Effect of pomegranate seed oil on egg production, egg quality and yolk fatty acid deposition in laying hen

Abstract Background Pomegranate seed oil (PSO) contains punicic acid as well as conjugated linolenic acid isomers, including α‐eleostearic and catalpic acids, along with phytosterols, especially β‐sitosterol, campesterol and stigmasterol, with lipotropic impact and egg fortifying effect in laying hens. Objectives The present experiment was designed to examine the effects of PSO on egg production, egg quality, blood lipids and yolk fatty acid deposition in laying hens. Methods A total of 360 Hy‐line laying hens (w‐80), at 25 weeks of age, were randomly allotted to five dietary treatments in a completely randomized design during a 10‐week period. Experimental treatments consisted of a basal diet or supplementation of 1, 2, 3 and 4 g PSO/kg to basal diet. Performance indicators and fatty acid composition of egg yolk were evaluated during different experimental periods. Blood lipid attributes were evaluated at the end of the experiment. Results Dietary supplementation of 4 g PSO/kg feed increased daily feed intake and egg production rate of laying hens (p < 0.05). There was not any significant influence of experimental treatments on egg quality, whereas an increasing trend observed in egg yolk colour of hens received dietary graded levels of PSO. Dietary supplemental 4 g/kg PSO increased the proportion of yolk poly‐unsaturated fatty acid (PUFA) concentration (p < 0.05). Furthermore, the PUFA to saturated fatty acid ratio increased after dietary supplementation of 1 or 4 g/kg PSO (p < 0.05). Serum concentration of cholesterol, triacylglycerol and low‐density lipoprotein decreased in response to the supplementation of more than 3 g PSO/kg in the feed (p < 0.05). Conclusions In conclusion, dietary supplementation with 4 g/kg PSO improved production rate and decreased blood lipids in laying hens. Moreover, dietary supplemental PSO modified yolk fatty acid deposition without detrimental effects on the egg quality.

certain nutrients for consumers (Surai & Sparks, 2001).Moreover, the possibility of using table eggs as a functional food to transfer conjugated fatty acid (CFA) to the egg yolk has already been verified (Hur et al., 2003).The CFAs are isomers of poly-unsaturated fatty acids (PUFA) with conjugated double bonds and are considered effective in prevention and treatment of chronic diseases.Furthermore, CFA has another conjugated form named conjugated linoleic acid (CLA) which shares beneficial physiologic properties against atherosclerosis, obesity, tumour and hypertension (Lee et al., 1994).However, incorporating CFA to the egg yolk might deteriorate sensory properties and shelf life of the egg (Dunn-Horrocks et al., 2011).Moreover, some parameters of the egg quality were compromised in response to the supplementation of hen diet with CLA oil, whereas quality was not affected when CLA was combined simultaneously with some other fatty acids (Kim et al., 2007).In this respect, plant seeds such as flaxseed or pomegranate seed oil (PSO) are commonly added to the diet of laying hen in order to fortify egg with alpha-linolenic acid and its long-chain anabolic derivative, which have shown health benefits in terms of inflammatory processes (Calder, 2017).Conjugated linolenic acid (CLnA) contains three conjugated double bonds as well as the carbon structure (18:3) of a-linolenic acid.Punicic acid (9cis, 11trans, 13cis-cLNA; 9c, 11t, 13c-CLNA) is a type of CFA contained in PSO at a level of about 72% (Suzuki et al., 2001).The PSO also contained cLNA isomers other than punicic acid, including α-eleostearic and catalpic acids, along with phytosterols, especially β-sitosterol, campesterol and stigmasterol, which are also considered to induce health beneficial effects.The PSO was reported to decrease hepatic triacylglycerol accumulation in obese hyperlipidemic rats (Arao et al., 2004) and decreased plasma cholesterol and low-density lipoprotein (LDL) concentration in rats fed with an atherogenic diet (Elbandy & Ashoush, 2012).On the other hand, improvement in egg production was reported when laying hen received diets supplemented with 2.5% PSO (Kostogrys et al., 2017).Moreover, successful transfer of CLA to the egg yolk was observed when layers received diets supplemented with PSO (Kostogrys et al., 2017) or mix of flaxseed and PSO (Ngo Njembe et al., 2021).Although research using CLA as a functional food is found in the literature, the effect of seed oils containing CLA isomers such as dietary punicic acid on egg deposition of laying hen is limited.Moreover, the effect of PSO on egg quality and blood lipids is less investigated.Thus, the present experiment was designed to investigate effects of dietary supplementation with 1, 2, 3 and 4 g PSO/kg on egg production variables, egg qualitative indices, serum biochemical attributes and yolk fatty acid deposition in laying hens.

MATERIALS AND METHODS
All animal work in the present study was conducted according to the comprehensive guidelines of the animal welfare adopted by Federation of Animal Science Societies (FASS) (2010).The experiment was conducted according to the regulations and guidelines established by this committee.

Preparation of pomegranate seed and pomegranate seed oil
The pomegranate was purchased from a local market as fresh Iranian pomegranate (Malas Saveh).The seeds were manually removed from arils and then air dried under ambient condition and finely ground in a grinder to pass 40-33 mesh.The seed powder was finally packed and stored at −18 • C. In order to prepare the PSO, pomegranate seeds were oiled by cold pressing method and applied in dietary treatments.

Active components and fatty acid composition of pomegranate seed oil
The fatty acid composition of PSO (Table 1) was determined by a direct method (O'Fallon et al., 2007) for fatty acid methyl ester synthesis using a gas chromatograph (YOUNG LIN ACME).Determination of total antioxidant activity applied through the methods described in the work of Madaan et al. (2011) and total phenolic contents (Table 2) assayed according to the method of Dewanto et al. (2002).

2.3
Birds, diets and experiment design consisted of five groups of birds fed a basal diet (CTL) or basal diet supplemented with either 1, 2, 3 or 4 g PSO/kg during a period of 10 weeks.
Iso-energetic and iso-nitrogenous experimental mash diets were formulated to be used in this experiment.Diet formulation was performed according to Hy-Line W-80 breeder recommendation guideline and according to their production phase (Table 3).Feed and water were ad libitum provided.The birds were exposed to continuous daily lighting for 17 h.Before the beginning of the trial, a 2-week pre-experiment period was carried out to ensure the eggs production rate was similar among the experimental groups.

Performance indicators and egg quality
Daily feed intake (DFI), egg production rate and egg weight were recorded for each replicate.Furthermore, egg mass (g egg/hen/day) and feed conversion ratio (g feed consumed/g egg mass) were also calculated.Collecting and weighting of eggs were carried out at the same time every afternoon.
A total of four fresh laid eggs/cages were collected from each treatment on the same day.After weighing the individual eggs, a force reader (Orka Food Technology) was used to break each egg to mea-sure eggshell breaking strength.Eggshell thickness is presented as an average value based on the thickness of eggshell measured at three different places (upper end, lower end and the middle) using a micrometer screw gauge (Suce Measuring Instrument Co., Ltd. ).An automatic egg quality analysis instrument was used to measure the haugh units and yolk colour (EMT-5200, Robotmation Co., Ltd.).The albumen was removed from shells, and shells plus membranes were weighed after 24 h of air drying.Specific gravity of the egg was measured by dipping the eggs in a predetermined salt solutions specific gravity ranging from 1.060 to 1.100 (Peebles & McDaniel, 2004).

Fatty acid composition of egg yolk
Total egg yolk lipids were extracted using the FAT Extractor TFE 2000, with liquid carbon dioxide as a solvent (Domagała et al., 2010).
After the extraction of fat (described above), the lipids were esterified according to the method described by de Man (1964).A volume of 0.1 mL of extracted fat was placed in the glass test tube of 2 mL capacity, and 0.5 mL of 0.025 M solution of sodium methylate was added.The mixture was heated in a closed tube at 60 • C until the mixture was clear.
The analysis of fatty acids was carried out using gas chromatograph Fatty acid methyl esters were identified by comparing their retention times with authentic standards (Sigma-Aldrich) and the Punicic Acid Standard (Larodan Fine Chemicals AB).The average laboratory sample was prepared from 20 randomly selected eggs.The analyses were performed in triplicates.

Statistical analysis
Data for recorded traits were subjected to the analysis of variance procedures appropriate for a completely randomized design using the General Linear Model procedure of SAS 9.2 (SAS Institute Inc.).For all statistical analyses, significance was declared at p ≤ 0.05, unless otherwise stated.The Tukey test was used for multiple treatment comparisons.Results are presented as least square means ± SEM.

Performance indicators and egg quality
Effects of experimental treatments on egg production and egg qualitative variables are shown in Tables 4 and 5, respectively.Increased DFI and egg production rate were observed as a response to 4 g dietary supplementation of PSO/kg compared to CTL diet (p < 0.05).Moreover, birds received 4 g/kg PSO in their feed displayed greater DFI and egg production than those fed 1 g/kg PSO supplemented diets (p < 0.05).
Egg quality was not affected by experimental treatments; however, an increasing trend observed in egg yolk colour of hens received graded levels of PSO by 7.4% compared to control group.

Serum biochemical parameters
As indicated in Table 6, the supplementation of PSO to the feed decreased serum triacylglycerol content (p < 0.05).Similarly, reduction in blood cholesterol concentration observed with dietary 3 or 4 g PSO/kg supplementation (p < 0.05).In comparison to CTL diet, serum HDL content increased in hens received 2-4 g PSO/kg feed (p < 0.05), whereas blood LDL concentration decreased with the inclusion of 3 or 4 g PSO/kg feed (p < 0.05).

Fatty acid composition of egg yolk
Fatty acids' profiles in the egg yolks are shown in Table 7. Dietary supplemental 4 g PSO/kg increased the proportion of yolk PUFA (p < 0.05).
Moreover, the PUFA to SFA ratio (PUFA/SFA) increased after dietary supplementation of 1 or 4 g PSO/kg (p < 0.05).Additionally, total proportion of PUFA and MUFA was greater in egg yolk of birds received 1 g PSO/kg diet (p < 0.05).Eicosapentaenoic acid (EPA; C20:5) concentration increased when greater than 2 g PSO/kg added to the feed (p < 0.05).Moreover, dietary supplemental 4 g/kg PSO caused higher yolk EPA content than the other PSO levels (p < 0.05).Furthermore, the yolk docosahexaenoic acid (DHA; C22:6) content decreased in response to the dietary supplementation of more than 3 g PSO/kg feed compared to CTL and lower levels of supplemental PSO (p < 0.05).

DISCUSSION
The PSO was evaluated for fatty acid profile, antioxidant activity (7.55%) and total phenolic (10.52 mg GAE/g) contents.All these values fell within the physiological range similar to the other Iranian  (2018).
Dietary supplementation of 4 g/kg PSO increased DFI and egg production rate of laying hens, suggesting the growth promotional function of PSO.Similarly, Arao et al. (2004) found an improvement in feed efficiency of obese rats received diets containing 1% PSO.Moreover, Chin et al. (1994) reported that CLA is a dietary factor that improves the growth and feed efficiency of rats.There are limited information on the effect of PSO on the performance of laying hens.In this respect, Kostogrys et al. (2017) showed that the supplementation of dietary 2.5% PSO improved DFI and laying rate of hens.Therefore, the increased rate of laying in response to PSO might be due to the improvement in palatability and consequently enhanced feed intake.
Active components of PSO also possess anti-oxidative properties, protecting body organs from undesirable effects of free radicals.On the contrary, Ngo Njembe et al. ( 2021) could not find any effect of dietary supplemental 7 wt% PSO mixed with 7.5% flaxseed oil on the laying performance of laying hens.Thus, further investigations are needed to evaluate the effect of PSO on laying performance.
As Kim et al. (2007) showed an improvement of egg quality in layers fed with a dietary combination of CLA and other fatty acids, we expected that using PSO as a source of CLnA and punicic acid might enhance egg quality.However, egg quality was not significantly affected using PSO, but there was a tendency to increase egg yolk colour with the consumption of higher PSO levels.This is in accordance with the work of Kostogrys et al. (2017) who reported a greater egg yolk colour in laying hens fed diets supplemented with 2.5% PSO.It is likely that higher levels of PSO supplementation compared to this trial made significantly darker colour.Reports have shown beneficial impact of feed additives on nutritional value of egg (Ahmad et al., 2012) which shows the opportunity to enrich the human food through dietary modification of poultry feed but the main obstacle is the subsequent reduction in egg quality (Franczyk-Żarów et al., 2008) and egg sensory properties (Kostogrys et al., 2017).This is in contrast to our data, suggesting that the inclusion of PSO did not induce any adverse effect on Haugh unit, specific gravity and shell thickness.It shows that PSO could keep proper quality of a typical fresh egg without any detrimental impact.
Fatty acid profile of the egg yolk could be affected by fatty acid composition of hen diet (Ahmad et al., 2012).Hence, blood lipid contents could be in part indicators of egg lipid concentrations.Data showed that serum cholesterol, triacylglycerol and LDL contents decreased in response to supplementation of more than 3 g PSO/kg in the feed.Generally, CFA are known as hypolipidaemic agents (Elbandy & Ashoush, 2012) and PSO containing conjugated octadecatrienoic fatty acids that is considered one of the isomers of CLN.The PSO was reported to decrease plasma total cholesterol and LDL cholesterol in rats receiving an atherogenic diet (Elbandy & Ashoush, 2012).Furthermore, a gradual reduction in serum total cholesterol and LDL of birds fed PSO and linseed oil combination observed in the work of Manterys et al. (2016).In contrast, Du and Ahn (2003) indicated that the dietary supplementation of 2% and 3% CLA increased plasma total cholesterol concentration in broiler chickens.On the other side, dietary PSO supplementation did not affect the serum lipid level of Otsuka longevans tokushima fatty rats but decreased triacylglycerol accumulation in their liver (Arao et al., 2004).Results on the effect of CFA on lipid metabolism are inconsistent and it seems that isomer configuration and amount of CLN and CLA are determining factors (Mirmiran et al., 2010).
Generally, an arrangement of lipids formed in the liver for yolk synthesis can be affected by diet (Walzem, 1996).Data obtained on the enrichment of eggs with PUFA is in agreement with findings of Kos- acid (Szymczyk & Szczurek, 2016).The egg yolk SFA content was not affected by the inclusion of PSO in the feed, whereas the PUFA to SFA ratio increased.Due to the fact that ω − 6 and ω − 3 are essential fatty acids in human diet, maintaining a proper ratio of ω − 6 and ω − 3 fatty acids not only improves performance but also prevents health risks (Alagawany et al., 2018).In the current experiment, the ω − 6 to ω − 3 ratio did not change in response to PSO supplementation to the feed, suggesting a lack of its detrimental impact on cardiovascular and neurodegenerative (Avallone et al., 2019) disease.

CONCLUSION
Dietary supplementation of PSO enhanced DFI, laying performance and tended to increase egg yolk colour of laying hen received diets containing 4 g/kg PSO.Serum cholesterol, triacylglycerol and LDL contents decreased following supplementation of more than 3 g PSO/kg in the feed, suggesting a hypolipidaemic effect of CFA in the PSO.Yolk deposition of EPA increased in birds fed 4 g/kg PSO added diets, whereas DHA decreased with the same supplemental level.Taken together, these data indicate that the dietary supplementation of 4 g/kg PSO could efficiently improve laying performance and beneficially modify egg fatty acid contents without any undesirable effects on the egg quality.
Trace GC Ultra (Thermo Electron Corporation) with a Supelcowax 10 column (dimensions 30 m × 0.25 mm × 0.25 mm).Helium as a gaseous phase was applied with the flow rate of 5 mL/min.The feeder had the temperature of 220 • C. The temperature of the column was kept for 3 min at 60 • C and then increased at a rate of 7 • C/min up to 200 • C and held at this temperature for 20 min.The detector had the temperature of 250 • C, and the split flow was 10 mL/min.Peak identification was possible with the aid of an external standard (FIM/FAME Supelco).
togrys et al. (2017) and Ngo Njembe et al. (2021) when birds fed on diets supplemented with PSO or a combination of PSO and flaxseed oil, respectively.In this regard, the deposition of DHA or EPA increased in the egg yolk of birds fed PSO supplemented diets.Similar data in animal products observed when concentrations of EPA and DHA increased in the breast muscle of broiler chickens fed diets containing punicic Fatty acid composition of pomegranate seed oil.
TA B L E 1 At the end of the experimental period, blood samples were collected from two birds of each cage and put in non-heparinized tubes by brachial vein puncture.Serum was separated via 2000 × g centrifuge of blood samples for 15 min (SIGMA 4-15 Lab Centrifuge).Individual serum samples were analysed for total cholesterol, triacylglycerol, high-density lipoprotein (HDL), LDL, calcium, phosphorus, iron and magnesium with a spectrophotometer using the kit package (Pars Azmoon Co.) according to manufacturer's guidelines.
Egg production variables of laying hens during 25-35 weeks age.Egg qualitative traits of laying hens.
TA B L E 4Note: Means in the same row with different superscripts (a and b) differ significantly (p < 0.05).*CTL: a basal diet; PSO1, PSO2, PSO3 and PSO4 represent a basal diet supplemented with 1, 2, 3 and 4 g/kg pomegranate seed oil, respectively.TA B L E 6 Serum biochemical parameters.pomegranatecultivars as indicated in the work ofDerakhshan et al.
Fatty acid profile of egg yolks.
TA B L E 7