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.

ABSTRACT

The objective of this study was to evaluate the antioxidant properties of serum and egg yolk in ISA Brown layer hens fed with mushroom (Flammulina velutipes) stembase (FVS). A total 150 hens of 30 wk old were grouped into 5 equal treatments with 5 replications of 6 hens each. Dietary treatments included basal diet as a control group; control diet including antibiotic (0.05% flavomycin) as an antibiotic group; 2% FVS fed group; 4% FVS fed group and 6%FVS fed group. The experimental duration was total 63 days, from 30 wk to 39 wk. Serum total antioxidant (T-AOC) was significantly higher (P<0.05) in 6%FVS than control; glutathione peroxidase (GSH-Px) was significantly higher (P<0.05) in FVS fed groups and control groups than in the antibiotic fed group; Malondialdehyde (MDA) was significantly lower (P<0.05) in FVS fed groups and antibiotic groups than in the control fed group. However, there was no significant difference were observed for serum total superoxide dismutase (T-SOD) in among experimental groups. 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 FVS fed groups than antibiotic group and control fed groups. Mushroom stembase can be used as a dietary supplement at 6% level to improve the antioxidant capacity of serum and egg yolk in ISA Brown laying hens.

Article Information

Received 22 June 2018

Revised 01 March 2019

Accepted 19 April 2019

Available online 07 October 2019

Authors’ Contribution

MC conducted this experiment work under the supervision of ZL and HS. YC and LJ helped in mushroom preparation, experimental diet and participated in the animal experiment. MC, YC and LJ worked with lab test. SM, MC and LJ helped in statistical analysis and formatting the manuscript.

Key words

Feed supplement, laying hens, mushroom stembase, serum antioxidant, yolk antioxidant

DOI: https://dx.doi.org/10.17582/journal.pjz/2020.52.1.sc6

* Corresponding author: songhuisk@jlau.edu.cn

0030-9923/2020/0001-0389 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Antioxidants 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 agro-industrial 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 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 240C 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 4oC (Legend Micro 17R centrifuge, Thermo Fisher, Germany) and were stored at -80oC 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.- The ingredients and chemical composition of experimental diets (g/kg).

Ingredients	Control	Antibiotic	2% FVS	4% FVS	6% FVS
Maize corn	577.0	576.5	562.0	545.0	533.0
Soyabean meal	263.0	263.0	259.0	258.0	254.0
Soyabean oil	31.0	31.0	30.0	28.0	24.0
FVW a	-	-	20.0	40.0	60.0
Lysine	1.0	1.0	1.0	1.0	1.0
Methionine	2.0	2.0	2.0	2.0	2.0
Dicalcium	30.0	30.0	30.0	30.0	30.0
Limestone	92.0	92.0	92.0	92.0	92.0
Common salt	2.0	2.0	2.0	2.0	2.0
Vit -mineral premix b	2.0	2.0	2.0	2.0	2.0
Antibiotics	-	0.5	-	-	-
Total	1000	1000	1000	1000	1000
Chemical analysisc
DM, (g/kg)	912.4	921.2	923.3	923.5	924.10
CP (g/kg)	168.3	168.0	169.1	168.8	169.3
Ca, (g/kg)	40.6	40.40	41.11	41.0	40.6
P, (g/kg)	7.10	7.05	7.15	7.20	7.20
EE, (g/kg)	51.4	52.1	52.10	51.30	51.60
CF, (g/kg)	25.6	25.5	29.60	33.7	37.90
Calculated analysis
ME (MJ/kg)	11.55	11.50	11.61	11.59	11.64
Lysine,(g/kg )	10.4	10.4	10.30	10.5	10.4
Methionine, (g/kg)	5.0	4.9	5.0	4.8	4.9
Cystine (g/kg)	2.6	2.6	2.7	2.5	2.6

a FVS= F. velutipes stembase at 2%, 4% and 6%. bProvided per kg of the complete diet: retinyl acetate, 4500 IU; cholecalciferol, 1200 IU; DL-α-tocopheryl acetate, 25000 IU; thiamin, 5000 mg; riboflavin, 20000 mg; phylloquinone,10000 mg; niacin, 45000 mg; pantothenic acid, 35000 mg; biotin, 1500 mg; folic acid, 3000 mg; cyanocobalamin, 40 mg; zinc, 45 mg; manganese 50 mg; iron, 30 mg; copper, 4 mg; cobalt, 120 μg; iodine, 1 mg; selenium, 120 μg. cDM, dry matter; CP, crude protein; Ca, calcium; P, phosphorus; EE, crude fat; CF, crude fiber; ME, metabolisable energy.

 

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 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).

 

Table II.- Chemical compositions and active ingredients of F. velutipes mushroom stem base (FVS).1

Chemical composition and active ingredients	Value
Moisture (g/kg)	116.0 ±0.85
Crude protein (CP, %Nx6.25) (g/kg)	137.5±0.75
Crude fiber (CF) (g/kg)	22.05±0.11
Ether extract (EE) (g/kg)	28.0±0.01
Ash (total minerals) (g/kg)	116.1±0.09
Organic matter (OM) (g/kg)	884.0±0.85
Nitrogen free extract (NFE) (g/kg)	498.0±3.45
Calcium (ca) (g/kg)	1.8±0.14
Available phosphorus (p) (g/kg)	6.4±0.28
Total phenolic content (mg, GAE/g)	6.87±0.25

1Values are expressed as the mean ± standard deviation (n=6).

 

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 (Ma et al., 2015; 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).

 

Tables III.- Effects of mushroom stembase on serum antioxidant in layer.

Parameters	Control	Antibiotic	2%FVS	4%FVS	6%FVS	SEM	P value
T-AOC	0.71a	1.14bc	1.45bc	1.33b	1.95c	0.124	0.01
MDA	19.71c	10.53a	15.36b	15.6b	13.62b	0.847	0.03
T-SOD	64.23	67.03	74.47	70.97	75.17	1.924	0.56
GSH-Px	878.68b	683.6a	767.21ab	865.57b	832.78b	22.984	0.01

T-AOC, total antioxidant capacity; MDA, malondialdehyde; T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase. 1data represent the mean value of 5 samples for each treatment (n=5). a, b,c means in the same row with different letters are significantly different at P<0.05. SEM, pooled standard error of the means.

 

Tables IV.- Effects mushroom stembase on egg yolk antioxidant in layer.

Parameters	Control	Antibiotic	2%FVS	4%FVS	6%FVS	SEM	P value
T-AOC	1.16a	1.15a	1.17a	1.74b	1.44a	0.074	0.02
MDA	18.11b	18.16b	14.83a	13.09a	13.71a	0.611	0.01
T-SOD	41.74a	42.79ab	51.98ab	58.11b	47.69ab	2.386	0.01

T-AOC, total antioxidant capacity; MDA, malondialdehyde; T-SOD, total superoxide dismutase. 1data represent the mean value of 5 samples for each treatment (n=5). a, b,means in the same row with different letters are significantly different at P<0.05. SEM, pooled standard error of the means.

 

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.

 

Acknowledgement

This work was supported by “Innovation Platform for Economic Fungi in Jilin Province” [Grant No. 2014-2016] Changchun, P.R. China.

 

Statement of conflicts of interest

Authors have declared no conflict of interest.

 

References

Beluhan, S. and Ranogajec, A., 2011. Fd. Chem., 124: 1076-1082. https://doi.org/10.1016/j.foodchem.2010.07.081

Chen, J., Yan, Li.,Yu, B., Chen, D., Mao, X., Zheng, P., Luo, J., and He, J., 2018. J. Anim. Sci., 96: 1108-1118. https://doi.org/10.1093/jas/skx078

Duan, X.X., Li, F., Mou, S.Y., Feng, J.W., Liu, P.F. and Xu, L.M., 2015. Poult. Sci., 94: 2938-2943. https://doi.org/10.3382/ps/pev283

Giannenas, I., Tontis, D., Tsalie, E., Chronis, EF., Doukas, D. and Kyriazakis, I., 2010. Resh. Vet. Sci., 89: 78-84. https://doi.org/10.1016/j.rvsc.2010.02.003

Han, X.J., Qin, P., Li, X.W., Ma, Q.G., Ji, C., Zhang, J.Y. and Zhao, H.L., 2017. Poult. Sci., 96: 3973-3980. https://doi.org/10.3382/ps/pex216

Kamran, S., Khushi, M. and Rubina, N., 2016. Pakistan J. Zool., 48: 1715-1719.

Kim, M.Y., Chuang, L.M., Lee, S.J., Ahn, J.K. and Kim, E.H., 2009. Fd. Chem., 113:386-393. https://doi.org/10.1016/j.foodchem.2008.07.045

Liu, W., Wang, J., Zhang, Z., Xu, J. and Xie, Z., 2014. Int. J. BioMacromol., 65: 446-453. https://doi.org/10.1016/j.ijbiomac.2014.01.062

Liu, Y., Zhang, B., Ibrahim, S., Gao, SS., Yang, H. and Huang, W., 2016. Carbohydrate polymers., 145:71-77. https://doi.org/10.1016/j.carbpol.2016.03.020

Ma, Z., Zhang, C., Gao, X., Cui, F. and Zhang, J., 2015. Int. J. BioMacromol., 73: 236-244. https://doi.org/10.1016/j.ijbiomac.2014.11.028

Mahfuz, S., Hui, S. and Zhongjun, L., 2017. Int. J. Poult. Sci., 16: 112-117. https://doi.org/10.3923/ijps.2017.112.117

Metin, D., Asuman, A.D., Köksal, K., Ecevit, E., Harun, C. and Mohammad, M.T., 2017. Pakistan J. Zool., 49: 129-137.

Moncol, J., 2007. Int. J. Biol. Cell Biol., 39: 44-84.

Mujahid, A., Akiba, Y. and Toyomizu, M., 2007. Poult. Sci., 86: 264-371. https://doi.org/10.1093/ps/86.2.364

National Research Council, NRC., 1994. Nutrient requirements of poultry, 9th rev. ed. Natl. Acad. Press, Washington, DC.

Rahman, K., 2007. Studies on free radicals, antioxidants, and co-factors. Clin. Interv. Aging., 2: 219-236.

Rahman, M.A., Abdullah, N. and Aminudin, N., 2015. Oxid. Med. Cell. Long., 10: 1-10. https://doi.org/10.1155/2015/403023

Rautou, P.E., Mansouri, A., Lebrec, D., Durand, F., Valla, D. and Moreau, R., 2010. J. Hepatol,. 53:1123-34. https://doi.org/10.1016/j.jhep.2010.07.006

Reis, F.S., Barros, L. and Martins, A., 2012. Fd Chem. Toxicol., 50: 191-197. https://doi.org/10.1016/j.fct.2011.10.056

Ser, H.L., Tan, L.T.H. and Palanisamy, U.D., 2016. Front. Microbiol.,7: 899. https://doi.org/10.3389/fmicb.2016.00899

SPSS, 15.0., 2006. Statistical software package for the social sciences SPSS, Int., USA.

Tang, C., Hoo, P.C.X., Tan, L.T.H., Pusparajah, P., Khan. T.M., Lee, L.H., Goh, B.H. and Chan, KG., 2016. Front. Pharmacol.,7: 474. https://doi.org/10.3389/fphar.2016.00474

Xia, Z., 2015. Carbohyd. Polym, 118: 41-43. https://doi.org/10.1016/j.carbpol.2014.10.074

Yang, W., Yu, J., Zhao, L., Ma, N. and Fang, Y., 2015. J. Funct. Fds, 18: 411-422. https://doi.org/10.1016/j.jff.2015.08.003

Zeng, X., Suwandi, J., Fuller, J., Doronila, A. and Ng, K., 2012. Fd. Sci. Technol. Int.,18: 367-379. https://doi.org/10.1177/1082013211427993

Zhao, X., Yang, Z. B., Yang, W. R., Wang, Y., Jiang, S.Z. and Zhang, G.G., 2011. Poult. Sci., 90: 1720-1727. https://doi.org/10.3382/ps.2010-01280