【作者】 任清长；

【导师】 杨红建；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 国内外研究表明,孕酮(P4)能够调节环境微生物的生长代谢。当奶牛进入妊娠期后,其血液中的孕酮水平迅速升高并维持较高水平直至分娩结束才降低。关于P4通过反刍动物的唾液和血液循环进入瘤胃后调节微生物生长代谢的研究,尚未报道。因此,本试验开展了以下研究：第一,采用5×5拉丁方随机试验,在饲喂相同精粗比(61：39)日粮的条件下,研究了中国北方常用的5种全混合日粮对瘤胃P4、睾酮和羟酮(ADD)昼夜浓度的影响。瘤胃P4、睾酮和ADD昼夜浓度变化范围分别为5.21～6.61ng/mL、1.37～1.61ng/mL和4.10～5.51ng/mL,并且其昼夜变化与瘤胃总VFA浓度变化呈正相关,相关系数分别为0.56、0.62和0.66(P<0.01)。不同的全混合日粮对瘤胃P4、睾酮和ADD浓度有显著的影响：与饲喂玉米秸组日粮相比,饲喂玉米秸+玉米黄贮；玉米黄贮+羊草；全株玉米青贮+羊草和全株玉米青贮+羊草+苜蓿日粮瘤胃中的P4浓度分别提高4.17%,5.44%,9.07%和34.48%(P<0.001);睾酮浓度分别提高8.10%,33.33%,54.05%和65.76%(P<0.001);ADD分别提高14.51%,49.84%,77.60%和95.26%(P<0.001)。结果提示,增加日粮蛋白和能量水平,可能提高瘤胃P4、睾酮和ADD浓度。第二,向培养液中添加P4,使其终浓度分别达到0,2,20,40,80和100ng/mL,分别培养瘤胃微生物6,12,24,36,48和72h,研究了瘤胃微生物对P4的转化。试验结果表明,瘤胃微生物能够将1mol的P4转化为0.55-0.99mo1的睾酮;生成的睾酮被进一步转化,生成0.39-0.82mol ADD.瘤胃微生物转化P4的速率和转化率分别与P4的添加浓度和培养时间有关：初始浓度越高,P4的转化速率越快(P<0.001)；培养时间越长,转化率越高(P<0.001)。试验结果提示,瘤胃可能是P4的一个重要的转化场所。第三,分别以高淀粉日粮和低淀粉日粮(200g/kg羊草+800g/kg玉米粉和800g/kg羊草+200g/kg玉米粉)为底物培养瘤胃微生物,同时向培养液中添加P4,研究了P4对瘤胃微生物消化代谢的调节。与对照组相比,P4添加组瘤胃微生物蛋白合成提高15.43%(P<0.001)：干物质降解率和降解速率分别提高6.57%(P=0.043)和19.91%(P<0.010)；总VFA浓度提高11.03%(P<0.010)。此外,P4能够提高乳脂和体脂前体物乙酸的摩尔比,降低生糖前体物的摩尔比。试验结果提示,当瘤胃P4浓度提高后,有利于乳脂前体物的合成。第四,体外培养瘤胃产甲烷菌,研究了P4对产甲烷菌消化代谢的影响。试验结果显示,P4可能抑制产甲烷菌的生长,减少甲烷的产生；降低纤维降解酶活和粗饲料底物的降解率以及总VFA浓度(P<0.05)。结果提示,当瘤胃P4的浓度提高后,有利于降低饲料的能量损失。第五,分离、纯化和培养瘤胃真菌Neocallimastix sp,研究了P4对Neocallimastix sp消化代谢的影响。P4能够促进Neocallimastix sp的生长代谢,提高纤维素降解酶活(P<0.010)、干物质、NDF和ADF的降解率(P<0.05)；提高总VFA、二氧化碳和氢气的产生(P<0.05)。试验结果提示,当瘤胃P4浓度提高后,有利于饲料的降解和总挥发酸的生成。总之,孕酮具有调节瘤胃发酵的功能并且可被微生物转化,改善日粮营养水平,能够提高瘤胃孕酮浓度。试验结果提示,瘤胃微生物宿主可能通过自身分泌的激素对瘤胃微生物的代谢活性进行调节。更多还原

【Abstract】 It has been reported that progesterone (P4) could regulate metabolite of environmental microorganisms. Progesterone will maintain high levels in blood serum when dairy cow become pregnant. However, information associated with about effect of P4on rumen microbes when it enter the reticulo-rumen through the wall of forestomach and saliva reproductive cycle are not well investigated. Thereby, the authors in the present study carried out following experiments:Firstly, a5×5Latin square experiment design was applied to investigate the effects of five total mixed rations (TMRs) which were formulated at same ratio of concentrate to forage (61:39) including different forage combinations on diurnal variation of P4, testosterone and ADD concentrations in rumen fluids of the lactating dairy cows. The whole experiment was completed in five periods and each period was carried out for18d, the cows were gradually adapted to the corresponding TMRs from d0to d15, and rumen fluids were intensively collected on d16, d17, and d18in each period. Over the24-h period, ruminal P4, testosterone and ADD concentration varied from5.21to6.61ng/mL,1.37to1.61ng/mL, and4.10to5.51ng/mL, respectively. The varyations were positively related to diurnal varyation in rumen total VFA concentration, with coefficiens of0.56(P<0.010),0.62(P<0.010), and0.66(P<0.001), respectively. The TMRs with different forage combinations hadt effect on ruminal concentrations of P4, testosterone and ADD (P<0.001). Compared with TMR including corn stover, the rumen P4concentration in TMR including corn stover plus ensiled corn stover; ensiled corn stover plus Chinese wild ryegrass hay; Chinese wild ryegrass(Leymus chinensis) hay plus whole corn silage; Chinese wild ryegrass hay plus whole corn silage plus alfalfa hay were increased by4.17%,5.44%,9.07%, and34.48%(P<0.001), respectively. Meanwhile, testosterone was increased by8.10%,33.33%,54.05%, and65.76%(P<0.001), respectively. ADD was increased by14.51%,49.84%,77.60%, and95.26%(P<0.001), respectively. The results indicate that increased of diary protein and energy leves may enhance ruminal P4, tstousterone and ADD concentration.Sencondly, A completely randomized design was applied to five runs of in vitro batch cultures with six P4dose rates of0(control),2,20,40,80and100ng/mL in culture fluids to investigate microbial transformation of P4. Within each run, five fermentations for each incubation interval of6,12,24,36,48and72h were arranged for each P4dosage treatment. Rumen microbes could translate1molar of P4into0.55to0.99molar testosterone, and the produced testosterone was further translated into0.39to0.82molar ADD. The transformation was related to intial P4concentration and incubation time, the high the initial P4concentration was, the quickly translated rate (P<0.001); and the longer incubated time, the more microbial transformation was (P<0.001). The results suggested that rumen should not be neglected as another important arena of P4metabolism.Thirdly, the effects of P4on microbial metabolism was investigated with high and low starch substrate in vitro batch culture.Rumen contents were collected from five lactating dairy cows. P4was added into culture fluids to achieve final concentration of0(control),2,20,40,80and100ng/mL. Compared with P4control, microbial crude protein was increased by15.43(P<0.001); dry matter degradation and rate were increased by6.57%(P=0.043) and19.91%(P<0.001), respectively. In addition, total VFA concentration was increased by11.03%(P<0.001). In the present study, molar proportion of milk and body fatty precursor were increased, whereas glucogentic VFA was decreased.The result in the present experiments showed that P4may be benefial for milk fatty synthesis.Fourthly, effects of P4on metabolite in methanogens was investigated. In this study, P4could inhibit growth of methanogens and decreased methano production (P<0.05). Meanwhile, fibrolytic enzymes were also decreased, which resulted in decrease in degradation of substrate (P<0.05) and production in total VFA (P<0.05), respectively. The result indicated that P4could inhibit metabolitic avtivity of methanogens, and the increase of P4in the rumen may be beneficial for reduce of energy loss.Fivthly, effects of P4on metabolite in pured fungi of Neocallimastix sp was investigated. In this study, P4could stimulate growth of Neocallimastix sp and increase fibrolytic enzymes activity, degradation of dry matter, NDF and ADF (P<0.05), which resulted in increases in total VFA concentration (P<0.05) and ratio of non-glucogentic to glucogentic acids, respectively. The enhanced Neocallimastix sp indicted that P4was benefial for degradation of feed and production of VFA.In brief, the results presented contribute important new knowledge about the P4transformation in rumen and effect of P4on metabolism of rumen microbes.The results showed that host may regulate microbial metabolite via its steroid hormone.更多还原