| In tropical and temperate regions,the environmental temperature often exceeds 30 °C,and poultry is susceptible to heat stress,which causes great economic losses,especially in laying period(about 300 days).Although it has been proved that heat stress affects the laying rate of laying hens,the mechanism of heat stress affecting the development of follicles in laying hens has not been fully understood.Follicular development is slow even atresia,may be related to the pathway of apoptosis.It is known that both the Fas L/Fas signaling and the TNF-α signaling can trigger apoptosis in various tissues.However,it has not been reported whether heat stress can induce follicular cell apoptosis by activating these two systems to reduce the laying rate.Therefore,studies on the mechanism of Fas L / Fas and TNF-α system in the effect of heat stress on the development of follicles in laying hens has important theoretical significance for alleviating heat stress and improving the performance of laying hens.Firstly,the heat stress model of layer was established,on the basis of the model,the effects of heat stress on the development of follicles,apoptosis of follicles,activation of Fas and TNF-α system,oxidative damage and secretion of stress-related hormones in laying hens after 5 days were analyzed.In addition,the performance of laying hens and recovery of follicles after heat stress relief were studied,which provide theoretical basis for relieving the influence of heat stress in clinic.The main results are as follows:(1)Establishment of heat stress model of laying hens.In order to establish the heat stress model of laying hens,32-week-old Highland brown laying hens were used to determine the heat stress pattern,temperature and time by laying rate,mortality,corticotropin releasing hormone(CRH)and corticosterone(Cort).First,the heat stress model and temperature were selected to confirm the continuous heat stress at 35-37 °C,and all the indexes of the experimental chickens met the requirements of the model.After 7 days of heat stress(S1-S7),it was found that the indexes of S5-day chickens were stable.Therefore,the heat stress model was established under the following conditions: 35-37 °C continuous heat stress for 5 days.(2)Effect of heat stress on the development of follicles in laying hens.According to the results of heat stress model,the effect of heat stress on the development of follicles in laying hens was examined.Statistics of follicles at different levels showed that the numbers of LWF and SYF in heat stress group were not significantly different from those in control group(P <0.05),but the numbers of LYF and hierarchical follicles decreased significantly(P <0.05).From the 3rd day of heat stress to the 6th day of recovery period,the laying rate in stress group was significantly lower than that in control group(P <0.05).From the 7th day of recovery period,laying rate gradually recovered to the level of the control group.Combined with the classification standard of this study,it is inferred that the significant decrease of laying rate in S3-5,R1-3,R4-6 time is due to the stress injury of hierarchical follicles,LYF and SYF.(3)Effects of heat stress on the apoptosis in ovarian granulosa cells of laying hens.Apoptosis is the main cause of follicular development inhibition.In this study,apoptosis of ovarian granulosa cells detected by TUNEL and flow cytometry to analyze the mechanism of heat stress on follicular development.The results showed that the apoptosis rate of SYF and LYF granulosa cells in heat stress group was significantly higher than that in control group(P < 0.05);the level of caspase-3 protein in SYF and LYF granulosa cells was significantly increased compared with the control group(P < 0.05);Bcl-2 gene expression and Bcl-2 / Bax ratio of SYF and LYF granulosa cells in stress group were significantly lower than those in control group(P < 0.05).In conclusion,heat stress significantly induced apoptosis in ovarian granulosa cells of laying hens.(4)Apoptosis pathway of ovarian granulosa cells induced by heat stress in laying hens.The mechanism of Hsps regulating apoptosis was explored by network pharmacology,and the protein levels of Fas and TNF-α were detected by Western and ELISA to explore the mechanism of heat stress induced apoptosis of ovarian granulosa cells.The results showed that: CDK1 and other HSPs family proteins were involved in the apoptosis of ovarian granulosa cells in laying hens;Fas L,TNF-α and TNFR1 protein of SYF and LYF granulosa cells in heat stress group were significantly increased(P < 0.05),and the concentrations of Fas L and TNF-α in follicular fluid were significantly increased compared with the control group(P < 0.05).Our results support that heat stress activated the Fas and TNF-α systems in ovarian granulosa cells of laying hens.(5)Heat stress-induced oxidative damage in ovarian granulosa cells of laying hens.Heat stress usually causes oxidative stress in cells,so we detected the SOD and MDA in serum and follicle of laying hens.The results showed that the level of TAS in the blood of the heat stress group was lower than that of the control group but the difference was not significant.Interestingly,the SOD level in blood,yolk and the rhubarb follicles in the heat stress group was significantly lower than that in the control group(P < 0.05).MDA level in the blood,small yellow follicles and Large yellow follicles was significantly increased compared with the control group(P < 0.05);These results support that heat stress caused oxidative stress in ovarian granulosa cells of laying hens.(6)The effect of heat stress on the secretion of heat stress-related hormones.In order to analyze the effect of heat stress on stress-related hormones,the levels of estradiol(E2)and progesterone(P4)in the follicles of laying hens,as well as the levels of CRH and corticosterone in serum and follicles were tested.The results showed that the concentration of E2 in SYF and LYF in the heat stress group was significantly lower than that in control(P<0.05);the concentration of P4 in LYF was significantly higher than that in control(P<0.05),while difference was not significant(P>0.05);the E2/P4 ratio in LYF in the heat stress group was significantly lower than that in control(P<0.05),which was consistent with the significant decrease in the E2/P4 ratio in mammalian atretic follicles.The CRH of serum,small yellow and rhubarb follicles in the heat stress group were significantly higher than those in the control group(P<0.05),in the meanwhile corticosterone levels were also significantly higher(P<0.05).(7)Study on the effect of heat stress relief on layer performance and follicular development.In order to analyze the recovery of layer production performance and follicular development after removing heat stress,the number of follicles,egg quality and corticosterone secretion of layers at all levels were tested.The results showed that the number of LWF and SYF in the stress group was not significantly different from that in the control group.LYF and Hie recovered to the control level at day R7;egg weight and eggshell strength were basically recovered but unstable at day R5,and recovered to control group level at day R11;Test of corticosterone showed that the level had returned to normal levels at R7 days though with some fluctuation,and returned to normal levels at R12;HE staining of small yellow follicles was performed on R7 and R12 days,with no edema and bubble degeneration on S5 days.It’s clear that follicle development and production performance are basically restored 7 days after heat stress is relieved.In summary,heat stress leads to the increase of CRH and corticosterone in the blood and follicles of laying hens,which in turn leads to oxidative stress in follicular,activates the Fas/Fas L,TNFα/TNFR1 signal pathways and reduces the E2/P4 ratio.Thence,heat stress causes apoptosis of follicular granulosa cells,resulting in obstruction or atresia of follicle development,and lower egg production rate.This study revealed the mechanism of heat stress on the follicle development of laying hens in some part,and provided basis for alleviating heat stress damage of laying hens and improving economic benefits theoretically. |
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