Effects of type and level of supplementation with dietary n-3 fatty acids on yolk fat composition and n-3 fatty acid retention in hen eggs

A marine algal oil (MAO) with a high docosahexaenoic acid content (46.7%) was evaluated for the production of n-3 fatty acid (FA) enriched eggs. To determine its effects, laying hens were fed one of two diets containing different amounts of MAO, or a control diet containing marine fish oil (MFO). The efficiency of retention of total n-3 FA in the yolk fat depended both on the type and level of dietary n-3 substrate. Per unit weight of dietary oil added, MAO was better than MFO in terms of total n-3 FA retention by the yolk fat. However, the efficiency of retention of total n-3 FA, expressed as g FA retained per g of FA intake, was higher for MFO than for MAO. Additional key words: layers, n-3 retention efficiency, n-3 sources, yolk fatty acid composition.

for LCn-3 FA to be reduced with increasing dietary concentration.However, González-Esquerra and Leeson (2000) report a linear response of yolk LCn-3 FA concentration for up to a 6% level of inclusion of menhaden oil.
The aim of the present study was to compare the yolk fat retention of LCn-3 FA [with a high DHA content (46.8%)] provided by MAO, at two levels of dietary inclusion.The results were compared to a control diet that included a commercial source of MFO.
The experimental birds were thirty six 58 week-old Warren laying hens housed individually in cages (33 cm × 41 cm).The trial lasted for 42 d, following a pre-experimental period of 14 d.Feed was restricted to 115 g d -1 while water was supplied ad libitum.No feed refusals were observed.The hens were provided with 15 h of light per day throughout the experiment.The temperature was maintained at about 21ºC.
The hens were randomly assigned to one of three dietary treatments, T1, T2 or T3.These diets were formulated by substituting the corresponding part of the base diet with 1.7% MFO, 0.77% MAO or 1.7% MAO.The concentrations of DHA and EPA were 22% and 8% respectively in the MFO, and 46.8% and 8.4% in the MAO.The proportions of C16:0, C18:0, C18:1, C18:2 and C18:3 were 18.3, 5.3, 16.0, 1.2 and 0.5%, and 2.6, 2.8, 6.8, 0.6 and 0.2% in the MFO and MAO respectively.Accordingly, diets T1 and T2 had a similar DHA content (0.36%) but differed in their EPA content (0.14% compared to 0.06% respectively).The EPA contents of diets T1 and T3 were similar (0.14%), but the DHA content of diet T3 (0.80%) was greater than that of diet T1 (0.36%).Table 1 shows the ingredients and chemical compositions of the base diet.
Hen-day egg production and egg weight per hen were recorded daily throughout the trial.The body weights of the birds were recorded at the beginning and end of the experimental period.All eggs produced during the third and last week of the experimental period were kept at 4ºC for less than seven days to determine the yolk weight and to analyse the yolk FA composition.The average values for each hen over these two weeks (twelve hens per treatment) were used in statistical analyses.The yolk lipids of three eggs (selected at random and pooled) per hen were extracted following the method of Folch et al. (1957).The fatty acid profiles (two analyses per replicate) of experimental fats and egg yolks 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; Walnut Creek, California, USA) equipped with a Supelco SP-2330 (30 m × 0.25 mm inside diameter) silica capillary column.The apparatus was programmed with an initial temperature of 170ºC for 20 min, allowing increases of 5.4ºC min -1 until a final temperature of 250ºC was reached.The temperature of the injector and detector was 250ºC.Hydrogen at 11.5 psi was used as the carrier gas.The calibration and identification of the peaks for the different FAs was performed by comparing retention times with that of a known standard (Qualimix Fich S. Ref. 89-5550 larodan; Malmoe, Sweden).
The efficiency of polyunsaturated n-3 FA retention was calculated as the ratio between the FA retained in the yolk fat (estimated as laying rate × yolk weight × yolk fat content × proportion of FA in the yolk fat) and FA ingested (estimated as feed intake x dietary fat content × FA concentration in dietary fat).An internal standard (trimargarolein; Sigma-Aldrich Corporation, St. Louis, Missouri, USA) was used to measure the concentration of total FA in the yolk and dietary fat.
The effects of inclusion of n-3 sources in the base diet were analysed (considering the experiment to have a completely randomised design) using SAS software (SAS Institute Inc., 1990).Non-orthogonal contrasts were used to test the effects of the different dietary levels of EPA (T1 vs. T2) and DHA (T1 vs. T3).
Further, the efficiency of retention (expressed as g FA retained/100 g FA ingested) of EPA was lower (P < 0.001) and that of DHA higher (P < 0.001) in treatment T1 than in T2.No effect of EPA addition was therefore found on the efficiency of retention of total n-3 FA.An increase in the dietary DHA concentration from 0.36% (treatment T1) to 0.80% (treatment T3) decreased the total yolk fat MUFA and C 20:4 n-6 contents by 4.3% and 15.7% respectively, but increased those of SAT, linolenic, EPA, DPA, DHA and total n-3 FA.The efficiency of retention of DHA and total n-3 FA was poorer (P < 0.001) in treatment T3 than in T1, whereas only a small difference (P = 0.1) was found for EPA retention eff iciency.As a consequence of the previous effects, total n-3 FA concentration per egg n-3 fatty acid supplementation in laying hens 211 Table 2. Effect of treatments on yolk fatty acid composition, n-3 fatty acid retention efficiency, and egg total n-3 concentration increased as a result of both EPA (from 191 mg in treatment T2 to 221 mg in treatment T1) and DHA addition (up to 250 mg in treatment T3).
Given that the total n-3 FA yolk fat concentration was 13.1% higher for MAO than for MFO at the same level of dietary inclusion, the present results confirm those of other authors (González-Esquerra and Leeson, 2001) indicating DHA to be more efficiently retained in the yolk fat than EPA.However, DHA retention eff iciency decreased greatly from 30.6% to 18.7% when the dietary MAO content increased from 0.77% to 1.70%.These values are lower than the mean efficiencies reported by González-Esquerra and Leeson (2001) at lower mean levels of DHA supplementation, but close to those obtained by Abril et al. (2000) at similar levels of DHA intake.
Table 2 shows that when dietary DHA content was increased by more than double (122%) but at a similar level of EPA (treatment T3 vs. treatment T1), the yolk DHA concentration only increased by 17%, but those of its metabolic substrates -linolenic, EPA and DPAincreased by 10.4%, 45.9% and 19.4% respectively.This suggests that a high yolk DHA concentration might reduce the activity of enzymes involved in the synthesis of LCn-3 FA.Consequently, the overall efficiency of DHA and total n-3 FA retention decreased sharply (by 51.0% and 36.9%respectively) in hens fed diet T3 compared to those fed diet T1.
The present results also indicate that an increase in dietary EPA at a low DHA concentration (0.37%) implies an increase in the yolk fat content of EPA, but also of DPA and DHA, which are synthesised from EPA.A similar effect has been described by Herber and Van Elswyk (1996).As a consequence, the overall efficiency of retention of EPA was 32.1% lower when increasing the dietary EPA content (treatment T1 vs. treatment T2) while that of DHA increased by 24.8%.
The efficiency of retention of FA n-3 in yolk fat appeared to be highly and negatively dependent on the level of dietary DHA supplementation.As a consequence, the total n-3 FA concentration of the egg yolk was higher with MAO than with MFO when equally included in the diet.However, a greater efficiency of retention of DHA and a greater amount of total yolk fat n-3 FA were observed with MFO than with MAO when compared at similar dietary levels of DHA or EPA.