Effect of Quercetin on Egg Quality and Components in Laying Hens of Different Weeks

You Ying， Han Chun-yan， Chaudhry Maria Tabassum， Li Ling， Yao Jia-ying， Wang Sheng-nan， Yang Jia-xin，Teng Nan， and Li Yao

College of Animal Sciences and Technology， Northeast Agricultural University， Harbin 150030， China

Effect of Quercetin on Egg Quality and Components in Laying Hens of Different Weeks

You Ying， Han Chun-yan， Chaudhry Maria Tabassum， Li Ling， Yao Jia-ying， Wang Sheng-nan， Yang Jia-xin，Teng Nan， and Li Yao*

College of Animal Sciences and Technology， Northeast Agricultural University， Harbin 150030， China

This trial was conducted to evaluate the effect of quercetin on egg quality and components in laying hens of different weeks. A total of 240 healthy Hessian laying hens at 29， 39-week-old with similar body weight and laying rate were randomly divided into four groups with six replicates of 10 each replicate， respectively. The treatments were fed with basal diet supplemented with 0，0.2， 0.4 and 0.6 g · kg-1quercetin for 8 weeks. The results showed that compared with the control， broken or soft shell rate significantly decreased at 0.2 and 0.4 g · kg-1quercetin and eggshell thickness significantly increased at 0.4 g · kg-1quercetin （P＜0.01） in laying hens at 39-47 weeks old； yolk protein significantly decreased at 0.6 g · kg-1quercetin （P＜0.05） in laying hens at 29-37 weeks old； while yolk protein significantly increased at three quercetin treatments in laying hens at 39-47 weeks old； yolk cholesterol significantly decreased by quercetin in laying hens at 29-37 weeks old （P＜0.05）； yolk total phospholipids significantly increased at 0.4 and 0.6 g · kg-1quercetin （P＜0.01） and yolk cholesterol significantly decreased at 0.6 g · kg-1quercetin （P＜0.05） in laying hens at 39-47 weeks old. In a word， quercetin affected egg quality and components to some extents in laying hens of different weeks， the older the hens became，the better improvement they would be. The optimum level of quercetin was 0.4 g · kg-1in the basal diet.

quercetin， laying hens， egg quality， egg components

Introduction

Eggs are the donor of main nutrients of chick embryo from the early development to hatching， a lot of nutrients are deposited in favor of embryonic development. In China， egg products， such as preserved and salted eggs， which have always been very popular，while the quality of the egg products is directly affected by egg quality. A large number of nutrients are used for egg formation in high-yielding laying hens， nutrient requirements also increase with the increasing of egg production， thereby laying hens are unable to slow down the attack of nutrient deficiencies resulting from growth and development， eventually lead to decrease the laying rate， thinning eggshell， poor egg quality because of lacking of calcium and proteins. Studies showed that strength and thickness of eggshell decreased with the increasing ages of laying hens （Ni et al.， 2007）. Flavonoids， widely presented in plants，exhibit a varieties of physiological activities， including anti-oxidation， anti-stress， lipid metabolic regulation，free radical scavengers， etc.， therefore， improving performance and egg quality of poultry. Also， flavonoids are a kind of mixture composed of a class of compounds with the structure of 2-phenyl chromone.Quercetin （3， 3'， 4'， 5， 7-pentahydroxyflavone）， one of the main components of flavonoids， played the role of anti-inflammatory （Hwang et al.， 2009）， antiviral（Murakami et al.， 2008； Shan et al.， 2009）， antiapoptosis （Cheng et al.， 2009； Hirpara et al.， 2009），anti-oxidation （Kebieche et al.， 2009； Romero et al.，2009）， anti-fibrillatio （Phan et al.， 2003）， skin enrichment and protection properties （Vicencentini et al.， 2011）， etc. Quercetin played a role in regulating lipid metabolism in animals， prevented the occurrence of non-alcoholic fatty liver caused by a high fat diet and alleviated atherosclerosis induced by a high cholesterol diet （Bhaskar et al.， 2013）； quercetin regulated fat anabolism and catabolism via inhibiting PPARγ expression in lipocytes of broiler （Li et al.，2013） and fat anabolism via regulating cAMP signal pathways （Ouyang et al.， 2013）， thereby reducing fat deposition in broilers. Additionally， quercetin possesses many similarities with estrogen in chemical structure，thereby plays a weak estrogen-like role. Estrogen promotes intestines to absorb nutrients， improves egg quality， regulates lipid metabolism， so it remains to be verified whether quercetin could improve egg quality and components and the optimal time of supplement. The previous studies showed that a certain dose of quercetin improved performance of laying hens via affecting cecal microflora of laying hens and promoting absorption of dietary nutrients， and the older the bird became， the better improvement the performance would be. To further investigate the effect of quercetin on egg quality and components in laying hens of different weeks old and the optimal time of supplement， Hessian laying hens at 29， 39-week-old were fed with basal diet supplemented with different levels of quercetin in this trial， it would provide a theoretical basis for quercetin as a new， functional feed additives.

Materials and Methods

Experimental animals and designs

A total of 240 healthy Hessian laying hens （29， 39 weeks old） with similar body weight and laying rate were taken from a commercial company （Yinong Poutry Limited Company， Harbin， China）. The hens were kept in wire cages in a temperature-controlled room at 22±1℃ and water was offered ad libitum. A 16 h light to 8 h dark lighting program was applied throughout 8 experimental weeks. This study was approved by the Animal Care and Use Committee of the University. Animals used in this trial were cared for under the guidelines stated in the Guide for the Care and Use of Agricultural Animal in Agricultural Research and Teaching of Heilongjiang Province. Total 240 laying hens were randomly divided into four groups with six replicates of 10 each replicate，respectively. The treatments were fed with basal diet supplemented with 0， 0.2， 0.4 and 0.6 g quercetin （97%，HPLC， Sigma-Aldrich Company St. Louis， Missouri，USA） per kilogram diet. Composition and nutrient levels of basal diet are given in Table 1.

Table 1 Composition and nutrient levels of basal diet （airdry basis， g · 100 g-1）

Sample collection and measurement

The number of broken or soft shell egg was recorded once a week during 8 experimental weeks. Egg quality was determined using two eggs from each replicate at the same time each week. Egg quality included egg shape index， Haugh unit values， eggshell thickness and eggshell relative weight. Yolk and albumen were separated and yolk was placed in open culture dishes（pre-weighted）.

Yolks were dried at 45℃ in an oven for 48 h to constant weight at the end of the experiment. Yolks were made into powder by grinding after drying to constant weight. Crude protein of yolk and albumen was determined using a Kjeldahl apparatus（KT-2300， FOSS）. Ether extract of yolk was determined using Soxhlet extraction method. Cholesterol and phospholipid of yolk were determined using a cholesterol kit （BIOSINO） and inorganic phosphorus kits （Nanjing Jiancheng Bioengineering Institute） with spectrophotometer （Shimadzu UV1206，Japan）.

Statistical analysis

The results were expressed as means and standard errors. Data was analyzed by One-way ANOVA analysis and Duncan's multiple comparisons using SPSS18.0 （IBM， SPSS， 2009）. Statistical significance was considered at P＜0.05.

Results

Effect of quercetin on egg quality in laying hens at 29-37 weeks old

Supplement of quercetin did not affect egg quality（P＞0.05） compared with control （Table 2）.

Table 2 Effect of quercetin on egg quality in laying hens at 29-37 weeks old

Effect of quercetin on egg quality in laying hens at 39-47 weeks old

Compared with control， broken or soft shell rate significantly decreased by 50.22% and 38.22% at 0.2 and 0.4 g · kg-1quercetin （P＜0.01）， and significantly decreased by 54.29% and 43.27% （P＜0.01） at 0.2 and 0.4 g · kg-1quercetin than that at 0.6 g · kg-1quercetin； eggshell thickness significantly increased by 8.45%（P＜0.01） at 0.04 g · kg-1quercetin； Haugh unit significantly increased by 7.06%， 5.57%， and 6.91%（P＜0.05） and yolk color increased by 7.08%， 6.84%，and 7.32% （P＜0.05）， respectively； while no significant differences in egg shape index， eggshell relative weight， eggshell strength and yolk weight/egg weight were observed （P＞0.05） （Table 3）.

Table 3 Effect of quercetin on egg quality in laying hens at 39-47 weeks old

Effect of quercetin on egg components in laying hens at 29-37 weeks old

Yolk protein significantly decreased by 3.13%（P＜0.05） and 5.65% （P＜0.01） at 0.4 and 0.6 g · kg-1quercetin； yolk cholesterol significantly decreased by 9.06% （P＜0.05）， 14.14% （P＜0.01）， and 7.94%（P＜0.05）， respectively， while no significant differences in albumen protein， albumen water， yolk water，yolk total phospholipids and yolk ether extract were observed （P＞0.05） compared with control（Table 4）.

Table 4 Effect of quercetin on egg components in laying hens at 29-37 weeks old （%）

Effect of quercetin on egg components in laying hens at 39-47 weeks old

Yolk protein significantly increased by 20.61%，21.64%， and 18.47% （P＜0.05）， respectively. Total phospholipids in yolk significantly increased by 8.23% and 8.73% at 0.4 and 0.6 g · kg-1quercetin （P＜0.01），yolk cholesterol significantly decreased by 12.84% at 0.6 g · kg-1quercetin （P＜0.05）， while no significant differences in albumen protein， albumen water， yolk water and yolk ether extract were observed （P＞0.05）compared with control （Table 5）.

Table 5 Effect of quercetin on egg components in laying hens at 39-47 weeks old （%）

Effect of quercetin on egg quality and components in laying hens of different weeks old

Haugh unit and yolk color of laying hens at 39-47 weeks old significantly increased with the increasing quercetin， but not in dose-dependent manner. In addition， Haugh unit and yolk color were higher in laying hens at 39-47 weeks than those at 29-37 weeks，it suggested that quercetin affected Haugh unit and yolk color， and the older the hens became， the better improvement the egg quality and components would be （Figs. 1 and 2）.

Fig. 1 Effect of quercetin on Haugh unit in laying hens of different weeks old

Fig. 2 Effect of quercetin on yolk color in laying hens of different weeks old

Eggshell thickness in laying hens at 39-47 weeks old significantly increased with the increasing quercetin，and which was thicker than that at 29-37 weeks old. Broken or soft shell rate in laying hens at 39-47 weeks old decreased firstly， but no significant difference with the increasing quercetin， which was lower than that in laying hens at 29-37 weeks old. It suggested that quercetin improved eggshell thickness and decreased broken or soft shell rate， and the older the hens became， the better improvement the egg quality and components would be （Figs. 3 and 4）.

Yolk cholesterol at 0.4 g · kg-1quercetin was thelowest in laying hens at 39-47 weeks old. Total phospholipids in yolk in laying hens at 39-47 weeks old significantly increased with the increasing quercetin. Ether extract in yolk in laying hens at 39-47 weeks old was lower than that at 29-37 weeks old（Fig. 5）.

Fig. 3 Effect of quercetin on eggshell thickness in laying hens of different weeks old

Yolk protein in laying hens at 39-47 weeks old significantly increased with the increasing quercetin，the trend was contrary to the laying hens at 29-37 weeks old. No significant difference in albumen protein， albumen water and yolk water was observed in laying hens of different weeks old （Fig. 6）.

Fig. 4 Effect of quercetin on broken or soft shell rate in laying hens of different weeks old

Fig. 5 Effect of quercetin on ether extract， total phospholipids and cholesterol of yolk in laying hens of different weeks old

Fig. 6 Effect of quercetin on albumen protein， yolk protein， albumen water and youlk water in laying hens of different weeks old

Discussion

Effect of quercetin on egg quality in laying hens

During late laying period， performance and egg quality decreased with the increasing age. Quercetin，a functional feed additive， effectively improved performance of livestock and poultry （Goliomytis et al.， 2014； Liu et al.， 2014）， data from the previous experiment showed that quercetin improved egg quality in hens during late laying period （Liu et al.，2013）. Therefore， the studies of the effect of quercetin on egg quality in laying hens are of practical significance. Egg shape index， one of the indicators in germplasm classification of chickens， which is different in certain breeds， is only related to package and transport. The results of this study showed that no significant difference in egg shape index was observed in laying hens of different weeks old. Eggshell protected egg contents from damage， prevented microbial invasion， and controled the exchange of water and gas during embryo developing period in vitro （Carrino et al.， 1996； Nys et al.， 2004）. Flavonoid such as daidzein and Minshan Trifolium pretense isoflavones significantly decreased broken or soft shell rate in laying hens during late laying period， increased eggshell thickness and strength （Ni et al.， 2007； Wang et al.， 2010）. A mass of calcium in high-yielding laying hens forms eggshell， and calcium requirements increased with the increasing egg production. Calcium deficiency decreased egg production， thinner eggshell occurred because of development of laying hens（Mcdanlel et al.， 1977）. Eggshell quality includes eggshell weight， eggshell relative weight， eggshell thickness and eggshell strength. Studies have shown that eggshell thickness was correlated to a permeation of Salmonella into a complete eggshell， it suggested that eggshell quality was important not only to egg producers， but also for the safety of consumers （Jones et al.， 2002）. Eggshell strength presented the role of resistance to breakage of the eggs， which was not only closely related to eggshell thickness， porous eggshell，eggshell membrane thickness， the mineral content of eggshell and protein matrix， but also to egg freshness and economic value （De Ketelaere et al.， 2002）. Eggshell thickness and eggshell strength of laying hens fed with 10 mg · kg-1T101 （soybean flavonoids as main ingredient） during late laying period were significantly improved （Zhou et al.， 2002）. In the experiment， 0.4 g · kg-1quercetin significantly increased eggshell thickness and 0.2， 0.4 g · kg-1quercetin significantly decreased broken or soft shell rate in laying hens at 39-47 weeks old. While no significant differences in eggshell strength， eggshell weight and eggshell weight/egg were observed in laying hens at 29-37 weeks old， this data was consistent with Suo's（2013） result.

Haugh unit is a universally important index of egg quality nowadays and also a measurement of egg protein quality and egg freshness. A high Haugh unit value indicates more protein viscosity and better protein quality. This study showed that Haugh unit in laying hens at 39-47 weeks old significantly increased，which was in agreement with the effect of soybean flavonoids in laying hens during late laying period （Ni et al.， 2007）. Yolk color， which is mainly influenced by genetic and feed coloring substances， did not affect the edible value of eggs， but influenced consumers' purchasing power to some extents. Pigment absorbed from intestines in poultry was hardly deposited in any tissues other than egg yolk， and chemically， the pigment uniformly deposited into yolk becomes very stable and unchanged （Cheng et al.， 2001）. In the experiment， supplement of quercetin increased yolk color in laying hens at 39-47 weeks old was consistent with the results of soybeans flavone and genistein（Gong et al.， 2009）， and the mechanism of quercetin on yolk color is needed to be further studied.

Effect of quercetin on egg components in laying hens

Eggs are rich in protein and almost all of the proteins can be absorbed by human body， therefore， it is veryimportant to explore the change of protein contents in eggs. Protein deposition in the egg reduces gradually with the more-needed protein for laying hens. The results of this study showed that 0.6 g · kg-1quercetin significantly decreased yolk protein in laying hens at 29-37 weeks old， while three levels of quercetin treatments significantly increased yolk protein in laying hens at 39-47 weeks old. Besides many nutrients in yolk， there is also cholesterol which may increase the risk of cardiovascular disease. Therefore，it is essential to explore the eggs with low cholesterol content in yolk. Flavonoids reduced cholesterol content in yolk to some extents （Lien et al.， 2008）. In our experiment， supplement of quercetin decreased yolk cholesterol in laying hens of different weeks old，it suggested that quercetin played an important role in lowering yolk cholesterol. Flavonoids raised hormone levels， such as 17-β estradiol and progesterone in poultry （Zhu et al.， 2004； Zhang et al.， 2008）； also，cholesterol acted as substrate for synthesis of a majority of steroid hormones in poultry， this result suggested that quercetin reduced cholesterol transporting into eggs via prompting the synthesis of cholesterol. The reduction of cholesterol in yolk by flavonoids may be due to the inhibition of β-hydroxy-β-methyl glutaryl coenzyme A reductase activity， thus inhibited the synthesis of cholesterol synthetase and reduced cholesterol synthesis （Kim et al.， 2003； Lee et al.，2003）. In addition， phospholipid in egg is very important to human health because of its functions of decreasing cholesterol， softening blood vessels， and lowering blood pressure. The studies showed that flavonoids， including green tea phenols， catechins and daidzein， improved phospholipid metabolism， inhibited the oxidation of phospholipids， and enhanced antioxidant capacity of the phospholipids （Naumov et al.，2010； Fu et al.， 2011）. The current study showed that supplement of quercetin increased the total phospholipids in yolk， it indicated that quercetin improved the lipid metabolism in egg and inhibited oxidation of the phospholipids， finally increased nutritional value of the eggs.

Effect of week old of laying hens on quercetin affecting egg quality and components

Egg quality and egg components decreased with the increasing age. According to statistics， the eggs which are unable to be sold resulting from the decline of egg quality during post-peak laying period accounted for 6% to 8%， the decreases of egg quality， laying rate and nutritional components of eggs led to huge economic losses and influenced poultry production（Qu， 2011）. Gradually， the function of glands declined with the decreasing secretion of reproductive hormone（gonadotropin estradiol and progesterone） during late laying period， thereby resulting in restriction of growth and development of follicles， and decreases of ovulation， egg quality and components.

The previous studies showed that flavonoids increased serum estrogen and improved the aged ovary （Liu et al.， 1998； Wang et al.， 2011）. The basal diet supplemented with 6 mg · kg-1daidzein increased serum estradiol （P＜0.05） （Zuo， 2002）， it suggested that flavonoids might play an estrogen-like effect on improving performance and egg quality via improving the levels of estradiol and other hormones. However，flavonoids exert estrogen-like effect and anti-estrogenlike effects depending on the hormone levels of body. While the endogenous estrogen is low， flavonoids serve as estrogen agonist to occupy ER； otherwise，flavonoids act as estrogen antagonist by competing with ER and produce a much weaker estrogen-like response. Reports showed that flavonoids reduced estrogen levels in vivo （Hocking et al.， 2000； Xue et al.， 2005； Liu et al.， 2007）， but no estrogen-like response was observed at higher estrogen levels. Quercetin， plays a weak estrogen-like role （Liu et al.，2011）， significantly increased serum estrogen in laying hens （Zhang et al.， 2013）. In addition， quercetin led to a significant increase of alkalinephosphatase （ALP）activity and phosphorylated extracellular signalregulated kinase （ERK） level in osteoblasts of arbor acres （AA） broilers' embryo， it indicated that quecertin played a weak estrogen-like role （Liu et al.， 2011）. Inthis experiment， the effect of quercetin on egg quality and components in laying hens was more significant at 39-47 weeks old than that at 29-37 weeks old， which was mainly due to the retrograde gland and lower estrogen in laying hens at 39-47 weeks old. Therefore，quercetin served as estrogen agonist to occupy ER and elevated estradiol levels of the body， thus regulated metabolism and utilization of nutrients. Eventually，quecertin improved egg quality and contents of egg nutrients.

Conclusions

A certain dose of quercetin improved egg quality and components， the older the hens became， the better improvement the egg quality and components would be. The optimum level of quercetin was 0.4 g · kg-1in the basal diet for improving egg quality， decreasing yolk cholesterol and increasing yolk phospholipid content.

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S831.5 Document code： A Article lD： 1006-8104（2015）-04-0023-10

Received 20 March 2015

Supported by Heilongjiang Department of Education （12541010）； Heilongjiang Department of Human Resources and Social Security （2014-2015）；Harbin Science and Technology Bureau （2015RQXXJ014）； Academic Team Construction of Northeast Agricultural University （2014-2017）

You Ying （1990-）， female， Master， engaged in the research of animal nutrition and feed science. E-mail: 18945058450@163.com

* Corresponding author. Li Yao， professor， supervisor of Ph. D student， engaged in the research of animal nutrition and feed science. E-mail: liyaolzw@163.com.