| Many special economic animals are seasonal animals. How to artificially change their breeding and make their quantity and quality to meet human needs is always the, main objective for the special economic animal industries. To find and use the right feed additives which can not only promote development and reproduction of special economic animals but also bring no food safety issues, thereby contributing to the development of special economic animal breeding industry, is an urgent problem to be solved for the special economic animal breeding industry. Many special economic animals are herbivorous animals. Looking for natural plant secondary metabolites from plants they feed and then making them as the feed additives is an effective way to promote the development of special economic animal industry.6-MBOA is a plant secondary metabolite derived from some grasses. It has been proven in some researches that6-MBOA can promote the development and reproduction of many animals, thus it is an ideal feed additive for special economic animals breeding. In this study, we selected the Leymus chinensis planted in experiment land which is the dominant plant in grasslands of Inner Mongolia for the object to study effects of different treatments on the content of6-MBOA in L.chinenisis as well as variation of6-MBOA content with ontogeny of L.chinenisis by method of HPLC to find the variation law and the factors influencing this variation. Then we chosed the Brandt’s vole breeded in captivity as the object to measure the hormones levels in blood and the transcription levels of the genes in hypothalamus and gonad related to reproduction, and check the morphology of gonad by using the method of Elisa test, RT-PCR, Western Blot, and paraffin slicing. Finally we studied short and long term impact of6-MBOA on the reproduction of adult Brandt’s vole and its effect on the sexual maturity of pubertal Brandt’s vole, based on which we probed the mechanism of6-MBOA work. Our studies can also provide a theoretical basis for6-MBOA as food additives of special economic animals breeding.(1) The content of6-MBOA in L.chinenisis gradually decreased from day7to day17after germination. The content on either day7or day8is significantly higher than day12and day17(P＜0.001), and there is no significant difference between day7and day8(P>0.05), the same is for the difference between day12and day17(P>0.05). One hour after treatment, the contents of6-MBOA in the control group and shear injury group were significantly lower than that in the chewing group and smearing saliva group (P＜0.05), but no significant difference was found between shear injury group and control group (P＜0.05). One day and five days after treatment, the content of6-MBOA in control group was both significantly lower than that in the other three groups (P＜0.001). Ten days after treatment, the content of6-MBOA in the control group was significantly lower than that in shear injury group and smearing saliva group (P＜0.001), but no significant difference was found between control group and chewing group (P>0.05). Repeated measurement analysis showed that the content of6-MBOA in the control group was significantly lower than that in the other three treatment groups (P＜0.001), the content of6-MBOA in shear injury group was significantly lower than that in smearing saliva group and chewing group (P<0.001, P=0.003), and the content of6-MBOA in smearing saliva group was significantly higher than that in chewing group (P=0.016).Based on the result of6-MBOA content variation, the study suggested that6-MBOA content of the whole plant of L.chinensis declined with ontogeny, under which we speculated that L.chinensis in Inner Mongolia grassland contains higher6-MBOA content in early spring germination stage, then it would soon decline significantly. This study found that the content of6-MBOA increased significantly after animal feeding and mechanical damage, and then drop to normal level, which shows that6-MBOA is a dynamic defensive substance of L.chinenisis. But the mechanical damage induced reactions on increasing of6-MBOA content in L.chinenisis lagged behind Brandt’s vole grazing induced reaction. So animals’ saliva may play an important role in the induced reaction of increasing of6-MBOA content in L.chinenisis. (2) In adult female Brandt’s vole treated by short term intraperitoneal injection of6-MBOA, E2level in the low dose group was significantly higher than that in the control group and the high dose group (P=0.026, P=0.002, respectively). FSH content in the control group was significantly lower than that in the middle dose group (P=0.008). MEL content in the middle dose group was significantly higher than that in the control group (P=0.050). In hypothalamic AVPV area, the mRNA of Kiss1gene in the low dose group was significantly higher than that in the control group, middle dose group, and high dose group(P<0.001, for three). In hypothalamic ARC area, the mRNA of Kissl gene in the control group was significantly lower than that in the low dose group, middle dose group and high dose group respectively (P<0.001, for three); the mRNA of GPR54gene in the control group was significantly lower than that in the low dose group (P=0.004); the mRNA of GnRH gene in the low dose group was significantly higher than that in both of the medium dose and high dose group (P=0.043, P=0.016, respectively). In ovarian, mRNA of StAR gene in the low dose group was significantly higher than that in the control group and the middle dose group (P=0.010, P=0.049, respectively); mRNA of CYP19gene in the middle dose group was significantly higher than that in the control group, low dose group, and high dose group (P＜0.001, P=0.044, P=0.005, respectively), and mRNA of CYP19gene in low dose group was also significantly higher than that in the control group (P=0.017); mRNA of CYP11A1gene in the high dose group was significantly higher than that in the control group and low dose group (P=0.002, P=0.003, respectively). In ovarian, StAR protein level in the low dose group was significantly higher than that in the control group (P=0.021); CYP19protein level in the middle dose group was significantly lower than that in the low dose group and high dose group (P=0.026, P=0.015, respectively); CYP11A1protein level in the low dose group was significantly higher than that in the control group and the high dose group (P=0.021, P=0.023, respectively).In adult male Brandt’s vole treated by short term intraperitoneal injection of6-MBOA, testosterone in the control group was significantly lower than that in the low dose and middle dose group (P=0.012, P=0.004, respectively), and testosterone in the high dose group was significantly lower than that in the middle dose group (P=0.036); FSH was the highest in the low dose group and significantly higher than that in the control group (P=0.004); MEL in the high dose group was significantly lower than that in the control group and the middle dose group (P= 0.002, P=0.039, respectively). In the hypothalamus AVPV area, mRNA of KiSS1gene in the low dose group was significantly higher than that in the control group, middle dose group, and high dose group (P＜0.001for three). In the hypothalamus ARC area, mRNA of KiSS1gene in the middle dose group was significantly higher than that in the control group and the high dose group (P=0.002, P=0.022, respectively); mRNA of GPR54gene in the control group was significantly lower than that in the low dose group and middle dose group (P=0.028, P=0.012, respectively); mRNA of GnRH gene in the high dose group was significantly lower than that in the low dose and middle dose group (P=0.043, P=0.019, respectively),In testis, mRNA of StAR gene in the low dose group was significantly higher than that in the control group and high dose (P=0.010, P=0.008, respectively); mRNA of CYP11A1gene in the middle dose group was significantly higher than that in the control group (P-0.022); transcription of CYP19gene in high dose group was significantly lower than that in the control group (P=0.020). In testis, StAR protein level in the low dose group was significantly higher than that in the control group (P=0.021); CYP19protein level in the middle dose group was significantly lower than that in the low dose group and high dose group (P=0.026, P=0.015, respectively); CYP11A1protein level in the low dose group was significantly higher than that in the control group and the high dose group (P=0.021, P=0.023, respectively).Based on the result of short term intraperitoneal injection of6-MBOA experiment for both male and female Brandt’s vole, these studies showed that a certain dose of6-MBOA can significantly increase the mRNA level of KiSSl gene in the hypothalamus, so that it can regulate the release hormone of GnRH, then promote the secretion of FSH. A certain dose of6-MBOA can also promote the transcription of StAR gene and CYP11A1gene in gonad. For CYP19gene,6-MBOA plays a positive effect in female animals, but plays a negative effect in male animals. So a certain dose of6-MBOA in the short-term can ultimately increase the synthesis and secretion of sex hormones and promote the breeding of Brandt’s vole.(3) In adult female Brandt’s vole treated by long term intraperitoneal injection of6-MBOA, the index of uterus in the high dose group was significantly lower than that in the low dose group and middle dose group (P=0.002, P=0.001, respectively); ovarian index in the high dose group was significantly lower than that in the control group, low dose group, and middle dose group (P=0.003,P=0.025, P＜0.001, respectively), and ovarian index in low dose group was significantly lower than the middle dose group (P=0.019). FSH in the middle dose group was significantly lower than that in the control group, low dose and high dose group (P=0.026, P=0.004, P=0.001, respectively). In hypothalamus AVPV area, the mRNA of KiSS1gene in the low dose group was significantly higher than that in the control group, middle dose group and high dose (P=0.004, P=0.005, P=0.002, respectively); tmRNA of GPR54gene in the control group was significantly lower than that in low dose and high dose group (.P=0.038, P=0.011, respectively). In ovarian, the mRNA of StAR gene in the high dose group was significantly lower than that in the control group and low dose group (P=0.027, P=0.017, respectively); mRNA of CYP11A1in the low dose group was significantly higher than that in the control group (P=0.034); mRNA of CYP19in the control group was significantly higher than that in the high dose (P=0.011). CYP19protein level in the control group was significantly higher than that in the high dose group (P=0.031); CYP11A1protein level in the low dose group was significantly higher than that in the control group (P=0.045).In adult male Brandt’s vole treated by long term intraperitoneal injection of6-MBOA, testis index in the control group was significantly higher than that in the middle dose group and high dose group (P=0.001, P=0.010, respectively), and it in low dose group was also significantly higher than that in the middle dose group (P=0.041). Plasma LH levels in the low dose group was significantly higher than that in the high dose group (P=0.049); FSH in the high dose group was significantly lower than that in the control group, low dose group and middle dose group (P=0.006, P=0.015, P=0.028, respectively); T level in the control group was significantly higher than that in the high dose group (P=0.037). In hypothalamus AVPV area, mRNA of KiSS1gene in the high dose group was significantly lower than that in the control group and the middle dose group (P=0.037, P=0.001, respectively), and low dose group was significantly lower than the middle dose group (P=0.021). In hypothalamus ARC area, mRNA of KiSS1gene in the control group was significantly higher than that in the low dose group, middle dose group, and high dose group (P=0.035, P=0.045, P=0.002, respectively); mRNA of GPR54gene in the high dose group was significantly lower than that in the control group, low dose group, and middle dose group (P=0.005, P=0.037, P=0.040, respectively); mRNA of GnRH gene in the control group was significantly higher than that in the low dose group, middle dose group, and high dose group (P=0.040, P=0.017, P=0.013, respectively). The mRNA of StAR gene in the control group was significantly higher than that in the low dose, middle dose, and high dose group (P=0.001, P＜0.001, P＜0.001, respectively); mRNA of CYP11A1gene in the control group was significantly higher than that in the low dose, middle dose, and high dose group (P<0.001,for three); mRNA of CYP19gene was significantly higher than that in control group, low dose, middle dose, and high dose group (P＜0.001, for three), and it in low dose group was also significantly higher than that in the middle dose group and high dose group (P=0.009, P=0.028, respectively). StAR protein level in the control group was significantly higher than that in the low dose group, middle dose group and high dose group (P=0.016, P＜0.001, P＜0.001, respectively), and it in low dose group was significantly higher than that in the middle dose group (P=0.006); CYP19protein level in the control group was significantly higher than that in the low dose group and high dose group (P=0.002, P=0.005, respectively), and it in middle dose group was also significantly higher than that in the low dose group and high dose group (P=0.010, P=0.028, respectively); CYP11A1protein level in the control group was significantly higher than that in the low dose group, middle dose group, and high dose group (P=0.002, P＜0.001, P＜0.001, respectively). Seminiferous epithelial thickness was significantly higher than that in control group, middle dose group, and high dose group (P=0.023, P＜0.001, respectively), and it in low dose group was significantly higher than that in the middle dose group and high dose group (P＜0.001, for both).Based on the result of long term intraperitoneal injection of6-MBOA experiment for both male and female Brandt’s vole, these studies show that the long-term application of6-MBOA on hypothalamus inhibited the KiSS1/GPR54system of male and female voles and it has more powerful inhibiting effect in male Brandt’s vole. The transcriptions and expressions of the StAR and CYP11A1genes of testis in testosterone synthesis pathway and StAR and CYP19gene of ovary in pathway of the estradiol synthesis were significantly inhibited by long-term application of6-MBOA. The growthes of testis of male and the ovary of female Brandt’s vole were both inhibited by the long-term application of some dose6-MBOA. Therefore, we speculate that the long-term application of6-MBOA has certain inhibiting effect on the reproduction of Brandt’s vole.(4) In pubertal female Brandt’s vole treated by30days intraperitoneal injection of6-MBOA, the opening time of cunnus of female pubertal Brandt’s vole in control group is much longer than that in low dose and middle dose groups (P=0.049, P=0.002, respectively), and the opening time in high dose group is much longer than that in the middle dose group (P=0.020). LH in control group is much lower than that in middle dose goup (P=0.046); FSH in low dose group is much higher than that in middle and high dose groups (P=0.034, P=0.045, respectively); E2in low dose group is much higher than that in control group, middle dose and high dose groups (P=0.008, P=0.047,P=0.002, respectively); mRNA of GPR54gene in low dose group is much higher than that in high dose group (P=0.045). mRNA of StAR gene in the low dose group was significantly higher than that in the control group and the high dose group (P=0.011, P=0.013, spectively); mRNAn of CYP11A1in the low dose group was significantly higher than that in the control group (P=0.032). StAR protein content in the control group was significantly lower than that in the low dose group (P=0.016, P=0.021, respectively). Paraffin sections showed that the ovary of the middle dose group had many large growing follicles.In pubertal male Brandt’s vole treated by30days intraperitoneal injection of6-MBOA, the weight of the pubertal male Brandt’s vole in the high dose group was significantly higher than that in the low dose group and middle dose group (P=0.031, P=0.041, respectively). LH in plasma was significantly lower than that in the low dose group and middle dose group (P=0.031, P=0.005, respectively); FSH in the control group was significantly lower than that in the low dose group and middle dose group (P=0.035, P=0.044, respectively). T in low dose group was significantly higher than that in the control group and high dose group (P=0.003, P=0.032, respectively). In AVPV area of hypothalamus, the mRNA of KiSSl gene in the control group was significantly higher than that in the middle dose and high dose group (P=0.029, P=0.043, respectively). The mRNA of the StAR gene in the testis in low dose group was significantly higher than that in the control group, middle dose group and high dose group (P＜0.001, P=0.026, P＜0.001). The middle dose group was also significantly higher than the high dose group (P=0.013). The mRNA of CYP11A1gene in the low dose group was significantly higher than that in the control group and the high dose group (P=0.036, P=0.026, respectively).The mRNAn of CYP19gene in the control group was significantly higher than that in the low dose group, middle dose group and high dose group (P=0.014, P=0.010, P=0.002,respectively), the low dose group was also significantly higher than the high dose group (P=0.040).The amount of StAR protein in the testicle in the low dose group was significantly higher than that in the control group and middle dose group (P=0.048).The diameter of the testicular song seminiferous tubule in low dose group was significantly lower than that in the other three groups (P<0.001),the middle dose group was also significantly lower than the high dose group (P=0.045).The thickness of the convoluted tubule’seminiferous epithelium in the low dose group was significantly thinner than the other three groups (P＜0.001, P=0.001, P＜0.001).Based on the result of30days intraperitoneal injection of6-MBOA experiment for both pubertal male and female Brandt’s vole, these studies show that a certain amount of6-MBOA can promote male puberty mice testis drop, make the transcription and translation of StAR and CYP11A1promote obviously in ovary, let the cunnus of female puberty Brandt’s vole open in advance. However, intraperitoneal injection of6-MBOA couldn’t promote the transcription of KiSS1gene and the growth of reproduction organ. Therefore, we consider that30days intraperitoneal injection of6-MBOA had little effect on the sex maturity of pubertal Brandt’s vole.(5) In conclusion, plant secondary metabolites analogous to6-MBOA were the defense substances for Leymus chinensis and were produced soon in the dynamic defense and induce defense processes in Leymus chinensis. The saliva of Brandt’s vole played an important role in the induce defense processes for6-MBOA increasing. Our study supported the hypothesis that the dynamic defense for plants to mammals is common. A certain dose of6-MBOA in the short-term can ultimately increase the synthesis and secretion of sex hormones and promote the breeding of Brandt’s vole, the long-term application of6-MBOA has certain inhibiting effect on the reproduction of Brandt’s vole and30days intraperitoneal injection of6-MBOA had little effect on the sex maturity of pubertal Brandt’s vole. So the6-MBOA may act as antagonist to melatonin to influence the expression of KiSSl gene in hypothalamus, this supported hypothesis that the6-MBOA is the antagonist to melatonin. Additionally,6-MBOA may act directly on the gnoad. |
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