Changes in egg production, egg quality and egg nutritional values of laying hens in response to dietary supplementation with green tea water extracts

ABSTRACT Our previous studies demonstrated that low-grade tea had some beneficial effects on egg production performance and egg quality. Extracts derived from low-grade tea had high levels of active ingredients. Thus, this study investigated the effects of green tea water extracts (GTWE) supplementation on egg production, egg quality and egg nutritional value of laying hens. A total of 192 laying hens were randomly allotted to four groups T0, T1, T2 and T3, which fed with basal diet supplemented with 0%, 0.3%, 0.5% and 1.0% GTWE, respectively. Results showed that 0.3% GTWE supplementation increased egg production and improved feed conversion ratio significantly, while 0.5% and 1.0% GTWE supplementation resulted in no significant effects. Furthermore, 1.0% GTWE had adverse effects on feed intake. GTWE treatment improved egg quality, as shown by the increased albumen height and Haugh unit. At the same time, GTWE treatment improved the egg nutritional value, as presented by the increased content of vitamin E and lecithin. On the other hand, GTWE treatment decreased cholesterol, triglyceride, and malonaldehyde levels. However, the strength and thickness of eggshell were negatively affected. The comprehensive analysis suggested low dose of GTWE could be used to developing a desired functional feed supplement.


Introduction
Egg is one of the most popular animal products in our daily life, and is a good source of protein and energy. Several researches focus on the nutritional improvement of eggs. Previous studies reported that addition of exogenous nutrients, such as B. subtilis (Liu et al. 2017), green tea (Yuan et al. 2016;Xia et al. 2018), saccharicterpenin  improved the egg quality and nutritional value. Thus, the dietary supplementation may serve as a pathway to improve the quality and nutritional value of eggs.
It is widely known that tea is a healthy beverage and has many health benefits. With the development of the tea industry, plenty of tea resource waste are generated in commercial tea garden. It contains high concentration of nutrients that can be used in feeding animals and especially poultry. It is important to take full advantage of tea by-products. In other words, it is urgent to regularly apply the utilization of tea waste leaves. How to convert tea by-products into feed additive has been a focus in today's research field. In our early experimentation, we found that low dose of green tea powder (1.0%) significantly improved quality and nutritional value of eggs. At the same time, the composition of eggs was improved. However, high doses of green tea powder decreased the production performance, which suggesting that low doses of green tea could be more appropriately applied as an ideal feed additive. Besides, tea extracts are derived from tea leaves, which contains higher levels of active ingredients than tea leaves of the same weight. Particularly, tea polyphenols (one common active ingredients in tea) have received widely concern for their antioxidant (Rodrigues et al. 2016), antibacterial (Kawarai et al. 2016), anticancer (Shih et al. 2016) properties. Up to now, although many studies investigated the effects of tea on egg production performance, the results were inconsistent and controversial (Uuganbayar et al. 2005;Panja 2007). In addition, few researchers dealt with the effects of tea by-products on quality and nutritional value of eggs. Thus, green tea water extracts (GTWE) were selected as the examined feed additive. This study investigated the effects of different doses of GTWE supplementation on production, quality and nutritional value of eggs.

Materials
One hundred and ninety-two healthy Xianju chickens (the average laying rate was 50%) were provided by Xianju Chicken Development Company (Huzhou, China). GTWE was extracted from Chinese low-grade green tea by our lab. High temperature extraction method is used to extract water-soluble substance (Zhang et al. 2018 Callipers (mm), Automatic egg analyser (Egg multi tester EMT-5200), Digital micrometre (karl deut sch Model-1061), Electronic balance (Mettler Toledo pL203-IC) and egg shell force gauge (Model-T1) were provided by Zhejiang Academy of Agricultural Sciences (Hangzhou, 310020, China).

Experimental design
The 192 Xianju chickens (20-week aged) were randomly allotted to four treatments, with three replicates per treatment and sixteen chickens in each replicate. Control group T0 fed with basal diet, test groups T1, T2 and T3 fed with basal diet supplemented with 0.3%, 0.5% and 1.0% GTWE, respectively. Studies included 7 day as a pre-experimental period that served as acclimation period and 67 day for the main experimental period. All the chickens had free access to water and feed. The hen house provided 16 h of light per day, with natural light during the day and artificial light from 1700 to 2400 HRS. The composition and main characteristics of the basal diet are shown in Table 1. The basal diet formulation was in accordance with the National Standards of People's Republic of China (NSPRC 2008a) and nutrients of the basal diet were analysed according to the standard method GB/T 18868-2002 of the National Standards of People's Republic of China (NSPRC 2002). The animal care and use protocol were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China, 1988) and the Standards for the administration of experimental practices (Jiangsu, China, 2008).

Sampling and handling
Ten eggs from each replicate at the end of this study were randomly selected and later used for egg quality analysis. The analyses were completed within 12 h. Three eggs from each replicate were randomly selected and later used for the egg nutrition analysis.

Egg production performance
Egg production including number and weight, feed intake and mortality were recorded daily. Average daily feed intake, average egg weight and feed-to-egg ratio were calculated at the end of the trial.

Egg quality
The egg shape index was measured (in mm) with a calliper, and vertical diameter·diameter-1 was calculated to determine the shape index. Haugh units, albumen height, and yolk color were measured with an automatic egg analyser (Egg Multi Tester, EMT-5200). Eggshell thickness was measured with a digital micrometre (karl deut sch MODEL-1061). Eggshell strength was measured with the eggshell force gauge (MODEL-T1). Egg and yolk weight were measured with the electronic balance (Mettler Toledo pL203-IC).

Statistical analysis
Data were reported as mean ± SEM, and all the data were analysed using the one-way ANOVA of SPSS17.0 in a randomized complete block design. When significant differences were found, Duncan's test was used for multiple comparisons. Pvalues <0.05 were considered significant.

Results and discussion
Analysis of egg production performance changes No mortality was observed during the experiment. The results of different levels of GTWE supplementation on egg production performance are shown in Table 2. Compared with group T0, the laying rate increased by 8.41% (P < 0.05), and the feed-toegg ratio decreased by 7.51% (P < 0.05) in group T1. The average egg weight decreased by 0.93% (P < 0.05), 0.64% (P > 0.05), 0.89% (P < 0.05) in groups T1, T2 and T3, respectively. The average daily feed intake decreased by 2.88% (P < 0.05) in group T3.
The results revealed that 0.3% GTWE treatment significantly improved egg production and feed conversion, while 0.5% GTWE had slight effects. Furthermore, 1.0% GTWE had adverse effects on the feed intake. In the case of the increased laying rate, but had no significant changes in feed intake, it was clear that 0.3% GTWE supplementation improved feed conversion ratio, and this phenomenon may be related to the action of tea (Yuan et al. 2016;Xia et al. 2018). Previous studies found that tea's functional ingredients had a lot of health benefits, such as anti-oxidation (Rodrigues et al. 2016), antibacteria (Kawarai et al. 2016), and contribute to the improvement of body healthy status (Lin and Lin 2010;Periandavan et al. 2017
The results suggested that GTWE supplementation had adverse effect on strength and thickness of eggshell, which was dose-dependent. These findings were consistent with Kojima and Yoshida's (2008) study, which showed that the addition of tea by-products decreased the eggshell strength. It could be related to insufficient nutrition absorption mentioned above; especially the inadequate absorption of calcium, or tea treatment altered the process of anabolism and catabolism during eggshell formation. Current studies proved that exterior nutrition could affect the material metabolism, and then cause some changes of egg quality Liu et al. 2017;Xia et al. 2018). Of course, there were some different results suggesting that tea resource treatment improved the eggshell thickness and egg specific gravity (Wei et al. 2012). Beyond that, we found that GTWE treatment improved the Haugh unit and albumen height of the eggs, and these findings were consistent with our previous studies (Xia et al. 2018). On the other hand, there were some contradictory results with that Panja's (2007) study, since they showed that Haugh unit, yolk color and albumen height were not significantly affected with increasing amounts of tea treatment. Although some inconsistent findings were reported, nutrition indeed could change the metabolism, and then alter egg quality (Olgun et al. 2009;Liu et al. 2016), suggesting that reasonable regulating exogenous nutrition may serve as a pathway to improvement in egg quality.
The results showed that GTWE treatment decreased the content of cholesterol, triglyceride and malonaldehyde. At the same time, it increased the vitamin E and lecithin of egg yolk and whole eggs which showed a dose-dependent potency. These findings were consistent with the studies of Panja (2007), Wei et al. (2012) and Xia et al. (2018). Current studies have shown that intake some exterior nutrients such as substances with physiological activity could affect and alter body metabolism, including energy metabolism and material metabolism (Oliveira et al. 2011). Tea polyphenols is one of the major categories of biological active compounds found in tea or tea extracts. They could reduce the lipid levels and increase the faecal excretion of total lipids and cholesterol depending on the dose (Matsumoto et al. 1998). Eventually, changes in body metabolism finally altered the egg composition and egg nutritional values Liu et al. 2017;Xia et al. 2018).

Conclusion
Low dose of GTWE (0.3%) treatment increased the egg production, decreased feed to egg ratio, but intermediate dose of GTWE (0.5%) had minor effects. More importantly, 1.0% GTWE had adverse effects on feed intake. GTWE treatment increased the quality and nutritional value of eggs, as shown by the increased values of albumen height and Haugh unit. In addition, the contents of vitamin E and lecithin increased, while the cholesterol, triglyceride and malonaldehyde decreased which was dose-dependent effect. However, it had adverse influence on strength and thickness of eggshell. By combing the changes of production performance, quality and nutritional value of eggs, low dose of GTWE had a great potential use for developing new kinds of feed additives. Values with different lowercased letters in the same row differ significantly (P < 0.05).