EFFECT OF DIETARY INCLUSION OF MARGARINE ON LAYING PERFORMANCE, EGG QUALITY, EGG YOLK FATTY ACIDS, AND SERUM LIPID METABOLITES IN LAYING HENS

In this study, feed consumption, feed conversion ratio, egg production, egg quality, and some serum lipid metabolites were evaluated in laying hens fed diets containing different inclusion levels of margarines with different degrees of saturation. A total of sixty 18-wk-old ATAK-S laying hens were distributed to 5 experimental groups, each consisting of 6 replicates with 2 laying hens in each replicate. In the experiment, two inclusion levels (5 and 10%) of margarine with two different degrees of saturation (17 and 35%) containing 60% crude fat were used. Control group was fed diet without the inclusion of margarine. Other groups were fed diets with 5 or 10% of 17% (M17-5 and M17-10) or 35% (M35-5 and M35-10) saturated margarines. All the groups were fed isocaloric and isonitrogenous diets. The experiment lasted for eight weeks. Feeding margarines at different inclusion levels had no effect on daily feed consumption, egg yield, feed conversion rates (dozen eggs/kg feed and kg eggs/kg feed), and serum triglyceride and cholesterol levels of laying hens. In addition, yolk color, albumen index, shape index, egg weight, Haugh unit, and yolk index were not affected across the groups. However, the addition of 35% saturated margarine with regardless of the inclusion level increased the total unsaturated fatty acids in the egg yolk of laying hens. In conclusion, egg yolk fatty acids can be manipulated by different dietary inclusion levels of margarines at saturation points.


INTRODUCTION
Margarine is obtained by the hydrogenation of vegetable oils (Li et al., 2019) that adds hydrogen atoms to the unsaturated bonds yielding a more saturated oil with triglycerides.Hydrogenation greatly modifies the physical and chemical properties of oils (Puprasit et al., 2020).However, this technique produces trans fatty acids (Li et al., 2019) known to adversely affect human health in terms of cardiovascular diseases, obesity, and cancer (Hu et al., 2017).Therefore, partial replacement of dietary saturated fatty acids (SFA) with their polyunsaturated counterparts (PUFA) is a cornerstone of numerous nutritional recommendations for protection against coronary heart disease (Virtanen, 2018;Karadağoğlu et al., 2019).
Oil improves the feed intake, palatability, immunity, and reduce the morbidity in poultry (Özdoğan and Sarı, 2001;Palmquist, 2009;Gopi et al., 2014;Stevanovic et al., 2018).Additionally, dietary fats are known to significantly alter the fatty acid composition of egg yolk (Rowghani et al., 2017;Skrtic et al., 2008) thereby allowing consumers to consume low-cholesterol eggs to control their blood cholesterol levels (Hoan and Khoa, 2016).Therefore, appropriate amount and type of dietary fat is significant for laying performance, lipid metabolism, and egg quality of laying hens (Gao et al., 2021).
It is widely accepted that margarine has adverse effects on human health due to the trans fatty acids that occur during the production of margarine (Hu et al., 2017).Studies involving animals on this subject could be more extensive since most studies have reported the use Journal of Animal & Plant Sciences,34(2): 2024, Page: 445-453 ISSN (print): 1018-7081; ISSN (online): 2309-8694 https://doi.org/10.36899/JAPS.2024.2.0730 of partially hydrogenated oils (older-type margarine).In new-generation margarine, some oil is fully hydrogenated (fully solidified) and mixed with non-hydrogenated (liquid) oil to adjust its softness.To the best of our knowledge, no study has been conducted so far on the use of new-generation margarine in laying hen diets.Therefore, this study was aimed to investigate the effects of different inclusion levels of dietary margarine containing two different saturated fatty acid contents on laying performance, egg quality, yolk fatty acids, and serum lipid metabolites of laying hens.

MATERIALS AND METHODS
Ethical statement: This study was conducted at Burdur Mehmet Akif Ersoy University, Turkiye as the coordinates 37°41'28" South latitude, 30°20'35" West longitude pursuant to the guidelines of the institutional Committee for Ethical Use of Animals approved vide protocol no.93773921-11.

Animals, experimental design, and diets:
The study was executed as a completely randomized design consisting of five experimental groups, each comprising of 6 replicates having 2 laying hens/replicate.One group serving as control was fed diet without the inclusion of margarine.Other groups were fed diets with 5 or 10% of 17% (M17-5 and M17-10) or 35% (M35-5 and M35-10) saturated margarines.The nutritional composition of margarines used has been summarized in Table 1.All the groups were fed isocaloric and isonitrogenous diets (Table 2) to meet or exceed the nutrient requirements in line with the recommendations of NRC (1994).Laying hens were housed in 2-storey and 2-compartment rearing cages designed for laying hens.The trial lasted for eight weeks.During the experiment, a 17-h L:7-h D lighting program was followed.Each replicate was subjected to group feeding.Laying hens were allowed ad libitum access to feed and water.
Productive performance and egg quality: Feed consumption was recorded on weekly basis.The amount of feed consumed to produce one kg and a dozen eggs were calculated.Laying hens were weighed at the commencement and at the end of the of the experiment.Live weight change was computed using the difference method.
Egg production was recorded continuously, eggs were weighed and stored at 15°C.External and internal egg quality was measured every two weeks using following methods: Egg size was measured in terms of width and length using a tripod micrometer.Yolk color, eggshell thickness, eggshell weight, shape index, albumen index, yolk index, and Haugh unit (HU) of 18 eggs from each group were evaluated.Shell thickness (mm) was measured from 3 different parts (upper and lower ends, and middle) using a micrometer.Egg yolk color was measured using the Roche Yolk Color Fan (The CIE standard colorimetric system, F. Hoffman-La Roche Ltd., Basel, Switzerland).Other egg quality measurements were calculated using the following equations: Where: H: albumen height W: egg weight Fatty acid composition of egg yolk and diets: Fatty acid composition of diets and egg yolks was measured from 12 eggs in each group using gas chromatography.Eggs were randomly selected, broken, and yolks were separated.Fat from diets and egg yolks was extracted by mixing with 20 ml solution of chloroform and methanol (2:1).Then, the organic phase was taken and evaporated.Fatty acid methyl esters (FAMEs) were prepared by saponification with sodium hydroxide followed by mixing with the solution of boron trifluoride in 35% methyl alcohol (methylated boron trifluoride).FAMEs were separated by the addition of n-hexane, vortexed, condensed, and filtration of the condensate.The condensate was injected into the GC-MS system (AGILENT 5975C&7890A GC, Hewlett Packard, Santa Clara, CA, US) fitted with 100 m × 0.25 mm × 0.2 μm column (HP-88 capillary column, Agilent J&W, Santa Clara, CA, US) under following conditions: initial temperature 60°C increased to 175°C with an increase in temperature of 13°C per minute followed by 4°C increments to 215°C that was maintained for 35 minutes.Finally, injector and detector temperatures were increased to 250ºC (Bardakçı and Seçilmiş, 2006).Fatty acid composition of diets is presented in Table 3.

Serum cholesterol and triglyceride levels:
At the end of the experiment, blood was collected from the wing vein, allowed to clot, and serum was separated by centrifugation at 3000 rpm for 10 minutes.Serum triglyceride and cholesterol were analyzed using commercial kits (Roche Diagnostics International Ltd., Risch-Rotkreuz, Switzerland) followed by the measurement of these metabolites in the plate reader (Roche COBAS Integra 800; Roche Diagnostics International Ltd., Risch-Rotkreuz, Switzerland).

Statistical analysis:
Data were analyzed by one-way ANOVA using the statistical software package SPSS (version 22.0, Armonk, NY, US) to assess the effect of diets containing two different types and doses of margarine on laying performance, egg production, egg quality, fatty acid profile of egg yolk, and serum cholesterol and triglyceride levels of laying hens.Following statistical model presented in equation 1 was applied: Where: Yij = phenotypic value of the trait for the jth group of laying hens belonging to ith type and inclusion levels of margarine in laying hen diets; µ = mean value of the trait for a given population; si = effect of ith type and inclusion levels of margarine in laying hens; eij = effect of experimental error.Duncan's multiple range test was applied as post-hoc test to separate the significantly different means.Significance of the results was assumed at 95% probability.Results were shown as mean ± SEM.

RESULTS
Laying performance and serum metabolites: This study showed that the addition of margarines at different inclusion levels had no effect on daily feed consumption, feed conversion rates (dozen eggs/ kg feed and kg eggs/ kg feed) during the experimental period.While weekly egg yield was not different among the groups in 1-4 weeks, it was lower in control and M17-5 groups at 5-8 weeks than in other groups (Table 4).Body weight of laying hens fed two doses of 35% saturated margarine was greater in comparison with other groups (P ≤ 0.001) (Table 5).Serum triglyceride and cholesterol levels were not different among the groups (Table 6).
Egg quality: Egg quality of laying hens fed diets with different inclusion levels of 17 or 35% saturated margarines remained unaffected in comparison with control group (Table 7).

DISCUSSION
Laying performance: The addition of fat to balance the energy level in the diet is a common practice in commercial animal feed industry.However, laying performance of hens relies on several factors such as breed, body weight, age, and energy level of the diet (Hoan and Khoa, 2016).In this study, it was expected that margarine with two different degrees of saturation may alter the nutritional composition of eggs and the performance of laying hens.This study showed that the addition of margarine with different saturation points at 5 or 10% dietary inclusion levels had no effect on feed consumption, egg yields, and feed conversion rates of laying hens.Similar findings were reported by Hosseini-Vashan and Afzali (2008) following the use of 0, 1.5, 3, and 4.5% palm oil in laying hen diets.Other studies reported that the addition of different fat levels in laying hen diets was not effective in the manipulation of egg weight (Grobas et al., 2001) and feed conversation (Lelis et al., 2009).On the contrary, Fouladi et al. (2021) reported that adding different fat levels to the diets of laying hens reduces feed intake.These findings might be attributed to the levels of added fat that increased the energy level of the ration thereby reducing egg production, egg weight, and feed intake (Agah et al., 2010).Hoan and Khoa (2016) reported that laying performance of laying hens such as egg production, egg weight, and feed intake were decreased in response to the supplementation of different dietary sesame oil levels but not feed conversion ratio.Another study showed that feed intake, egg production, egg weight, egg yolk weight, liver mass, and cholesterol levels of liver and egg yolk were not affected in laying hens fed diets enriched with plant sterols (Liu et al., 2010).
Egg quality: Egg quality attributes like shape index, breaking strength, shell thickness, weight, yolk color, yolk index, flow index, Haugh unit, and egg yolk fatty acid composition depend significantly on the composition of diet rather than energy levels.Vegetable oils, widely used in animal diets, are rich in polyunsaturated fatty acids.Most of these fatty acids significantly relate to the growth and production performance of laying hens (Hoan et al., 2016).In the present study, shape index, yolk index, albumen index, yolk color, Haugh unit, and egg weight remained unaffected across the groups.Similarly, Kucukersan et al. (2010) reported that different oils added to laying hen diets affect the egg production and egg weight but not feed consumption and efficiency.Rowghani et al. (2017) reported that addition of 3% or 5% rapeseed oil to the basal diet of 24-week-old Hy-Line Brown chickens had no significant effect on egg weight.Hosseini-Vashan and Afzali (2008) reported that Haugh unit score, yolk color index, yolk index, egg shape index, shell weight, shell thickness, and egg density of laying hens were not affected by the addition of 0, 1.5, 3, and 4.5% dietary palm oil.Likewise, Hoan and Khoa (2016) described that increasing levels of sesame oil in laying hen diets did not affect the shape index, breaking strength, or shell thickness.Similarly, Liu et al. (2010) reported that laying hens receiving diets enriched with plant sterols had no effect on the Haugh unit of eggs.
In our study, serum cholesterol and triglyceride levels remained unaffected across the groups.Likewise, Sim and Bragg (1977) reported that the addition of cholesterol to the basal diet including 8% safflower oil caused a significant increase in serum cholesterol levels, while in combination with hydrogenated coconut oil, it did not change the serum cholesterol level.
In the present study, inclusion of margarines with different degrees of saturation significantly altered the fatty acids composition of egg yolk.In addition, total UFA levels were higher when laying hens were fed dietary margarine with 35% degree of saturation.Besides these, there was no defined relationship between MUFA composition of egg yolks, and the type of margarine added to the diet.Several studies have reported that egg quality and yolk fatty acid profile can be modified by the inclusion of lipid sources in the diets of laying hens (Wu et al., 2005;Ribeiro et al., 2007;Oliveira et al., 2010).These differences may be related to the content of saturated and unsaturated fatty acids in the diets and genetic differences in the metabolism of each chicken.Oliveira et al. (2010) reported that, regardless of the source of lipids and the age of the laying hens, the percentage of trans fat in the egg yolk is meagre.The main problem with trans-fat is that despite being plantbased and unsaturated, it is metabolized more like saturated fat and converted to cholesterol, which is one of the contributing causes of cardiovascular diseases (Oliveira et al., 2010).In our study, C18:2 and C18:3 concentrations were proportional to their levels in the diet.Because these fatty acids play an essential role in human health and are the precursors of longer-chain fatty acids such as the n-3 and n-6 series, it is desirable to have these fatty acids in balanced concentrations in the egg yolk.Also, mammals do not synthesize these fatty acids and are obtained only from the diet (Oliveira et al., 2010).For linoleic acid (C18:2 n-6), laying hens fed diets containing 5% of 35% saturated margarine and control diets had higher concentrations in the yolk, followed by those in M17-5, M17-10, and M35-10 groups.The highest concentration of alpha-linolenic acid (C18:3 n-3) was seen in M17-5 and control groups followed by M35-10 and M17-10 groups.Shafey et al. (1992) also confirmed that adding vegetable oils to the diets of laying hens increases the linoleic acid concentration in the egg yolk.

Conclusions:
In conclusion, inclusion of different dietary levels of margarines with different degrees of saturation may not affect the laying performance, serum fat metabolites, and egg quality of laying hens.However, dietary margarines with different degrees of saturation at different inclusion levels may alter the fatty acid composition of egg yolk.Further studies elucidating the hepatic metabolites of fats and liver fatty acids may enlighten the possible net anabolism and net catabolism of fatty acids involved in the final fatty acid composition of eggs.