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Production of n-3PUFA Enriched Eggs By Feeding Various Dietary Ratios of n-
6 to n-3 Fatty Acids and Vitamin A Levels to the Laying Hens in Hot Climate
Shakeel Ahmad1, Ahsan-ul-Haq2, Muhammad Yousaf2, Zahid Kamran1, Ata-ur-
Rehman3 and Muhammad U. Sohail4
1University College of Veterinary and Animal sciences, The Islamia University of
Bahawalpur 63100, Pakistan.
2Department of Poultry Science, University of Agriculture, Faisalabad 38040, Pakistan.
3Research Support Section, Pakistan Science Foundation, Islamabad 44000, Pakistan.
Department of Physiology, Government College University Faisalabad 38000, Pakistan.
Running Title: Production of n-3 PUFA enriched eggs
---------------------------
Correspondence : Dr. Shakeel Ahmad, University College of Veterinary & Animal Sciences,
Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Punjab, Pakistan. (E-mail:
drshakeel@iub.edu.pk)
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
Advance View Proofs
Abstract
In the present study, the effect of various dietary ratios of linoleic acid (LNA) to α-
linolenic acid (ALA), and vitamin A (Retinyl Acetate) on egg quality and some blood
metabolites was examined in laying hens kept at high ambient environmental
temperature. Three hundred and sixty laying hens were fed rations supplemented with
different combinations of canola oil and linseed oil to have LNA to ALA dietary ratios
of 20:1, 10:1, 4:1, 2:1, 1:1 and 1:2, each with 3000 IU or 10000 IU vitamin A/kg of diet
(6x2 factorial; under Completely Randomized Design). Various blood metabolites, egg
cholesterol, egg triglycerides and egg-yolk fatty acids were determined during the last
week of the study. The results showed that serum triglycerides, serum total- and low
density lipoprotein-cholesterol, and blood glucose were similar (P > 0.05) in laying
hens; fed on various dietary LNA/ALA ratios and vitamin A levels. Consequently, egg
cholesterol and egg triglycerides were also similar (P > 0.05) for the all dietary
treatments. However, Serum high density lipoprotein-cholesterol increased (P = 0.045)
with the decrease in dietary LNA/ALA ratio. The n-3 polyunsaturated fatty acids
(PUFA) in egg yolks from laying hens were increased (P < 0.001) while, the n-6 PUFA
and the ratio of n-6 to n-3 fatty acids were decreased (P < 0.001) with the decrease in
dietary LNA/ALA ratio. It was recommended that a dietary ratio of 4:1 or less between
LNA and ALA was quite beneficial for the production of eggs by the hens with
desirable quantities of n-6 and n-3 PUFA that are suitable for human consumption.
Key words: egg quality, hens, hot climate, PUFA, vitamin A
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Introduction
Polyunsaturated fatty acids (PUFA) are the structural components of cell
membranes in the human and animal body. Among PUFA, linoleic acid (LNA; n-6
PUFA) and α-linolenic acid (ALA; n-3 PUFA) hold the prime importance as these are
the precursors of long chain PUFA and, eventually, of eicosanoids (Simopoulos, 2000).
These eicosanoids play a vital role in various physiological activities in the body
including regulation of fat and cholesterol metabolism. It is now well recognized that
the intake of n-6 PUFA in large quantities increases the risk of coronary heart diseases.
The eicosanoids of n-6 origin are involved in pro-inflammatory and pro-aggregatory
activities which result in vasoconstriction in the body (Petrovic et al., 2012). On the
other hand, the eicosanoids originating from n-3 PUFA have anti-inflammatory and
anti-aggregatory activities which result into vasodilatation (Chow, 2008). The n-3
PUFA also play a role in reducing plasma triglycerides (TG), LDL-cholesterol, blood
pressure and protect the body from coronary heart diseases and auto immune disorders
(Simopoulos, 1991). In the body, LNA and ALA compete for the same enzymes
required for their desaturation and elongation and conversion into eicosanoids. Hence;
the ratio between n-6 and n-3 PUFA in the diet appears to be more important for the fat
and cholesterol metabolism in the body than the absolute concentrations of n-6 or n-3
PUFA (Sijben et al., 2000). Therefore, there is a need to regulate the intake of n-6 and
n-3 PUFA in balanced proportions by human. But, the diet of modern age community is
very high in n-6 PUFA. This is due to the industrial production of animal feed from
grains rich in n-6 PUFA making the meat and eggs rich in n-6 PUFA but poor in n-3
PUFA (Simopoulos, 2000). The intake of n-6 and n-3 PUFA in a ratio of 1 to 5:1 is
recommended (EFSA, 2009). The amount of n-3 PUFA can be increased in a desired
The Journal of Poultry Science
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ratio to n-6 PUFA in the foods from animals by supplying these PUFA through their
diets (Ahmad et al., 2013). In this regard, poultry eggs can serve the purpose if these
have balanced quantities of n-6 and n-3 PUFA. The dietary fatty acids are absorbed by
the chicken and deposited in their tissues and products without any considerable change
in composition (Coetzee and Hoffman, 2002) and the fatty acids composition of the
produced reflects the fatty acid composition of the diet taken. So; n-6 and n-3 PUFA
deposition in the eggs in a ratio suitable for human consumption can be regulated by
offering the diets to the laying hens with balanced quantities of n-6 and n-3 PUFA. In
PUFA enriched eggs, the control of lipid per-oxidation is required especially during
summer months. Supplementation of hens’ diet with vitamin A higher than National
Research Council (NRC, 1994) recommendations of 3000 IU/Kg of diet not only
increase the content of this vitamin in the eggs (Mendonca et al., 2002) but, also, can
prevent any possible lipid per-oxidation in the eggs and egg products during storage
specially in hot summer months. Higher Vitamin A supplementation can also restore the
normal activity of reproductive organs in laying hens kept at high ambient
environmental temperature (Lin et al., 2002).
In the past, various dietary sources have been supplemented to the laying hens for the
production of n-3 PUFA-enriched eggs but the supplementation of optimum quantity
and the optimum ratio of n-6 to n-3 PUFA required for the production of best quality
eggs have, yet, not been worked out for the chicken diets. Moreover, the most of the
previous trials on n-3 PUFA supplementation to laying hens for the production of
PUFA-enriched eggs were conducted in the temperate climates and little data is
available for such trials in tropical or sub-tropical climates. The present study was
planned to observe the effects of various dietary LNA/ALA ratios and vitamin A levels
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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on various blood metabolites and egg quality in laying hens kept in hot climatic
conditions.
Materials and Methods
Birds, housing, and experimental diets
Three hundred and sixty White Leghorn laying hens at 40th week of age were
randomly divided into 36 replicates. These replicates were randomly allotted to 12
treatment groups (three replicates per treatments) which were fed layer rations
supplemented with various combinations of canola oil and linseed oil to have LNA to
ALA dietary ratios of 20:1, 10:1, 4:1, 2:1, 1:1 and 1:2, in combination with 3000 or
10000 IU vitamin A (Retinyl Acetate) per kg of diet (6 × 2 factorial, Completely
Randomized Design). Commercially available raw canola oil and linseed oil were
purchased from the market and supplemented in the diets. The hens were kept in cages
(2 birds/cage) providing 0.093 m2 floor space area to each, and at a high ambient
environmental temperature (diurnal temperature: average min. 29.3 °C, average max.
37.4 °C) throughout the experimental period of 12 weeks. The light regime was 16L:8D
for all treatment groups. The layer diets for all treatment groups were isonitrogenous
and isocaloric formulated according to the recommendations of NRC (1994) for the
dietary needs of laying hens. The rations were mixed on weekly basis. The hens under
all treatment groups had ad-libitum access to feed and water throughout the experiment.
All the procedures were approved by the Animal Welfare and Ethical Use Committee of
the University.
Data collection
During the last week of the trial, blood samples were taken from two birds per
replicate via wing vein to determine the serum concentration of TG, total-cholesterol,
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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LDL-cholesterol, HDL-cholesterol, and blood glucose. In the last week, three eggs per
replicate were collected and yolks were separated, pooled and homogenized, and frozen
till the fatty acids determination by gas chromatography.
Laboratory analyses
Feed ingredients were analyzed for proximate components (Association of Official
Analytical Chemists, 1990) prior to formulation of the diets. Ingredients and chemical
composition of the layer rations are shown in Table 1. Serum TG levels (mg/dl) were
determined on a photometric system microlab-300 (Vital Scientific N.L. 6950 AC
Dieren, The Netherlands) by calorimetric enzymatic test using glycerol-3-phosphate-
oxidase as described by Cole et al. (1997). Serum cholesterol levels (mg/dl) were
determined on an automated photometric system microlab-300 (Vital Scientific N.L.
6950 AC Dieren, The Netherlands) by “CHOD-PAP” enzymatic photometric test
method as described by Artiss and Zak (1997). Blood glucose concentrations (mg/dl)
were determined by an automated Apparatus “Selectra E”. Egg cholesterol and egg TG
(mg/dl) were extracted according to the methods described by AOAC (1990).
Fatty acids analysis
The fatty acid contents of egg yolks were determined by gas chromatography.
The yolk lipids were extracted according to the method described by AOAC (1990) by
using chloroform and absolute alcohol (1:1). Fatty acids were converted into fatty acid
methyl esters (FAME) according to the method described by Chin et al. (1992). The
FAME were separated and quantified by a gas chromatograph (Varian 3900) using a
fused silica capillary column (30 metre, 0.25 mm diameter) and were expressed as
percentage of total FAME.
Statistical analyses
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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The data were analyzed by two-way Analysis of Variance Technique (ANOVA) using
General Linear Model (GLM) and means were compared by Tukey’s honestly
significant difference test (Minitab 13.1, Minitab Inc., State College, PA).
Results
The production performance in laying hens was not according to the genetic
potential of the birds and seemed to be suppressed by the heat-stress effects. The
feeding of various LNA/ALA ratios and vitamin A levels did not affect the feed intake
and egg production in laying hens. Hen-day egg production %age and hen-housed egg
production %age were similar (P > 0.05) for various LA/LNA ratios and vitamin A
levels in the hens’ diet and ranged between 78.16 to 81.5% and 77.40 to 81.5% for
various treatment groups, respectively. Although the feed intake remained similar
statistically (P > 0.05), a slight decrease in feed intake (up to 1%) was observed
numerically with the decrease in dietary LA/LNA ratio. Similarly, no change was
observed in the feed conversion ratio per dozen of eggs by various dietary treatments
and ranged between 1.45 to 1.52. The egg weight responded positively (P < 0.05) to n-3
PUFA feeding. The heaviest eggs (61.83 g) were produced by the hens in treatment
group with LA/LNA ratio of 1:2 and the lowest egg weight (57.0 g) was recorded for
the hens at LA/LNA dietary ratio of 10:1. However, egg weight for both levels of
vitamin A was recorded with no difference (P > 0.05).
Various LNA/ALA ratios did not change (P > 0.05) the serum TG, total-
cholesterol, LDL-cholesterol, and blood glucose levels in the laying hens (Table 2). The
serum HDL-cholesterol level in laying hens was increased (P = 0.045) with the
decrease in dietary LNA/ALA ratio (Table 2). Vitamin A supplementation did not affect
(P > 0.05) the serum cholesterol and blood glucose levels in laying hens. The egg
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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cholesterol and TG contents were similar (P > 0.05) in laying hens fed various dietary
LNA/ALA ratios or vitamin A levels in the diet (Table 3).
The percentage of oleic acid (OA; 18:1, n-9), LNA, arachidonic acid (AA; 20:4, n-6)
and total n-6 PUFA in the yolk fatty acids was decreased (P < 0.05) with the decrease in
dietary LNA/ALA ratio (Table 4). The percentage of ALA, eicosapentaenoic acid
(EPA; 20:5, n-3), docosapentaenoic acid (DPA; 22:5, n-3), docosahexaenoic acid
(DHA; 22:6, n-3) and total n-3 PUFA in the egg-yolk fatty acids was increased almost
linearly (P < 0.001) with the decrease in dietary LNA/ALA ratio (Table 5). The ratio of
n-6 to n-3 PUFA was also decreased linearly (P < 0.001) with the decrease in
LNA/ALA ratio in the hens’ diet (Table 5). However; supplementation of Vitamin A at
a level higher than NRC recommendations did not affect the egg yolk fatty acids. No
significant (P > 0.05) interaction was observed between various dietary LNA/ALA
ratios and vitamin A levels for the egg-yolk fatty acids.
Discussion
Blood metabolites
Serum total-cholesterol, LDL-cholesterol and TG levels were almost similar for
the all dietary LNA/ALA ratios and vitamin A levels fed to the laying hens. Serum
HDL-cholesterol was significantly lower in the hens at dietary LNA/ALA ratio of 2:1
when compared to other groups. This observation seemed to be exceptional: as other
dietary groups were similar with each other for serum HDL-cholesterol. Murata et al.
(2003) reported no influence of dietary sunflower oil or canola oil on plasma total-
cholesterol, and HDL- and LDL-cholesterol in laying hens. Roy et al. (2008) also
reported that feeding diets rich in n-3 PUFA to the broilers did not alter the serum total
cholesterol, HDL- and LDL cholesterol. Similar to the present study, linseed oil
The Journal of Poultry Science
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doi: 10.2141/jpsa.0120169
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supplementation as ALA source in the diet did not alter the plasma TG level in the birds
(Febal et al., 2008). The results of present study indicated that blood glucose level of the
hens had no relation with dietary PUFA. Crespo and Esteve-Garcia (2003) reported no
change in serum insulin and blood glucose in fasted broilers given the diets having
sunflower oil and linseed oil and suggested that PUFA have no visible influence on
insulin hence on glucose metabolism, accordingly, in birds. Febal et al. (2008) also
found similar results for glucose level in PUFA-fed broilers.
Egg cholesterol and egg triglycerides
The egg cholesterol and egg TG contents were not influenced by the different
dietary LNA/ALA ratios or vitamin A levels. Numerically, there was slight decrease in
egg cholesterol and egg TG contents but this decrease was negligible. It could be
suggested that egg cholesterol and egg lipids had a resistance to any change in their
contents by inclusion of n-3 PUFA in the diet of hens. Hargis (1988) concluded that the
inability to reduce egg-yolk cholesterol markedly is possibly due to natural selection
pressure to maintain a certain level of cholesterol in the egg for use by the developing
embryo. Along with feeding unsaturated fatty acids, lipogenesis would decrease but
cholesterol biosynthesis in the liver will not change, therefore the extent of egg
cholesterol content would not change (Weiss et al., 1967). Previous reports had
indicated that plasma cholesterol concentration is not related to egg yolk lipid level,
though synthesized in the liver and transported by the blood (Sutton et al., 1984).
Similar findings on the effect of dietary fat on egg yolk cholesterol were reported by
Shafey et al. (1998) who reported that dietary fat had little effect on cholesterol
deposition in the egg yolk. Many other researchers found no effect of dietary n-3 PUFA
on egg cholesterol and TG contents (Jiang et al., 1991; Murata et al., 2003; Silke et al.,
The Journal of Poultry Science
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2008). The results of present study also suggested no relation of vitamin A with the egg
lipids.
Egg-yolk fatty acids
The results of the present study showed that OA contents of egg yolks varied
with the decrease in LNA/ALA ratio of the diet while, vitamin A did not affect the yolk
OA contents. The visible decrease in OA contents was observed only for the diets
having LNA/ALA ratio of 1:1 and 1:2. The diets with ratios of 10:1, 4:1 and 2:1 had
high percentage of canola oil than linseed oil, and since canola oil is a rich source of OA,
it favored the deposition of OA in the yolks. While, the diets with LNA/ALA ratio of
1:1 and 1:2 had mostly linseed oil in them and linseed oil is a rich source of ALA and
LNA and low in OA contents. That is why, the OA content of yolks was decreased in
those treatment groups. The decreased OA contents in egg yolks might also be due to
the inhibitory effects of PUFA against Δ9-desaturase enzyme activity, preventing the
formation of OA from its precursors (Garg et al., 1988). The present trial confirmed the
results of previous studies in which supplementation of n-3 PUFA in the hens’ diet
caused a reduction in OA content of yolks (Jiang et al., 1991; Bou et al., 2004; Souza et
al., 2008; Ahmad et al., 2010).
The LNA, AA and total n-6 PUFA deposition in the egg yolks of laying hens
were decreased with the decrease in dietary LNA/ALA ratio. The lowest level of n-6
PUFA was observed in the egg yolks of the layers at the diets with LNA/ALA ratio of
1:1 and 1:2. These diets were fortified mostly with linseed oil and rich in ALA: it is
possible to suggest that it was ALA which might limit the deposition of LNA and AA in
the yolks. ALA intake is associated with inhibitory effects on the regulation of AA
metabolism (Simopoulos, 1999). Jiang et al. (1991) suggested that when hens received
The Journal of Poultry Science
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doi: 10.2141/jpsa.0120169
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diets high in ALA or n-3 PUFA, the conversion of LNA to AA was reduced due to the
competition between n-3 and n-6 PUFA for the same enzymes for their biosynthesis and
consequently AA content decreased. The metabolic pathways for LNA and ALA
compete for the ∆-6 desaturase enzyme in the desaturation and elongation to provide
longer-chain PUFA (Brenner, 1981), and increase in dietary ALA may inhibit the
conversion of LNA to longer chain n-6 PUFA (AA) in the tissues. A decrease in the
dietary n-6/n-3 PUFA ratio is more effective in limiting n-6 PUFA elongation than an
increase in the total amount of n-3 PUFA consumed (Hayek and Reinhart, 1998).
Mazalli et al. (2004) observed that the inclusion of linseed oil, with high ALA content,
decreased the synthesis of AA from LNA in the laying hens. Similarly, decreasing n-
6/n-3 PUFA ratio by supplementing various fish and rapeseed oil levels in the diets of
laying hens resulted into egg yolks with reduced LNA, AA and total n-6 PUFA
deposition (Kralik et al., 2008).
Results of the present trial indicated that ALA, EPA, DPA, DHA and total n-3
PUFA in the egg yolks were increased almost linearly with the decrease in LNA/ALA
ratio of the diet. The inclusion of vitamin A at higher level did not influence the yolk n-
6 or n-3 PUFA. From the results, it could be suggested that the increase in ALA in the
diet favoured the deposition of ALA and its long chain derivates in the egg yolks. It is
possible that a limited, but effective, formation of EPA, DPA and DHA may take place
from ALA inside the body of chicken (Ayerza and Coates, 2000). The results of the
present study confirmed the finding of Kralik et al. (2008) who reported an increase in
the content of ALA, EPA and DHA as well as total n-3 PUFA in egg yolks from laying
hens on rations with decreased n-6/n-3 PUFA ratios and confirmed that laying hens
have the ability to synthesize EPA and DHA in the limited amounts if given enough
The Journal of Poultry Science
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amounts of ALA in the diet. Bou et al. (2004) found an increase in yolk n-3 PUFA with
ALA supplementation in the diet of hens and also suggested that the efficiency of
deposition seemed to be proportional to the diets’ n-3 PUFA contents.
In the present study, the n-6 PUFA were not decreased consistently with the
decrease in LNA/ALA ratio of the diet but n-3 PUFA increased linearly with the
decrease in dietary LNA/ALA ratio. This resulted into a linear decrease in n-6/n-3
PUFA ratio in the egg yolks with the decrease in dietary LNA/ALA ratio. The n-6/n-3
PUFA ratio is the most important factor which determines the functional value of the
eggs. Kaminska et al. (2001) reported that hens who were fed diets with n-6/n-3 PUFA
ratio from 13:1 (wheat-based) to 38:1 (maize-based) laid eggs with n-6/n-3 PUFA ratios
from 11:1 to 21:1. In the present study, the n-6/n-3 PUFA ratio decreased up to 1.3:1 in
eggs from hens fed with dietary LNA/ALA ratio of 1:2, fulfilling the recommendation
for functional food. The current results are in line with the findings of Ajuyah et al.
(2003) who found that dietary n-6/n-3 PUFA ratios of 37.12, 4.21 and 0.98 resulted n-
6/n-3 PUFA ratios of 28.36, 2.83 and 0.89 in the egg yolks of layers, respectively. The
results of the present study indicated that the response of heat-stressed laying hens to
dietary PUFA was similar to that reported earlier for the hens kept at temperate or
controlled climates. So it might be suggested that the laying hens can be provided the
diets high in PUFA for the production of PUFA enriched eggs, without any limitations,
in hot climates.
It was concluded from the results of the present study that a dietary ratio of 4:1 or less
between LNA and ALA fed to the hens was quite beneficial for the production of eggs
with n-6/n-3 PUFA ratio of 4.24 or less. The production of eggs with desirable n-6/n-3
PUFA ratio offers an alternative choice of food to the health conscious consumers
The Journal of Poultry Science
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around the globe. The eggs which have been considered an atherogenic food, now, can
be consumed safely if enriched with balanced quantities of n-6 and n-3 PUFA.
References
Ahmad S, Ahsan-ul-Haq, Yousaf M and Nawaz H. Effect of feeding canola oil and
vitamin A on the fatty acid profile of egg yolks in laying hens. Pakistan Journal of
Nutrition 9:191-194. 2010.
Ahmad S, Ahsan-ul-Haq, Yousaf M, kamran Z, Rehman AU, Sohail MU and Samad
HA. Effect of canola oil and vitamin A on egg characteristics and egg cholesterol in
laying hens during hot summer months. Pakistan Veterinary Journal 33:346-349. 2013.
Ajuyah AO, Wang Y, Sunwoo H, Cherian G and Sim JS. Maternal diet with diverse
omega-6/omega-3 ratio affects the brain docosahexaenoic acid content of growing
chickens. Neonatology 84:67-73. 2003.
AOAC. Official Methods of Analysis. 16th ed. Association of official analytical
chemists. Arlington VA. 1990.
Artiss JD and Zak B. Measurement of cholesterol concentration, In: Handbook of
Lipoprotein Testing (Rifai N, Warrnic GR and Dominiczak MH eds.) pp. 99-114.
AACC Press. Washington DC. 1997.
Ayerza R and Coates W. Dietary levels of chia: Influence on yolk cholesterol, lipid
content and fatty acid composition for two strains of hens. Poultry Science 79:724-739.
2000.
Bou R, Guardiola F, Tres A, Barroeta AC and Codony R. Effect of dietary fish oil, α-
tocopheryl acetate, and zinc supplementation on the composition and consumer
acceptability of chicken meat. Poultry Science 83:282-292. 2004.
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
Advance View Proofs
Brenner RR. Nutritional and hormonal factors influencing desaturation of essential fatty
acids. Progress in Lipid Research 20:41-47. 1981.
Chin SF, Liu W, Storkson JM, Ha YL and Pariza MW. Dietary sources of conjugated
dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. Journal of
Food Composition and Analysis 5:185-197. 1992.
Chow CK. Fatty acids in Foods and Their Health Implementations. 3rd edition. Marcel
Dekker. New York. 2008.
Coetzee GJM and Hoffman LC. Effects of various dietary n-3/n-6 fatty acid ratios on
the performance and body composition of broilers. South African Journal of Animal
Science 32:175-183. 2002.
Cole TG, Klotzsch SG and McNamara J. Measurement of Triglyceride Concentration.
In: Handbook of Lipoprotein Testing (Rifai N, Warrnic GR and Dominiczak MH eds.)
pp. 115-126. AACC Press. Washington DC. 1997.
Crespo N and Esteve-Garcia E. Polyunsaturated fatty acids reduce insulin and very low
density lipoprotein levels in broiler chickens. Poultry Science 82:1134-1139. 2003.
EFSA. Scientific opinion of the panel on dietetic products, nutrition and allergies on a
request from European Commission related to nutrition claims concerning omega-3
fatty acids, monounsaturated fat, polyunsaturated fat and unsaturated fat. The EFSA
Journal 253:1-29. 2009.
Febal H, Mezes M, Plfy T, Herman A, Gundel J, Lugasi A, Balogh K, Kocsis I and
Blazovics A. Effect of dietary fatty acid pattern on growth, body fat composition and
antioxidant parameters in broilers. Journal of Animal Physiology and Animal Nutrition
92:369-376. 2008.
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
Advance View Proofs
Garg ML, Sebokova E, Wierzbicki E, Thompson ABR and Clendinin MT. Differential
effects of dietary linoleic and linolenic acid on lipid metabolism in rat tissues. Lipids
23:847-852. 1988.
Hargis PS. Modifying egg yolk cholesterol in the domestic fowl: a review. World’s
Poultry Science Journal 44:17-29. 1988.
Hayek MG and Reinheart GA. Utilization of ω3 fatty acids in companion animal
nutrition. In: The Return of ω3 Fatty Acids Into the Food Supply (Simopoulos AP. ed)
pp. 176-185. Karger AG. Basel. 1998.
Jiang Z, Ahn DU and Sim JS. Effects of feeding flex and two types of sunflower seeds
on fatty acid composition of yolk lipid classes. Poultry Science 70:2467-2475. 1991.
Kaminska B, Gasior R and Skraba B. Modification of polyunsaturated fatty acid
contents in yolk lipids using various cereals and blended animal fat in hens’ diets.
Journal of Animal Feed Science 10:255-260. 2001.
Kralik G, Gajcevic Z and Skrtic Z. The effect of different oil supplementations on
laying performance and fatty acid composition of egg yolk. Italian Journal of Animal
Science 7:173-183. 2008.
Lin H, Wang LF, Song JL, Xie YM and Yang QM. Effect of dietary supplemental
levels of vitamin A on egg production and immune responses of heat stressed laying
hens. Poultry Science 81:458-465. 2002.
Mazalli MR, Faria DE, Salvador D and Ito DT. A comparison of the feeding value of
different sources of fats for laying hens: 1. Performance characteristics. Journal of
Applied Poultry Research 13:274-279. 2004.
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
Advance View Proofs
Mendonca CX, Almeida CRM, Mori AV and Watanabe C. Effects of dietary vitamin A
on egg yolk retinol and tocopherol levels. Journal of Applied Poultry Research 11:73-
378. 2002.
Murata LS, Ariki J, Machado CR, Silva L and Rezende MJM. Effect of oils sources on
blood lipid parameters of commercial laying hens. Brazilian Journal of Poultry Science
5:203-206. 2003.
NRC. Nutrient Requirements of Poultry. 9th ed. National Academy Press. Washington
DC. 1994.
Petrovic M, Gacic M, Karacic V, Gottstein Z, Majiza H and Madic H. Enrichment of
eggs in n-3 polyunsaturated fatty acids by feeding hens with different amount of linseed
oil in diets. Food Chemistry 135:1563-1568. 2012.
Roy R, Singh S and Pujari S. Dietary role of omega-3 polyunsaturated fatty acid
(PUFA): a study with growing chicks, Gallus domesticus. International Journal of
Poultry Science 7:360-367. 2008.
Shafey TM. Effects of dietary retinol and sunflower oil on the performance and on the
lipoprotein, lipids, cholesterol and retinol concentrations of plasma and egg laying hens.
Arab Gulf Journal of Science Research 16:331-345. 1998.
Sijben JWC., Degroot H, Nieuwland MGB, Schrama JW and Parmentier HK. Dietary
linoleic acid divergently affects immune responsiveness of growing layer hens. Poultry
Science 79:1106-1115. 2000.
Silke HS, Nutztierethologie FG and Kleintierzucht U. Effect of genetic types with two
types of dietary fats on performance and egg yolk fatty acids in laying hens. European
Poultry Science 72:177-184. 2008.
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
Advance View Proofs
Simopoulos AP. Essential fatty acids in health and chronic disease. American Journal of
Clinical Nutrition 70:560-569. 1999.
Simopoulos AP. Human requirement for n-3 polyunsaturated fatty acids. Poultry
Science 79:961-970. 2000.
Souza JG, Costa FPG, Queiroga RCRE, Silva JHV, Schuler ARP and Goulart CC. Fatty
acid profile of eggs of seamy-heavy layers fed feed containing linseed oil. Brazilian
Journal of Poultry Science 10:1590-1599. 2008.
Sutton CC, Muir WM and Mitchell GE. Cholesterol metabolism in laying hen as
influenced by dietary cholesterol, caloric intake and genotype. Poultry Science 63:972-
80. 1984.
Weiss JF, Naber EC and Johnson RM. Effect of dietary fat and d-thyroxin on the
incorporation of acetate1-14C into egg yolk lipids. Journal of Nutrition 93:153-160.
1967.
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Table 1. Ingredients and nutrient composition of layer diets
Diets R1
a & R2
b R3
a& R4
b R5
a& R6
b R7
a& R8
b R9
b& R10
b R11
a& R12
b
LNA1:ALA2 20:1 10:1 4:1 2:1 1:1 1:2
Ingredients ________________________g/kg _______________________
Maize 687.0 597.6 520.0 520.0 480.0 310.0
Rice broken 0.0 61.0 121.0 115.0 133.5 240.0
Soybean meal 140.0 179.0 200.0 210.0 241.0 255.0
Fish meal 52% 56.0 0.0 0.0 0.0 0.0 0.0
Corn gluten 60% 35.0 60.0 47.5 40.0 20.0 10.0
Canola oil 0.0 10.0 16.0 10.0 3.0 0.0
Linseed oil 0.0 0.0 3.5 12.0 28.5 56.0
Wheat bran 0.0 0.0 0.0 0.0 0.0 34.0
Limestone 69.0 70.0 70.0 72.0 72.0 72.8
Di-calcium Phosphate 7.8 17.1 17.0 16.6 17.0 17.0
L-Lysine 0.6 1.6 1.2 0.9 0.4 0.0
DL-Methionine 0.3 0.6 0.7 0.7 0.9 1.0
Sodium chloride 1.0 0.1 0.2 0.7 0.9 0.9
Sodium bicarbonate 0.25 0.3 0.3 0.3 0.3 0.3
Vit./min premix3 2.5 2.5 2.5 2.5 2.5 2.5
Total 1.0 1.0 1.0 1.0 1.0 1.0
Nutrient Composition ____________________________________
Crude Protein (%) 17.00 17.00 17.00 17.00 17.00 17.00
ME (MJ/Kg) 12.14 12.14 12.14 12.14 12.14 12.14
Ether Extract (%) 3.33 3.57 4.22 4.43 5.15 6.99
Crude Fibre (%) 2.59 3.27 3.81 3.81 4.08 5.20
Ca (%) 3.25 3.27 3.26 3.29 3.28 3.24
Av.P (%) 0.40 0.41 0.40 0.41 0.42 0.40
Lysine (%) 0.90 0.92 0.89 0.90 0.91 0.92
Methionine (%) 0.38 0.39 0.37 0.40 0.38 0.37
Threonine (%) 0.64 0.67 0.63 0.65 0.64 0.66
LNA (%) 1.60 1.60 1.60 1.60 1.60 1.60
ALA (%) 0.08 0.16 0.40 0.80 1.60 3.20
3Vitamin/mineral premix, provided per kilogram of diet: Cholecalciferol, 1,250 IU; Vitamin E (dl-alpha-
tocopheryl acetate), 12 IU; menadione, 2.5mg; riboflavin, 6 mg; calcium pantothenate, 8 mg; niacin, 15
mg; pyridoxine 2 mg; folic acid, 1 mg; cinocobalamine, 7µg; Mn, 50 mg; Zn, 55 mg; Fe 40 mg; Cu, 4
mg; I, 2 mg; Co, 0.3 mg; ethoxiquin, 150 mg.
a rations containing 3000 IU/kg of diet vitamin A:
b rations containing 10000 IU kg of diet vitamin A;
1 LNA = Linoleic Acid;
2 ALA = Α-linolenic Acid
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Table 2. Effect of LNA/ALA ratios and vitamin A on serum triglycerides, serum HDL-,
LDL-, and total cholesterol, and blood glucose in laying hens
T.G1 HDL-Chol.2 LDL-Chol.3 T. Chol.4 B. Glucose5
Diet (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl)
LNA:ALA
20:1 167.15 50.83ab 72.46 137.60 259.92
10:1 163.70 53.02ab 70.40 137.70 254.90
4:1 162.36 50.83ab 72.72 137.70 257.08
2:1 166.71
49.55b 73.74 137.80 251.05
1:1 156.47 53.41ab 69.37 136.40 262.10
1:2 168.42 55.34a 68.47 138.40 259.90
SEM 4.117 1.298 3.298 2.687 2.686
Vit. A (IU/kg diet)
3000 163.90 51.80 71.49 137.50 256.70
10,000 164.33 52.53 70.89 137.70 258.22
SEM 2.377 0.749 1.904 1.552 1.551
ANOVA _______________________Probabilities________________________
LNA:ALA 0.362 0.045 0.843 0.998 0.084
Vit. A 0.900 0.497 0.825 0.922 0.476
LNA:ALA×Vit. A 0.295 0.847 0.954 0.871 0.326
a-b means within a column with different superscripts differ significantly (P < 0.05)
1Triglycerides; 2HDL-cholesterol; 3LDL-cholesterol; 4Total cholesterol; 5Blood glucose
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Table 3. Effect of various LNA/ALA ratios and vitamin A on egg cholesterol and egg
triglycerides in laying hens
Egg cholesterol Egg triglycerides
Diet (mg/dl) (mg/dl)
LNA:ALA
20:1 361.70 10.43
10:1 358.00 10.52
4:1 334.80 10.44
2:1 339.70 10.53
1:1 341.00 10.35
1:2 343.30 10.39
SEM 9.413 0.047
Vit. A (IU/kg diet)
3000 345.80 10.42
10,000 347.00
10.46
SEM 5.435 0.027
ANOVA ________________________Probabilities______________________________
LNA:ALA 0.289 0.062
Vit. A 0.881 0.339
LNA:ALA×Vit. A 0.438 0.928
Means within a column with no superscripts differ non-significantly (P > 0.05).
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Table 4. Effect of various LNA/ALA ratios and vitamin A on egg yolk oleic acid (OA),
linoleic acid (LNA), arachidonic acid (AA) contents and total n-6 PUFA (% of
total Fatty acids) in laying hens
OA LNA AA Total n-6 PUFA
Diet (%) (%) (%) (%)
LNA:ALA
20:1 41.41c 13.26a 1.94a 15.20a
10:1 44.42a 13.67a 1.58b 15.25a
4:1 43.11b 12.24b 1.83a 14.08b
2:1 43.26ab 13.65a 1.80a 15.48a
1:1 41.01c 13.32a 0.79c 14.12b
1:2 34.78d 12.09b 0.63c 12.71c
SEM 0.245 0.173 0.043 0.190
Vit. A (IU/kg diet)
3000 41.37 13.00 1.45 14.44
10,000 41.29 13.09 1.41 15.50
SEM 0.142 0.099 0.025 0.110
ANOVA ________________________Probabilities______________________________
LNA:ALA 0.000 0.000 0.000 0.000
Vit. A 0.715 0.525 0.372 0.706
LNA:ALA×Vit. A 0.995 0.923 0.720 0.954
a-d means within a column with different superscripts differ significantly (P < 0.001).
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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Table 5. Effect of various LNA/ALA ratios and vitamin A on egg yolk α-linolenic acid
(ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA),
docosahexaenoic acid (DHA) and total n-3 PUFA (% of total Fatty acids), and n-
6:n-3 in laying hens
ALA EPA DPA DHA n-3 PUFA n-6:n-3
Diet (%) (%) (%) (%) (%)
LNA:ALA
20:1 0.95f 0.07d 0.16d 0.44e 1.62f 9.37a
10:1 1.42e 0.09cd 0.11d 0.65de 2.28e 6.69b
4:1 1.92d 0.12c 0.23c 1.04d 3.32d 4.24c
2:1 2.93c 0.14c 0.34c 1.62c 5.03c 3.07d
1:1 3.89b 0.20b 0.40b 2.96b 7.46b 1.89e
1:2 5.09a 0.31a 0.77a 3.54a 9.72a 1.31e
SEM 0.053 0.011 0.022 0.097 0.105 0.138
Vit. A (IU/kg diet)
3000 2.72 0.15 0.33
1.67 4.87 4.42
10,000 2.68 0.15 0.34 1.76 4.94 4.45
SEM 0.031 0.006 0.013 0.056 0.061 0.08
ANOVA ________________________Probabilities______________________________
LNA:ALA 0.000 0.000 0.000 0.000 0.000 0.000
Vit. A 0.408 0.588 0.359 0.283 0.469 0.784
LNA:ALA×Vit. A 0.936 0.542 0.974 0.360 0.611 0.968
a-f means within a column with different superscripts differ significantly (P < 0.001).
The Journal of Poultry Science
J-STAGE Advance Publication Date:September 25, 2013
doi: 10.2141/jpsa.0120169
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