The Antioxidant Status of Serum and Egg Yolk in Layer Fed with Mushroom Stembase (Flammulina velutipes)

Mo Chen1, Shad Mahfuz1, 2, Yan Cui1, Liying Jia1, Zhongjun Liu3 and Hui Song1, 4* 1School of Life Science, Jilin Agricultural University, Changchun, Jilin, P.R. China. 2Department of Animal Nutrition, Sylhet Agricultural University, Sylhet-3100, Bangladesh. 3College of Chinese Medicine Materials, Jilin Agricultural University, Changchun, Jilin, P.R. China. 4Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Changchun 130118, China. Article Information Received 22 June 2018 Revised 01 March 2019 Accepted 19 April 2019 Available online 07 October 2019

A ntioxidants are a group of compounds that inhibit oxidation and reduce free radicals directly or indirectly in body system (Liu et al., 2014). Oxidation and generation of free radicals are part of the normal body metabolites in the host organisms. In any environmental stress, these highly reactive chemicals can be overproduced and may cause abnormalities and dysfunction of the body system (Liu et al., 2014). Excessive free radicals can affect animal performance and even lead to the development of diseases (Moncol, 2007). In addition, Mujahid (2007) and Zhao (2011) reported that the antioxidant status can improve production performance in birds. Oxidative stress in the body can be relieved by exogenous supplemental antioxidants (Rahman, 2007). However, several synthetic antioxidants have shown potential side effects such as liver damage and carcinogenesis, especially long-term use of synthetic antioxidants (Rautou, 2010). Therefore, exploring safe and natural antioxidants to defend against oxidative stress has become a body of research in recent years.
The conventional use of butylated hydroxyanisole and butylated hydroxytoluene as synthetic antioxidants may have a public human hazard, so it is necessary to discover natural antioxidant products (Ser et al., 2016).
Different herbs have been used as chemotherapeutics in the poultry industry (Kamran et al., 2016). Besides, Metin et al. (2017) reported that natural plants and agroindustrial bye products can be used as potential ingredients in poultry industry.
Flammulina is a kind of edible mushroom that can provide key nutrients such as protein, vitamins, minerals, unsaturated fatty acids and fiber (Reis, 2012;Mahfuz et al., 2017). In addition, F. velutipes mushroom is a good source of protein (Kim, 2009). The main amino acids in F. velutipes are methionine, valine, isoleucine, leucine, lysine, phenylalanine and threonine, L-glutamic acid, L-alanine, glycine and L-lysine (Beluhan and Ranogajec, 2011). Yang (2015), Ma et al. (2015) and Xia et al. (2015) have reported that the polysaccharides in F. velutipes have good antioxidant properties. The various proportion of F. velutipes mushroom have been reported as a potential source of antioxidants (Tang et al., 2016).
As the production of F. velutipes is increasing year O n l i n e

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by year, that leads to the availability of the stem waste material. This sty focuses on the utilization of mushroom stembase materials. Here the effect of feeding mushroom stembase on antioxidant status of serum and egg yolk in laying hens, has been evaluated.

Materials and methods
Experimental chickens (ISA Brown) were purchased from Changchun Octavia farms and F. velutipes stembase (FVS) was collected from the local domestic mushroom farm in Changchun city, Jilin, China.
A single factor completely randomized design was used to select 150 healthy, 210-day-old ISA brown laying hens and were randomly divided into 5 groups. Each group had 5 replicates and each had 6 hens. The control group was fed the basal diet, the antibiotic group was fed with the basal diet with the addition of antibiotic, (flavomycin, 0.05%) and the 2%FVS, 4%FVS and 6%FVS fed groups were added with 2%, 4%, and 6% of the ground mushroom stembase on the basal diet. The experimental period is 63 days. The basal dietary nutrition level of laying hens is based on the nutritional requirements of laying hens in the NRC (1994). Feeds and water were provided ad libitum in the whole trial period. The hen house temperature was 24 0 C and a 17 h light with 7 h dark period was maintained throughout the whole experimental period.
The mushroom stembase sample was prepared (0.01mm) by laboratory high speed universal sample grinder (Huanghua xinxing electric Appliance Co, Hebei, China). Six representative samples in triplicate were obtained and analyzed for proximate components dry matter (DM), crude protein (CP), crude fiber (CF), ether extract (EE), total minerals (Ash) including calcium and phosphorus following the method of AOAC. Nitrogen was determined using an FP-528 nitrogen determinator (LECO Corporation, USA). Total phenolics content was measured according to the method of Giannenas et al. (2010). The value was expressed as mg of gallic acid equivalents (GAE) per gram of dry weight (mg GAE/g) on dry weight) basis. The analyzed nutritional composition of the experimental diets and FVS were presented in Table I  and Table II, respectively.
At the end of the experiment at d 273 (39 wk), blood samples were obtained via wing vein from 5 hens per experimental group. Serum was obtained by centrifuged at 3000 × g for 20 min at 4 o C (Legend Micro 17R centrifuge, Thermo Fisher, Germany) and were stored at -80 o C until measuring serum antioxidant properties. Similarly, at the end of the experimental period at d 273(39 wk), fresh eggs were selected (5 eggs per treatment) and egg yolks were collected for the test. Malondialdehyde (MDA) content, total superoxide dismutase (T-SOD) activity, glutathione peroxidase (GSH-Px) activity, and total antioxidant capacity (T-AOC) in serum and egg yolk were measured using commercial kits provided by Nanjing Jiancheng Bioengineering Institute, Nanjing, China (MDA A003-1, T-SOD A001-1, GSH-Px A005,T-AOC A015-3). Table I  One-way analysis of variance (ANOVA) was performed using SPSS 15.0. The results were expressed as the mean plus SEM value (standard error), and each set of data was processed using Duncan's multiple comparison method.

Results and discussion
Serum antioxidant T-AOC was significantly O n l i n e

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higher (P<0.05) in 6%FVS than control; GSH-Px was significantly higher (P<0.05) in all levels of mushroom fed groups and control groups than antibiotic fed groups; MDA was significantly lower (P<0.05) in all levels of mushroom fed groups and antibiotic groups than control fed groups. However, there was no significant difference were observed for T-SOD among experimental groups (Table III). For yolk antioxidant T-AOC, and T-SOD were significantly higher (P<0.05) in 4%FVS than the control group and antibiotic fed groups; MDA was significantly lower (P<0.05) in all levels of mushroom fed groups than antibiotic group and control fed groups (Table IV).
This study assumed that mushroom contains different bioactive components especially phenolic compound, the amino acids that may role on improving the antioxidant capacity. The phenolics compounds, has the antioxidants activities, was discovered from the mushroom F. velutipes (Rahman et al., 2015). The various proportion of F. velutipes mushroom has been reported as a potential source of antioxidants (Tang et al., 2016). Form some past studies it has been known that the polysaccharides and oligosaccharide present in F. velutipes mushroom shows the antioxidant function Xia, 2015). Liu et al. (2016) reported that different polysaccharides originated from F. velutipes mushroom residue were purified and the antioxidant function has been considered in the study. Zeng et al. (2012) stated that F. velutipes mushroom holds the higher phenolic with the highest antioxidant activities. Besides, F. velutipes mushroom was found to exhibit vitamin-C that may play a role in antioxidant function (Tang et al., 2016). In a recent study by Han et al. (2017) found that dietary inclusion of yeast selenium and selenite could improve the activity of glutathione peroxidase in serum, and improve the activity of superoxide dismutase in the liver in chicken. Adding polyphenols in feed could increase the activity of glutathione peroxidase and superoxide dismutase in serum and improve the total antioxidant capacity of serum in weaned pigs (Chen et al., 2018). In addition, adding L-arginine in the diet of laying hens could improve the total antioxidant capacity and reduce the concentration of malondialdehyde in egg yolk and serum (Duan et al., 2015).

Conclusion
The present study explored that FVS could be a good source of natural feed supplement as well as antioxidant that can improve both serum and yolk antioxidant properties. Focus on quality eggs production and sound health status, F. velutipes mushroom stem base can be used as a natural supplement as well as a substitute for antibiotic on layer production.