| In recent years,the high-yielding cows in intensive dairy farms in China are generally in a state of negative energy balance(NEB)after calving,and the anestrus dominated by inactive ovaries(IO)due to NEB has become a major reproductive obstacle faced by large-scale dairy farms.Brain-derived neurotrophic factor(BDNF)has been found to play an important role in the development of follicles,but its role in the IO of dairy cows has not been previously reported.Therefore,this study will clarify the role of BDNF in postpartum IO caused by NEB in dairy cows through four experiments in vitro and in vivo,so as to provide scientific basis for further research on the molecular mechanism and new prevention and treatment strategies of postpartum IO in dairy cows.(1)The relationship between BDNF and energy balance and ovarian quiescence.In a large-scale farm in Heilongjiang Province,120 dairy cows with 2?3 parities were selected for blood energy index detection,clinical disease,estrus and B-ultrasound examination.Fifty days after calving,the cows were divided into the positive energy balance natural estrus group(PEB-E,n=15)and the negative energy balance ovarian rest group(Neb-IO,n=15)by two groups of dairy cows blood energy metabolic index,clinical diseased,estrus behaviors and B ultrasound examination.Through two groups of dairy cows breeding performance,energy metabolism index,hepatic function index and blood hormone indexes of independent sample t-test and single factor variance analysis,the results showed that the breeding performance,blood levels of E2,INS,IGF 1 and LP were markedly decreased,and of GH was significantly increased in NEB-IO group of dairy cows,showing that NEB may result in IO by causing cow hormone disorder.Cohort study M-H2 test was used to determine the relationship between energy negative balance and levels of BDNF postpartum 20 d and BDNF and the IO postpartum d 50 in two groups of cows,the results showed that postpartum 20 d cow NEB were strong positive correlated with decreased BDNF,postpartum 50 d low blood levels of BDNF were strong positive correlated with inactive ovary,suggesting that NEB,low blood levels of BDNF can promote the occurrence of postpartum IO of cows.Through ROC analysis of BDNF and Glu in blood at 20 d and 50 d postpartum,the results showed that BDNF and Glu at 20 d and 50 d postpartum could be used as early warning indicators of IO in dairy cows,among of them the early warning values at 20 d postpartum were BDNF < 1354.70 ng /L and Glu < 2.48 mmol/L,he early-warning values at 50 d postpartum were BDNF < 1421.77ng/L and Glu < 2.28 mmol/L.Through two groups of cows(each 3 head)HE staining and FSH,BDNF and Trk B immunohistochemical test of ovaries,the results showed that NEB-IO groups of ovarian follicular dysplasia was more,granular cell membrane layer of tertiary follicles,FSH,BDNF and Trk B in NEB-IO group were decreased significantly,showing that low levels of BDNF inhibits NEB-IO group of cows follicular granulosa cell proliferation.(2)The effect of BDNF on the growth,proliferation and hormone secretion of dairy cow granulocyte under low glucose conditions.Model for cows follicular granulosa cells and by Western blot test of expression level of BDNF and Trk B in the normal and low glucose medium,combining BDNF secretion of ELISA detection,the results showed that the expression of BDNF secretion was significantly decreased in low glucose cultivation of follicular granulosa cells,and the expression of Trk B no difference,showing that low glucose reduce the expression and secretion of BDNF and follicular granulosa cells.Western blot was used to detect the expression levels of CCNA1,CCND1,CCNE2,CDK1,St AR and HSD3B1 in normal and low glucose cultures,the results showed that the expression levels of cyclin and steroid hormone synthesis protein in follicular granulosa cells cultured in low glucose were weaker than those cultured in normal culture,indicating that low glucose inhibited the proliferation and hormone secretion of follicular granulosa cells.CCK8,cell count,cell cycle test,and medium E2 and P4 tests were used to determine the optimal dose and time of BDNF addition in normal and low glucose medium,the results showed that 100 ng/m LBDNF addition for 24 h had the best effect on follicular granulosa cell proliferation and hormone secretion.Cell cycle and CCK8 were used to detect follicular granulocyte cells in normal and low glucose cultures with K252 a and BDNF+K252a,the results showed that the ratio of S phase and cell activity of cells in the two groups were significantly lower than that of the BDNF group under low glucose and normal culture conditions(P<0.01),indicating that BDNF,through its main receptor Trk B,can play an active role in the proliferation and viability of follicular granulocyte cells in dairy cows under low glucose and normal culture conditions.(3)Effects of low glucose on proliferation and hormone secretion of follicular granulosa cells promoted by BDNF through PI3K/AKT signaling pathway.Western blot was used to detect the phosphorylation of AKT in follicular granulosa cells cultured with low glucose by LY294002(PI3K/AKT inhibitor),K252 a and BDNF,the results showed that both LY294002 and K252 a could reduce the phosphorylation level of BDNF on AKT,indicating that BDNF activated the PI3K/AKT pathway through Trk B.CCK8,cell cycle and Western blot were used to detect the activity,cell cycle,cyclin and steroid hormone synthesis protein levels of follicular granulosa cells under low glucose and normal medium with LY292004 and BDNF,the results showed that S phase cell viability,cell cycle in BDNF group under low sugar and normal cultivation were significantly higher than the control group,the proportion of LY292004 group and LY292004 + BDNF,BDNF group of cell cycle protein and steroid hormone synthesis related protein expression were significantly higher than the control group,LY292004 + BDNF group of cell cycle protein and steroid hormone synthesis related protein expression of BDNF treatment group were significantly lower than the control group,showing that BDNF in low sugar and normal cultivation conditions can promote granulosa cell proliferation and steroid hormones by PI3K/AKT.(4)The effect of low glucose on BDNF promoting follicular granulosa cell proliferation and hormone secretion through MAPK/ERK signaling pathway.Western blot was used to detect the ERK1/2 phosphorylation level of PD98059(MAPK/ERK inhibitor),K252 a and BDNF on low-sugar follicular granulosa cells,the results showed that PD98059 and K252 a could both reduce the ERK1/2 phosphorylation level stimulated by BDNF,indicating that BDNF activated the MAPK/ERK pathway through Trk B.CCK8,cell cycle and Western blot were used to detect the activity,cell cycle,cyclin and steroid hormone synthesis protein levels of follicular granulosa cells under low glucose and normal medium with PD98059 and BDNF,the results showed that S phase cell viability,cell cycle in BDNF group were significantly higher than the control group under the condition of low sugar and normal cultivation,the proportion of PD98059 and PD98059 + BDNF group,BDNF group of cell cycle protein and steroid hormone synthesis related protein expression were significantly higher than the control group,PD98059 + BDNF group of cell cycle protein and steroid hormone synthesis related protein expression of BDNF treatment group were significantly lower than the control group,showing that BDNF in low sugar and normal cultivation conditions can promote granulosa cell proliferation and steroid hormones by MAPK/ERK.Conclusions: This study determines blood clinical pathology characteristic of postpartum IO,and NEB,establishes low blood level of BDNF as high risk factors of IO and the blood BDNF and Glu as early warning indicators of IO,and confirms that BDNF-Trk B through PI3K/AKT,MAPK/ERK two ways promotes granulosa cell proliferation and secretion of steroid hormones,it provides a new direction for the future in-depth revealing molecular mechanism and control of IO in dairy cows. |
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