| The experiment was conducted to study the effects of different contents of Flammulina velutipes stembase(FVS) as dietary supplements on immune function of broilers. A total of 270 one-day-old Arbor Acre(AA) broilers were randomly divided into five groups, with three replications of eighteen chickens each. The experimental groups were control group(basic diet), antibiotic group(basic diet + 5mg/kg flavomycin), low dose group(basic diet +2% content mushroom stembase), middle dose group(basic diet +4% content mushroom feet), and high dose group(basic diet +6% content mushroom stembase). The experimental period was 42 days. The detection indexes were production performances, immune organ indexes, observation of the morphology of immune organs(Paraffin section), peripheral T and B lymphocyte proliferation rates(MTT assay), serum cytokines IL-2, IL-4, IFN-γ assay(ELISA assay) and serum immunoglobulin IG-A, IG-G, IG-M potency assay(ELISA assay), Newcastle disease(Newcastle Disease, ND) antibody titers to evaluate the effects of FVS on immune function of broilers. The mechanism of FVS was explored to improve the immune function of broilers by determining TCR, CD28, BCR, CD21, PTEN, PI3 K, AKT gene expression(q-PCR) in spleen tissues, and protein expression level of PI3 K and the phosphorylation level of AKT in spleen tissues by Western Blot methods.The results are as follows:1. Effect of FVS on the production performance of BroilersAt the whole period, 2%, 4% and 6% FVS groups of broilers average daily gain weight was significantly higher than control group(P < 0.05) and has no significant differences with antibiotic group(P > 0.05); At the early stage of the experiment, broilers feed and meat ratio of each dose FVS group was significantly lower than that of control group(P < 0.05) and has no significant differences with antibiotic group(P > 0.05); broilers survival rate of each dose FVS group have been increased in varying degrees. The results show that, FVS can improve the production performance of broilers. 2. Effect of FVS on immune organs of broilersAt the age of 21 d, 2% FVS group thymus indexes and 42 d old bursal indexes were significantly higher than that of control group(P <0.05), 4% FVS group thymus indexes were significantly higher than that of control group(P <0.05), 6% FVS group 42 d thymus index and spleen index were significantly higher than control group(P <0.05); 4%FVS group of broilers’ thymus indexes were significantly higher than that of control group at 21 d(P < 0.05), at age of 42 days, thymus indexes and spleen indexes were significantly higher than that of control group(P < 0.05); 6%FVS broilers thymus index and spleen index were significantly higher than those of control group at 42 days old(P < 0.05); By histological observation, the immune organs of broilers of each group were well developed. This shows that FVS can promote the development of immune organs without adverse effects. 3. Effects of FVS on cellular immune function of broilersAt the age of 22-28,29-35,36-42 days, broilers T and B cell proliferation rate of each dose FVS group was significantly higher than that of control group(P < 0.05); At the age of 22-28,36-42 days 22-28,36-42 days of age, each dose FVS can significantly improve the content of IL-2(P < 0.05); While at the day of 15-21,22-28,6%FVS group’s IFN- γ contents were significantly increased(P < 0.05). This shows that, FVS can improve the cellular immune function of broilers. 4. Effects of FVS on humoral immune function of broilersDiets added with FVS can significantly improve broiler’s serum ND-HI antibody titers(P < 0.05) at early growth; At the age of 22-28 and 36-42 d, FVS could improve Ig A titers significantly(P <0.05), and Ig G titers at 7-14 and 22-28 d(P <0.05). At age of 22-28 days 6%FVS group and antibiotics groups’ Ig M contents were significantly higher than that of other experimental group(P < 0.05). The results showed that FVS can improve the humoral immune function of broilers. 5. Effects of FVS on the expression of TCR, CD28, BCR and CD21 m RNA of cell receptorsAt the age of 21 d, each dose group of FVS’s TCR m RNA expressions were up-regulated, while at 42 days of age, 2%, 4%, 6%FVS groups’ TCR m RNA expressions were significantly increased(P<0.05); At age of 21 and 42 days, each dose group of FVS’s CD28 m RNA expressions were significantly up-regulated(P<0.05) CD28 m RNA expression of 4% and 6%FVS group was up-regulated(P<0.05); At the age of 21 d and 42 d, 2%, 4%, 6%FVS groups’ BCR m RNA expression were up-regulated(P<0.05); 21 and 42 days of age, each dose group of FVS’s CD21 m RNA expressions were up-regulated, And at the age of 21, 4%, 6%FVS groups were significantly higher Than that of control group(P<0.05), at the age of 42 d, 2%, 4%, 6%FVS groups’ CD21 expressions were significantly higher than that of control group(P<0.05). This shows that in the process of proliferation of T and B lymphocytes, T cell receptor TCR and CD28 receptor, B cell receptor BCR and CD21 played an important role in signal transduction. 6. Effects of FVS on the expression of m RNA of PI3 K, Akt and PTEN in PI3K/Akt signaling pathwayAt the age of 21 day, 4% and 6%FVS groups’ PI3 K and Akt expressions were significantly increased(P < 0.05), and at 42 d, the expression of PI3 K of 2%, 4% and 6% FVS were significantly up-regulated(P < 0.05); At the age of 21, 2%, 4%FVS’s PTEN expressions were down regulated, when at 42 d, 2%, 4% and 6% FVS groups’ PTEN m RNA expressions were significantly reduced(P < 0.05). The results indicated that PI3 K / Akt signaling pathway was activated.by FVS’s synergistic stimulation. 7. Effects of FVS on the expression of protein of PI3 K and the phosphorylation level of Akt in PI3K/Akt signaling pathwayAt 21 d and 42 d, the PI3 K protein expression was up-regulated in each dose group of FVS, and the phosphorylation level of Akt was increased at the age of 21 and 42 days, which further demonstrated the activation of PI3K/Akt signaling pathway.In conclusion, FVS can improve the production performance, the humoral and cellular immunity of broilers, and by CD28, TCR, BCR and CD21 of T, B cell receptors to start signal transduction, and transmit signals through the PI3K/Akt pathway, eventually leading to cell proliferation and secretion of cytokines, enhancing the immunity of broilers. |
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