it may therefore be premature to conclude the lack of any effect of these treatments on production traits.Previous work has shown that CLA supplementation at higher levels for longer periods leads to a reduction in the weight gain of hens (Jones et al., 2000;Álvarez et al., 2004a).
The present results show that combined supplementation with 3 g kg -1 CLA and 30 g kg -1 HOSO leads to the production of eggs containing 9 g CLA per kg of yolk fat.This impaired several sensorial quality traits compared to eggs produced by hens receiving the control diet with no added CLA, although they were classed as acceptable by the panellists (above 5.75 on a scale from 0 to 10).The inclusion of 3 g CLA kg -1 increased the degree of saturation of the FAs in the yolk fat, but the simultaneous addition of 30 g kg -1 HOSO allowed a high yolk C 18:1 content (403 g kg -1 ) to be maintained and the firmness of the yolks was similar to that of control eggs.Other characteristics related to low consumer acceptance, such as changes in yolk and albumen pH or yolk moisture (Ahn et al., 1999;Du et al., 1999;2000;Álvarez et al., 2004b, 2005), were not affected by the addition of CLA to the control diet.
In the present study, the average yolk CLA concentration obtained in diets supplemented with 3 g CLA kg -1 (8.74 g kg -1 ) was within the 3 to 22 g kg -1 range found in ruminant products (meat/milk etc.) (Chin et al., 1992;Dhiman et al., 1999).It was also higher than that previously obtained (6.82 g kg -1 ) in eggs from hens fed diets containing 2 g kg -1 of CLA and 30 g kg -1 of HOSO (Álvarez et al., 2005), although the sensorial evaluation was slightly worse.
Linoleic conjugated acid was retained in the yolk fat with an average efficiency of 13%.This tended to decrease (P = 0.10) with the level of fat supplementation of the basal diet.This agrees with previous observations (Raes et al., 2002;Álvarez et al., 2004b, 2005), in which lower levels of total fat supplementation of the basal diet were provided (Álvarez et al., 2004 a,b, 2005).
The retention of isomer c 9 ,t 11 was greater than that of t 10 ,c 12 , as recorded in previous studies (Du et al., 1999;Jones et al., 2000;Aydin et al., 2001;Raes et al., 2002;Álvarez et al., 2004a,b, 2005), in which this was related to differences in the rate of its catabolism.Using the data of the present work plus those published by Álvarez et al. (2004a,b, 2005) [which were recorded using a similar methodology and under similar experimental conditions (n = 26)], a regression analysis was performed in order to predict the concentration of both isomers in the yolk fat of eggs from CLA-supplemented hens. Figure 1 shows the equations obtained.These indicate a linear and quadratic effect (P < 0.001) of dietary CLA content on the retention of both isomers, and a higher rate of deposition (around double) for c 9 ,t 11 than for t 10 ,c 12 CLA.The results of the present study also suggest that some tt-CLA isomers were formed in the hens from c9,t11 or t10,c12 dietary CLA, since tt isomers were not detected in the CLA source.
Dietary supplementation with CLA did not interfere with the synthesis of total long-chain n-3 FA since the increasing retention of C 22:5 n-3 was compensated for by a decreasing trend for C 22:6 n-3 .A positive relationship between CLA addition and C 22.5 n-3 yolk retention is also reported by Raes et al. (2002) and Álvarez et al. (2004b).Dietary supplementation with CLA reduced yolk retention of C 20:4 n-6 , which suggests that CLA favours the use of elongases and desaturases for n-3 rather than for n-6 long-chain FA metabolism, as reported by Álvarez et al. (2004b).
Increasing the supplement of HOSO from 30 to 35 g kg -1 had little effect on yolk fat concentrations of CLA or other FAs, including C 18:1 .Nor did it significantly affect the sensorial quality of the eggs laid.Álvarez et al. (2005) reported a reduction in yolk CLA concentration, an increase in yolk C 18:1 content, and improved sensorial quality following an increase in HOSO supplementation from 10 to 30 g kg -1 .Similar trends were observed in this work, although the effects did not reach statistical significance due to the smaller variation in dietary C 18:1 content among treatments.According to the present results, high levels of dietary C 18:1 supplementation might lead to a reduction in the efficiency of retention of C 18:1 in yolk fat, and to reduced elongase and ∆4 desaturase activity [enzymes required for the synthesis of longchain n-6 and n-3 FA (C 22:4 n-6 and C 22:6 n-3 )].
Dietary supplementation with 17 g FO kg -1 led to an increase in the yolk fat concentration of long-chain n-3 FAs, especially C 22:6 n-3 .The efficiency of retention for total n-3 FAs was close to 50%, decreasing at the higher HOSO supplementation level.Supplementation with FO reduced the yolk retention of long-chain n-6 FA, reflecting a reduced availability of the enzymes required for its synthesis (these are shared with the n-3 FA series).The addition of FO had an overall positive effect on yolk PUFA content (by 18%, P < 0.05) and tended to reduce the firmness of hardboiled eggs, although the differences did not reach statistical significance.
The inclusion of FO in diets with CLA and HOSO supplements greatly impaired the sensorial quality of the eggs produced; these were deemed unacceptable for human consumption by the panellists (values < 5), although the level of FO supplementation was close to that used in the commercial production of n-3 enriched eggs.
An excess of dietary FO or other sources of n-3 FA, such as flaxseed, has long been recognized to reduce egg sensorial quality (Van Elswyk, 1997;Noble, 1998;González-Esquerra and Leeson, 2001;Surai and Sparks, 2001).It has been suggested that adverse flavours are promoted by oxidative deterioration of the egg lipids.Long-chain n-3 FAs are highly susceptible to peroxidation because of their long length and many double bonds.The inclusion of marine algal oil in the diet seems to have less of a negative effect on sensorial scores despite its high C 22:6 n-3 concentration.This has been related to its high natural carotenoid content which might help to stabilize yolk lipids (Herber-McNeill andVan Elswyk, 1996, 1998).In the present study, the antioxidant properties of CLA might have helped to reduce the negative effect of FO addition, but failed to produce eggs double-enriched in CLA and n-3 FA that were of acceptable sensorial quality.Similarly, the supplementation of diets with high levels of vitamin E (100 IU kg -1 ) and a high flaxseed content (200 g kg -1 ) results in a further reduction of overall egg acceptability (Leeson et al., 1998).Deodorization of FO to reduce its content of volatile compounds produced via lipid oxidation neither appear to improve it (González-Esquerra and Leeson, 2000).The reasons for the poorer acceptability of eggs enriched in long-chain n-3 FA and/or CLA therefore remain unclear.
The greater influence of C 22:5 n-3 than other n-3 FAs on the presence of off-flavours would also explain the lower impact on egg sensory quality of marine algae compared to FO, as the inclusion of marine algae in the diet leads to an increase in C 22:6 n-3 retention in yolk content, whereas the concentration of C 22:5 n-3 remains almost unchanged (Herber-McNeill and Van Elswyk, 1996;Cachaldora et al., unpublished).
The results of the present work indicate that the combined addition of appropriate levels of CLA and HOSO to the diet of laying hens allows the production of eggs with a moderate CLA content (9 g kg -1 yolk fat) and an acceptable sensorial quality.However, the study failed to obtain commercially viable eggs double-enriched in CLA and long-chain n-3 FA, since the negative effects of FO on sensorial quality were additive with those of CLA.Further research is needed to attain this objective by modifying the supplementation-induced levels of and/or source of n-3 FA.

Table 1 .
Ingredients and chemical composition of the base diet (g kg -1 as fed basis)

Table 2 .
Fatty acid profiles of the ether extracts of the experimental diets (g kg -1 ) SFA: total saturated fatty acids.MUFA: total monounsaturated fatty acid.

Table 4 .
Effect of the different diets on the sensorial and rheological properties (yolk firmness) of hardboiled eggs 1