山羊瘤胃原虫与细菌吞噬关系和微生物AA变化机制的研究
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摘要
瘤胃微生物蛋白质是反刍动物小肠氨基酸(AA)的重要组分。微生物的任何变化,如微生物的区系、AA-N比例、AA组成等的变化都将影响十二指肠AA的供给。在过去的研究和生产中一直认为瘤胃微生物AA是恒定的。然而1992年Clark等综合比较众多研究者的研究结果提出了瘤胃微生物AA是可变化的观点,此后至今,由于其对宿主不可或缺的重要性而使得微生物AA变化与否的论题一直为本领域研究与争论的焦点。为此,本课题结合in vitro与in vivo法,并引入荧光标记细菌技术以及SSCP、克隆测序等分子生物学技术,系统地探讨微生物蛋白质AA的变化规律及其机制,试验共分九个部分进行。
试验1荧光标记瘤胃细菌方法的建立和原虫吞噬细菌速率的研究
以4只装有永久性瘤胃瘘管的徐淮山羊提供的瘤胃液制备荧光标记瘤胃细菌,用于瘤胃原虫的摄食实验,研究山羊瘤胃原虫吞噬细菌的速率。试验设置清洗原虫祛除瘤胃自由细菌的荧光全标组(WFLB)及未清洗原虫保留瘤胃自由细菌的荧光标记组(FLB)。结果表明: WFLB组、FLB组吞噬速率分别为:398.40 cells/(cell h)、230.40 cells/(cell h),换算为细菌N为:2.15 pg N/(cell h)、1.24 pg N/(cell h);每天每头山羊瘤胃内蛋白微循环中细菌N循环量WFLB组、FLB组分别估计为:103.20 mg N/(d头)、59.50 mg N/(d头),或者细菌蛋白循环量分别为0.645 g/(d头)和0.372 g/(d头);结果同时表明,荧光标记技术可以应用于瘤胃原虫对细菌吞噬速率的研究。
试验2日粮精粗比对山羊瘤胃内微生物蛋白质微循环影响的研究
本试验以4只装有瘤胃瘘管的山羊,研究不同精粗比日粮对山羊瘤胃微生态中原虫和细菌区系以及微生物蛋白微循环的影响规律。试验设置精(玉米-豆粕):粗(稻草)分别为10:90、30:70、50:50、70:30的四种比例日粮(A、B、C、D),采用4×4拉丁方设计进行动物试验,并结合荧光标记瘤胃细菌技术测定瘤胃原虫对细菌的吞噬速率。结果表明:日粮精粗比显著影响微生物细胞的密度。原虫的密度以C组最高;细菌和原虫的密度以都A组最低。日粮精粗比也显著影响原虫吞噬的速率,A、B、C、D四组吞噬速率分别为:429.50 cells/(cell h)、366.74 cells/(cell h)、389.48 cells/(cell h)、402.20 cells/(cell h),换算为对细菌N的吞噬速率分别为:2.319 pg N/(cell h)、1.98 pg N/(cell h)、2.103 pg N/(cell h)、2.172 pg N/(cell h)。每天每头山羊由于原虫的吞噬造成的细菌N的循环量分别估算为:136.49 mg N/(d头);369.02 mg N/(d头);485.99 mg N/(d头);440.56 mg N/(d头),或者菌体蛋白循环量为0.853 g Pr/(d头)、2.306 g Pr/(d头)、3.370 g Pr/(d头)和2.754 g Pr/(d头),以C组菌体蛋白循环量最大,细菌周转率最高(3.07 %)。
试验3精粗比对瘤胃发酵、原虫种群结构及吞噬速率的影响
在试验2的基础上,进一步研究在不同精粗比例配合日粮条件下山羊瘤胃发酵、原虫种群结构以及吞噬速率的变化规律。结果表明:日粮精粗比对瘤胃发酵有影响。以30:70组微生物活力较强、NDF降解率较高、瘤胃pH值也相对波动较小;精粗比对瘤胃原虫结构也有影响。50:50、70:30日粮组内毛虫和等毛虫的比例显著高于10:90、30:70组;而双毛虫和头毛虫则相反。内毛虫与双毛虫和头毛虫的吞噬速率分别为361.90、606.30和607.50 cells/(cell h),内毛虫的吞噬速率显著低于双毛虫和头毛虫(P =0.000),表明不同种属原虫吞噬细菌速率间有显著差异,但随日粮精粗比变化的规律基本一致。回归分析表明吞噬速率与日粮结构间呈三次方的曲线关系(Y=-724.53X+563.15X2-124.666X3+646.833,R2=0.97864)。另外,多元回归分析表明吞噬速率与微生物细胞密度有线性关系(R2=0.839)。方程为:Y=445.514-3.078X1+1.864X2(Y为吞噬速率;X1为细菌密度;X2为原虫密度)。
试验4碳水化合物结构对瘤胃发酵和微生物群体结构的影响
以3只瘘管山羊作为瘤胃液供体,用体外法研究底物碳水化合物结构对瘤胃发酵及微生物群体特征的影响。底物可溶性淀粉/纤维素比例为:100:0、70:30、50:50、30:70、0:100。结果表明:30:70组微生物产量和纤维降解率最高,发酵状态最佳;微生物蛋白产量与淀粉/纤维有三次方的曲线关系,细菌蛋白:Y=0.2410+0.0855X-0.0371X2+0.0029X(3R= 0.7397);原虫蛋白:Y=0.2276+0.0853X-0.0380X2+0.0030X3(R=0.7370)。各组分别在8、16、24、24和24 h出现最大微生物产量(P<0.01)。另外,微生物AA-N比例在各组间也有显著差异(P<0.05)。微生物区系原虫与细菌的比例总体上随淀粉水平下降呈先上升后降低,以50:50组最高;而且PCR-SSCP图谱反映了区系内部类群因底物的改变而改变。原虫分类计数结果表明随淀粉水平的下降,内毛虫与等毛虫的比例下降,而双毛属与头毛亚科原虫的比例增加,与SSCP的结果一致地表明其内部类群的改变。对提取DNA的质、量的检测表明:微生物分离平均获得率为53.29 %;细菌DNA的提取率(45.40 %)显著低于原虫(56.10 %)(P<0.05);微生物分离后DNA提取率为26.13 %;所提取的基因组DNA大小在20 kb以上,适合于后续研究的分子操作。综合以上试验结果可认为底物的变化引起了微生物发酵和其群系特征的变化,结果还证实PCR-SSCP适合于瘤胃微生物混合群体多样性研究,同时也验证了ITS1区段是研究瘤胃原虫的良好素材,从而基本确立了本课题瘤胃微生物群体研究的方法体系。
试验5不同分子形式氮源对瘤胃微生物蛋白产量和类群多样性的影响
本试验的主要目的是研究不同分子形式氮源在人工瘤胃体外培养条件下对瘤胃发酵、微生物蛋白合成和类群多样性的影响。以3只瘘管山羊为瘤胃液供体,底物设计为:氯化铵、寡肽混合物(<3, 000 Da)、小肽混合物(<500 Da)、游离氨基酸混合物。结果表明:pH在6.40-6.90之间变化,游离氨基酸组pH均值最低为6.61,氯化铵组的最高为6.79;氨氮浓度变化范围为11.60-29.45 mg/100 ml,均值以寡肽组最低为15.70 mg/100ml,游离氨基酸组最高为19.15 mg/100 ml;氯化铵组的细菌与原虫蛋白产量皆最低(0.1522、0.1179 mg/ml)(P<0.01),而两肽组的微生物蛋白产量则相对较高;另外原虫与细菌比值以氯化铵组最低为77.49 %,小肽组最高为104.50 %(P<0.01)。同时AA-N比例在各组间也有显著差异,以游离氨基酸组最低(P<0.05)。SSCP指纹图谱表明微生物类群内部种属也发生了变化,并以氯化铵组微生物的多样度最低。以上研究结果揭示了氮源的分子形式显著影响瘤胃发酵、微生物蛋白合成和微生物的类群结构。
试验6特定AA缺省对体外培养瘤胃微生物生长限制性的研究
本试验采用底物移除法研究特定氨基酸在人工瘤胃体外培养条件下对瘤胃微生物生长及其发酵特征的影响。以3只瘘管山羊作为瘤胃液供体,底物为:全量必需氨基酸组(A),组氨酸(B)、赖氨酸(C)、蛋氨酸(D)和支链氨基酸(E)的缺省组。结果表明:培养液pH值在5.90-6.80之间变化,均值以E组最高为6.54;培养液氨氮浓度变动范围为10.99-30.51 mg/100ml,均值以A组最高为17.85 mg/100 ml;底物对微生物生长的限制程度不同。以支链氨基酸缺省对微生物蛋白合成的限制最大,其细菌与原虫及其微生物蛋白的产量皆最低(0.1389、0.1772和0.3161 mg/ml)(P<0.01),相对于A组微生物蛋白下降了44.52 %。底物对细菌和原虫影响不同。原虫与细菌比值以C组最低(89.12 %),E组最高(127.60 %)(P<0.01)。同时底物还引起了微生物AA-N比例的变化(P<0.01),以A组最低(16.89),E组最高(18.76)。另外遗传指纹分析提示微生物区系内部类群也因底物而发生了变化。综合以上试验结果可认为特定氨基酸对微生物生长及微生物发酵都有一定的影响。
试验7蛋白质补充料对体外培养瘤胃微生物区系和发酵的影响
本试验以不同蛋白质补充料为底物进行体外培养,主要探讨蛋白质补充料对瘤胃微生物发酵和区系特征的影响。试验采用3只瘘管山羊作为瘤胃液供体,底物为:A(羽毛粉)、B(玉米蛋白粉)、C(豆粕)和D(鱼粉)。结果表明:pH值在5.80-6.80之间变化,均值以C组最低为6.19,A组最高为6.61;氨氮浓度变动范围为3.68-12.01 mg/100ml,均值以A组最低为5.49 mg/100ml,C组最高为9.95 mg/100ml;微生物蛋白产量在各组间差异显著或极显著(P<0.05、P<0.01),以D组最高(0.6513 mg/ml),A组最低(0.5289 mg/ml),C组细菌蛋白量(0.3309 mg/ml)为四组之最高。底物对微生物区系有选择作用。原虫与细菌比值以A组最高(107.00 %),C组最低(84.30 %)。SSCP图谱分析表明微生物区系内的类群结构也因底物发生了改变。另外底物还引起了微生物AA-N比例的变化,以D组最高(17.75),C组最低(16.48)(P<0.01)。综合以上试验结果认为不同蛋白质补充料体外培养条件下其瘤胃微生物发酵和微生物区系特征有明显不同。
试验8、9混合日粮蛋白质对徐淮山羊瘤胃微生物结构及AA组成的影响
试验8以4只瘘管山羊作为试验动物,采用4×4拉丁方设计进行试验。研究由羽毛粉(A)、玉米蛋白粉(B)、豆粕(C)和鱼粉(D)等不同蛋白质饲料配合的混合日粮对瘤胃发酵、微生物群体结构、微生物蛋白(MCP)产量及其AA组成模式的影响规律。在第一期正试期开始时分别从四只羊瘤胃中采集瘤胃液,用于相应日粮作为培养底物的预先体外培养试验,以确保复杂与昂贵的体内试验的有效性和必要性。结果表明,由不同蛋白补充料所配制混合饲料底物对瘤胃发酵、微生物类群结构及AA-N影响与单纯的蛋白补充料相比,在底物样本间差异程度上虽然有所降低,但影响规律影响基本一致,并且微生物类群结构及AA-N仍有明显或显著变化,表明进一步开展体内试验是必要和有意义的。
试验9继而开展的104天的4×4拉丁方体内试验结果表明:瘤胃液pH值在5.60-6.80之间变化,均值以A和C组较高,B和D组较低(P<0.05),A和C、B和D的pH均值虽相近,但随时间的动态变化模式相差较大;氨氮浓度变动范围为6.77-21.67 mg/100ml,均值以A组最低为11.08 mg/100ml,C组最高为15.04 mg/100ml,各组随时间动态变化模式也有所不同;日粮蛋白显著影响MCP产量,以C、D组微生物蛋白较高,A、B组较低(P<0.05);虽然C、D组微生物蛋白相当,但C组细菌蛋白产量却显著高于D组(P<0.05)。原虫与细菌区系比例在组间差异显著,豆粕组(81.27 %)显著低于其他三组(P<0.05)。进一步克隆测序分析表明细菌中的类R.flavefaciens、R.bromii、Roseburia faecalis等8群系在组间差异显著;原虫中除Diplodinium外其他4类群都有显著差异。微生物的AA-N比例在各组间也有显著差异,并以C组最低,D组最高,并且与细菌(原虫)蛋白质呈负(正)相关关系。研究同时发现部分种类AA含量在微生物区系间和同一区系内不同组间差异显著。原虫蛋白的Val比例高于细菌,而细菌蛋白的Lys则高于原虫。细菌蛋白的Arg;原虫蛋白的Met、Leu和His在各组间差异显著。差异性AA的变化与微生物类群的变化有一定的关联。综上可见混合日粮蛋白质对瘤胃发酵、微生物类群结构和微生物AA组成都有一定的影响。
The most important sources of AA at the duodenum of ruminants are the microbial crude protein (MCP) synthesized in the rumen, any change in microbes or MCP, such as, microbial flora, microbial AA-N ratio, and AA profile of microbes etc., is bound to affect AA supply to small intestine, and consequently influences animal performance. For a long time, it is well accepted that ruminal microbial AA is constant; moreover, it is common to use certain AA to direct ruminant practice. However, later studies reveal that, microbial AA is not constant. A review by Clark et al. (1992) indicated great differences in the AA composition of rumen bacteria, and followed by a continuous debate about this subject. Unfortunately, whether the microbial AA change or not, are still controversial issues till now. Therefore, in vitro and in vivo experiments were conducted using varied sources in this study, and aimed to illustrate whether microbial AA change, and how microbial AA change with diets. The Fluorescence-Labeled Rumen Bacteria Techniqe (FLRB) was developed to determine grazing rate of protozoa on bacteria in rumen, and the molecular technique including SSCP, cloning and sequencing etc., were also introduced into this paper. This dissertation was described in the following nine sections.
Trail 1 Establishment of Fluorescence-labeled Rumen Bacteria Technique and Study on the Grazing Rate of Protozoa
Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled rumen bacteria. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows: one group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid (WFLB); the other group was bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid (FLB). The result showed that, the bacterial predation rates of rumen protozoa were 398.40 cells/(cell h) for the group WFLB, 230.40 cells/(cell h) for the group FLB; When the corresponding values expressed as bacteria-N were: 2.15 pg N/(cell h) for the group WFLB and 1.24 pg N/(cell h) for the group FLB respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.20 mg N/(d head) for the group WFLB, 59.50 mg N/(d head) for the group FLB, respectively. It was estimated that protein recycling were 0.645 g Pr/(d head) for the group WFLB and 0.372 g Pr/(d head) for the group FLB, respectively. And finally, the fluorescence-labeled rumen bacteria technique (FLRB) would be a potential assay for determination of bacterial predation rate by rumen protozoa.
Trail 2 Effects of Dietary Concentrate to Forage Ratio on Microbial Protein Recycling in the Rumen of Goats
In this study, the main aims were to investigate effects of dietary concentrate to forage ratio on microbial protein recycling in the rumen of goats using a technique of FLRB, developed in trail 1. 4×4 Latin squares were conducted by using 4 Xuhuai goats with permanent cannulas, and diets were divided into A (10:90), B (30:70), C (50:50), and D (70:30) by varying concentrate to forage ratios, which concentrated food were corn-soybean meal, while forage were straw in this experiment. The result showed that, rumen micro-ecosystem was shifted heavily by concentrate to forage ratio. The highest protozoal density were recorded by group C which dietary concentrate to forage ratio was set as 50:50, whereas densities of both protozoa and bacteria were lowest for the group A; grazing rates of rumen protozoa on bacteria were, respectively: 429.50 cells/(cell h), 366.74 cells/(cell h), 389.48 cells/(cell h), and 402.20 cells/(cell h) for group A, B, C, and D. When the corresponding values expressed as bacteria-N were: 2.319 pg N/(cell h), 1.98 pg N/(cell h), 2.103 pg N/(cell h), and 2.172 pg N/(cell h) respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat with different diets, there were 136.49 mg N/(d head), 369.02 mg N/(d head), 485.99 mg N/(d head), and 440.56 mg N/(d head) for group A, B, C, and D respectively. It was estimated that, protein recycling of rumen bacteria were 0.853 g Pr/(d head) , 2.306 g Pr/(d head), 3.370 g Pr/(d head), and 2.754 g Pr/(d head) respectively, with group C recording the highest protein recycling of rumen bacteria and turnover rates (3.07 %).
Trail 3 Effects of Dietary Concentrate Levels on Rumen Fermentation, Protozoal Profiles and Grazing Rates
The objectives of present studies were to demonstrate the effects of dietary concentrate to forage rate on rumen fermentation, protozoal structure and grazing rates in goats’rumen. The experimental animal and desigh were the same as trial 2. The result showed that, rumen fermentation was shifted heavily by concentrate to forage ratio. High microbial activity and high NDF degradability were observed in group B, and pH was stable in this group, comparatively. It was also observed that, protozoal dynamics was modulated by dietary structure. Percentages of Entodiniinae and Isotrichidae were higher for diets which concentrate level were high, whereas percentages of Diplodiniinae and Ophryoscolecinae were higher for diets which forage level were high. The predation rates were 361.90, 606.30, and 607.50 cells/(cell h) for Entodiniinae, Diplodiniinae, and Ophryoscolecinae respectively, and marked difference was found among genus (P=0.000), however, the change rule of grazing rate with dietary structure seemed to be similar across genus. Regression analysis revealed that, there were strong cubic relationship between predation rates and dietary structure (Y=-724.53X+563.15X2-124.666X3+646.833, R2=0.97864). Additionally, multivariate regression analysis revealed the existence of linear correlations between predation rates and cell densities (protozoa and bacteria) (R2=0.839). The equation was presented bellow: Y=445.514-3.078X1+1.864X2 (Y- predation rate; X1 - bacteria density; X2 - protozoa density).
Trail 4 Effects of Different Structure of Carbhyborate on Rumen Fermentation and Microbes characteristics in vitro
Three goats fisted with cannulas were used to investigate the effects of rations in different starch to cellulose ratio on rumen fermentation and microbial characteristics in vitro. Substrates were designed by varying the level of starch/cellulose ratio as follows: 100:0, 70:30, 50:50, 30:70, 0:100. The results showed that: Cellulose degradability and microbial biomass were highest when starch/cellulose ratio in the culture was set to 30:70; The regression analysis between microbial protein and substrate were: Bacteria: Y=0.2410+0.0855X-0.0371X2 +0.0029X3 (R=0.7397); Protozoa: Y=0.2276+0.0853X-0.0380X2+0.0030X3 (R=0.7370) (Y: microbial protein, mg/ml; X: Starch/Cellulose), respectively. Significant differences were found in microbial AA-N ratio between groups (P<0.05); Furthermore, protozoa to bacteria ratio had a tendency to increase firstly, followed by decline when starch/cellulose ratio decreased, and the highest record falling in the group provided ration containing starch to cellulose ratio of 50:50; SSCP analysis further revealed that microbial structure was shifted by substrates; and also, cell-counting of protozoa showed that, Entodinium and Isotricha decreased, whereas Diplodinium and Ophryoscolecinae increased according to the decrease of starch/cellulose ratio, and revealed that the profile of protozoa was subjected to substrates, which agreed with SSCP. Additionally, the average recovery rate of microbes after detaching was 53.29 %; the DNA extraction ratio from bacteria (45.40 %) were significantly less than protozoa (56.10 %); the size of DNA fragment extracted from all the samples were larger than 20 kb; and fit for advanced research. It was therefore concluded that carbohydrate structure influenced both rumen fermentation and microbial characteristics. And finally SSCP has a potential for research on characterizing rumen mixed microbes, ITS1 is also good at research on protozoal diversity. So far, a measurement system was developed for the research on microbial characteristics in this trail.
Trail 5 Effects of Different Nitrogen Compounds MCP Yields and Microbial Diversity of Rumen
In this study, the main aims were to investigate effects of different N compounds on rumen fermentation, MCP yields and microbial diversity in vitro, using rumen liquor provided by 3 goats. Four treatments were NH4Cl, mixed oligo-peptide (<3, 000 Da), mixed oligo-peptide (<500 Da), and free amino acid respectively. Results showed that, the recorded pH-value ranged between 6.40 and 6.90, the average pH-value was lowest for the group with free amino acid, and highest for the group with NH4Cl in the culture. The varied range of NH3-N concentration was from 11.60 to 29.45 mg/100ml. The mean concentration of NH3-N was lowest in mixed oligo-peptide, and the highest was found in free amino acid. It was also observed that, yields of microbial protein varied with substrates, with lowest record dropping in NH4Cl, and microbial yields of both two peptides groups were comparatively higher. The protozoa to bacteria ratio was also shifted by substrates, and oligo-peptide recorded the highest peak, while the opposite was found for NH4Cl (P<0.01). Additionally, significant differences were found in microbial AA-N ratio between groups, with the lowest record falling in the group whose substrate was free amino acid (P<0.05). Furthermore, microbial diversity was demonstrated in SSCP fingerprint clearly, revealed that the structure of bacteria or protozoa community was altered by substrates, and the diversity in NH4Cl was much low. It could be concluded that, both rumen MCP yields and microbial structure were modified by different N compounds.
Trail 6 Study on Effects of Certain Amino Acids-Removal on the Growth of Rumen Microbe in vitro
The objectives of this study were to determine the effects of certain amino acids on rumen fermentation and microbial growth in vitro. Three goats fitted with cannula were used to provide rumen liquor, and the removal method was introduced into current experiment. Treatments were, respectively, total essential amino acid (A), His-removal (B), Lys-removal (C), Met-removal (D), branch chain amino acid (BCAA)-removal (E). Results showed that, the pH-value ranged between 5.90 and 6.80, and the highest mean value was observed in the group E (6.54). Concentration of NH3-N ranged between 10.99 to 30.51 mg/100ml, with the highest mean value dropping in the group A (17.85 mg/100ml). It was also observed that, yields of microbial protein and limiting degree on microbial growth were varied with treatments, with the group E demonstrating the lowest record (0.1389, 0.1772 and 0.3161 mg/ml for bacteria, protozoa, and microbes respectively) (P<0.01). The microbial yield of this group, comparing with the group A, decreased by 44.52 %. And also, the response to the substrate of protozoa differed from bacteria, and significant differences were found in the protozoa to bacteria ratio between groups. The group C interpreted the lowest data (89.12 %), and the reverse was true for the group E, displayed the highest value (127.60 %) (P<0.01). Additionally, microbial AA-N ratio was shaped by substrates, and lowest in the group A (16.89), while highest in the group E (18.76) (P<0.01). Further genetic fingerprint analysis revealed that, microbial profile was modified by substrates within bacteria or protozoa community. In conclusion, the rumen fermentation and microbial growth responded differently to certain amino acid, based on current in vitro trail.
Trail 7 Effects of Different Protein Supplement on Rumen Fermentation and Microbial Community in vitro
Certain protein supplement were used as substrates in this trail, and aimed to detect the characteristics of rumen fermentation and microbial community in vitro, by three goats fitted with cannula. Treatments: A (Plume meal), B (Corn gluten meal), C (Soybean meal), D (Fish meal). Results showed that, the pH-value ranged between 5.80 and 6.80, and the lowest mean value was found in the group C (6.19), while the highest occurred in the group A (6.60). NH3-N concentration ranged between 3.68 to 12.01 mg/100ml, with the group A recording the lowest average NH3-N concentration (5.49 mg/100ml), while the group C (9.95 mg/100ml) writing the highest value. Also, yields of microbial protein were varied with treatments (P<0.05, P<0.01), and the lowest record was observed in the group of A (0.5289 mg/ml), the other way round, the highest was found in the group of D (0.6513 mg/ml), it should be noted that the group C showed the highest bacteria yield (0.3309 mg/ml). It was further observed that, the protozoa to bacteria ratio was lowest in group C (84.30 %), the opposite was found for group A (107.00 %), interpreting the highest peak. Further SSCP analysis revealed that, profiles of both bacteria and protozoa subjected to substrates. Additionally, significant differences were found in microbial AA-N ratio between groups, and AA-N ratio was lowest (or highest) in the group of C (or D) (P<0.01). In a word, protein stuff brought out the changes, which were not just in microbial fermentation and flora, but in microbial AA-N ratio.
Trail 8, 9 Effects of Dietary Protein on Microbial Community and AA Composition of Rumen
Trail 8 The objectives of these parts were to investigate how rumen fermentation, microbial community, microbial protein (MCP) yields, and AA composition of MCP changed with dietary protein. Four goats fitted with rumen cannula, were used in a 4×4 Latin square design. And formula diets were divided into 4 groups according to their nitrogen source, which was, respectively, plume meal (A), corn gluten meal (B), soybean meal (C), and fish meal (D). At the beginning of first experimental period, rumen liquor was collected from each goat, and used for in vitro culture with the corresponding mixed diet, before the animal experiment. This pre-experiment aimed to ensure the necessity and validity of the complex and expensive animal experiment. The results showed that, marked differences were found in fermentation parameters, microbial diversity, and AA-N ratio etc. across groups, although the degree of variation had a somewhat decline, compared with the results of sole culture of protein supplement (trail 7). And these results indicated that, it was necessary and significant to carry out animal experiment using these mixed diet.
Trail 9 The results of the following 104-day in vivo experiment were described here. The recorded pH-value ranged between 5.60 and 6.80, and mean pH value of group A and C were high, the reverse was true for group B and D (P<0.05), the most interesting thing was that, although the mean pH value of group A and C (B and D) seemed to be similar, the change patterns of pH with time differed from each other; Concentration of NH3-N ranged between 6.77 to 21.67 mg/100ml, with the group of A interpreting the lowest average NH3-N concentration (11.08 mg/100ml), while highest peak droping in the group of C (15.04 mg/100ml). Yields of microbial protein were also varied with diets; microbial protein of the group C and D were much higher than that of the group A and B comparatively (P<0.05). And surprisingly, the microbial protein yield of group C and D were similar, but the bacterial protein yield of group C was significantly higher than that of group D. As for microbial community, protozoa to bacteria ratio had notable differences across groups, with group C recording the lowest value of 81.27 %, through a comparison with 3 other groups (P<0.05). Further cloning and sequence analysis revealed that, significant differences were found in 8 kinds of bacteria, such as: R.flavefaciens, R.bromii, and Roseburia faecalis etc., and also, there were marked differences in 4 protozoal genuses except Diplodinium. The microbial AA-N ratio was also alerted by dietary protein. Group C recorded the lowest valve, and the opposite was found for group D, recorded the highest peak. Furthermore, multiple regression analysis showed that AA-N ratio had a negative (positive) correlation with bacteria (protozoa) protein. It was also observed that, significant differences were found between protozoa and bacteria in AA content for some, but not all, amino acids. Within each microbial fraction, additionally, significant differences in the content of AA were found between rations. Protozoal Valine content was higher than bacterial, while Lysine content was high in bacteria. For bacteria, significant difference was detected in Arginine only; for protozoa, Methionine, Leusine, and Histidine showed significant differences. And the variations of AA were related to the changes of microbial fractions in some degree. All in all, rumen fermentation, microbial profile, AA-N content, and AA composition of ruminal MCP were all modified by dietary protein.
试验1荧光标记瘤胃细菌方法的建立和原虫吞噬细菌速率的研究
以4只装有永久性瘤胃瘘管的徐淮山羊提供的瘤胃液制备荧光标记瘤胃细菌,用于瘤胃原虫的摄食实验,研究山羊瘤胃原虫吞噬细菌的速率。试验设置清洗原虫祛除瘤胃自由细菌的荧光全标组(WFLB)及未清洗原虫保留瘤胃自由细菌的荧光标记组(FLB)。结果表明: WFLB组、FLB组吞噬速率分别为:398.40 cells/(cell h)、230.40 cells/(cell h),换算为细菌N为:2.15 pg N/(cell h)、1.24 pg N/(cell h);每天每头山羊瘤胃内蛋白微循环中细菌N循环量WFLB组、FLB组分别估计为:103.20 mg N/(d头)、59.50 mg N/(d头),或者细菌蛋白循环量分别为0.645 g/(d头)和0.372 g/(d头);结果同时表明,荧光标记技术可以应用于瘤胃原虫对细菌吞噬速率的研究。
试验2日粮精粗比对山羊瘤胃内微生物蛋白质微循环影响的研究
本试验以4只装有瘤胃瘘管的山羊,研究不同精粗比日粮对山羊瘤胃微生态中原虫和细菌区系以及微生物蛋白微循环的影响规律。试验设置精(玉米-豆粕):粗(稻草)分别为10:90、30:70、50:50、70:30的四种比例日粮(A、B、C、D),采用4×4拉丁方设计进行动物试验,并结合荧光标记瘤胃细菌技术测定瘤胃原虫对细菌的吞噬速率。结果表明:日粮精粗比显著影响微生物细胞的密度。原虫的密度以C组最高;细菌和原虫的密度以都A组最低。日粮精粗比也显著影响原虫吞噬的速率,A、B、C、D四组吞噬速率分别为:429.50 cells/(cell h)、366.74 cells/(cell h)、389.48 cells/(cell h)、402.20 cells/(cell h),换算为对细菌N的吞噬速率分别为:2.319 pg N/(cell h)、1.98 pg N/(cell h)、2.103 pg N/(cell h)、2.172 pg N/(cell h)。每天每头山羊由于原虫的吞噬造成的细菌N的循环量分别估算为:136.49 mg N/(d头);369.02 mg N/(d头);485.99 mg N/(d头);440.56 mg N/(d头),或者菌体蛋白循环量为0.853 g Pr/(d头)、2.306 g Pr/(d头)、3.370 g Pr/(d头)和2.754 g Pr/(d头),以C组菌体蛋白循环量最大,细菌周转率最高(3.07 %)。
试验3精粗比对瘤胃发酵、原虫种群结构及吞噬速率的影响
在试验2的基础上,进一步研究在不同精粗比例配合日粮条件下山羊瘤胃发酵、原虫种群结构以及吞噬速率的变化规律。结果表明:日粮精粗比对瘤胃发酵有影响。以30:70组微生物活力较强、NDF降解率较高、瘤胃pH值也相对波动较小;精粗比对瘤胃原虫结构也有影响。50:50、70:30日粮组内毛虫和等毛虫的比例显著高于10:90、30:70组;而双毛虫和头毛虫则相反。内毛虫与双毛虫和头毛虫的吞噬速率分别为361.90、606.30和607.50 cells/(cell h),内毛虫的吞噬速率显著低于双毛虫和头毛虫(P =0.000),表明不同种属原虫吞噬细菌速率间有显著差异,但随日粮精粗比变化的规律基本一致。回归分析表明吞噬速率与日粮结构间呈三次方的曲线关系(Y=-724.53X+563.15X2-124.666X3+646.833,R2=0.97864)。另外,多元回归分析表明吞噬速率与微生物细胞密度有线性关系(R2=0.839)。方程为:Y=445.514-3.078X1+1.864X2(Y为吞噬速率;X1为细菌密度;X2为原虫密度)。
试验4碳水化合物结构对瘤胃发酵和微生物群体结构的影响
以3只瘘管山羊作为瘤胃液供体,用体外法研究底物碳水化合物结构对瘤胃发酵及微生物群体特征的影响。底物可溶性淀粉/纤维素比例为:100:0、70:30、50:50、30:70、0:100。结果表明:30:70组微生物产量和纤维降解率最高,发酵状态最佳;微生物蛋白产量与淀粉/纤维有三次方的曲线关系,细菌蛋白:Y=0.2410+0.0855X-0.0371X2+0.0029X(3R= 0.7397);原虫蛋白:Y=0.2276+0.0853X-0.0380X2+0.0030X3(R=0.7370)。各组分别在8、16、24、24和24 h出现最大微生物产量(P<0.01)。另外,微生物AA-N比例在各组间也有显著差异(P<0.05)。微生物区系原虫与细菌的比例总体上随淀粉水平下降呈先上升后降低,以50:50组最高;而且PCR-SSCP图谱反映了区系内部类群因底物的改变而改变。原虫分类计数结果表明随淀粉水平的下降,内毛虫与等毛虫的比例下降,而双毛属与头毛亚科原虫的比例增加,与SSCP的结果一致地表明其内部类群的改变。对提取DNA的质、量的检测表明:微生物分离平均获得率为53.29 %;细菌DNA的提取率(45.40 %)显著低于原虫(56.10 %)(P<0.05);微生物分离后DNA提取率为26.13 %;所提取的基因组DNA大小在20 kb以上,适合于后续研究的分子操作。综合以上试验结果可认为底物的变化引起了微生物发酵和其群系特征的变化,结果还证实PCR-SSCP适合于瘤胃微生物混合群体多样性研究,同时也验证了ITS1区段是研究瘤胃原虫的良好素材,从而基本确立了本课题瘤胃微生物群体研究的方法体系。
试验5不同分子形式氮源对瘤胃微生物蛋白产量和类群多样性的影响
本试验的主要目的是研究不同分子形式氮源在人工瘤胃体外培养条件下对瘤胃发酵、微生物蛋白合成和类群多样性的影响。以3只瘘管山羊为瘤胃液供体,底物设计为:氯化铵、寡肽混合物(<3, 000 Da)、小肽混合物(<500 Da)、游离氨基酸混合物。结果表明:pH在6.40-6.90之间变化,游离氨基酸组pH均值最低为6.61,氯化铵组的最高为6.79;氨氮浓度变化范围为11.60-29.45 mg/100 ml,均值以寡肽组最低为15.70 mg/100ml,游离氨基酸组最高为19.15 mg/100 ml;氯化铵组的细菌与原虫蛋白产量皆最低(0.1522、0.1179 mg/ml)(P<0.01),而两肽组的微生物蛋白产量则相对较高;另外原虫与细菌比值以氯化铵组最低为77.49 %,小肽组最高为104.50 %(P<0.01)。同时AA-N比例在各组间也有显著差异,以游离氨基酸组最低(P<0.05)。SSCP指纹图谱表明微生物类群内部种属也发生了变化,并以氯化铵组微生物的多样度最低。以上研究结果揭示了氮源的分子形式显著影响瘤胃发酵、微生物蛋白合成和微生物的类群结构。
试验6特定AA缺省对体外培养瘤胃微生物生长限制性的研究
本试验采用底物移除法研究特定氨基酸在人工瘤胃体外培养条件下对瘤胃微生物生长及其发酵特征的影响。以3只瘘管山羊作为瘤胃液供体,底物为:全量必需氨基酸组(A),组氨酸(B)、赖氨酸(C)、蛋氨酸(D)和支链氨基酸(E)的缺省组。结果表明:培养液pH值在5.90-6.80之间变化,均值以E组最高为6.54;培养液氨氮浓度变动范围为10.99-30.51 mg/100ml,均值以A组最高为17.85 mg/100 ml;底物对微生物生长的限制程度不同。以支链氨基酸缺省对微生物蛋白合成的限制最大,其细菌与原虫及其微生物蛋白的产量皆最低(0.1389、0.1772和0.3161 mg/ml)(P<0.01),相对于A组微生物蛋白下降了44.52 %。底物对细菌和原虫影响不同。原虫与细菌比值以C组最低(89.12 %),E组最高(127.60 %)(P<0.01)。同时底物还引起了微生物AA-N比例的变化(P<0.01),以A组最低(16.89),E组最高(18.76)。另外遗传指纹分析提示微生物区系内部类群也因底物而发生了变化。综合以上试验结果可认为特定氨基酸对微生物生长及微生物发酵都有一定的影响。
试验7蛋白质补充料对体外培养瘤胃微生物区系和发酵的影响
本试验以不同蛋白质补充料为底物进行体外培养,主要探讨蛋白质补充料对瘤胃微生物发酵和区系特征的影响。试验采用3只瘘管山羊作为瘤胃液供体,底物为:A(羽毛粉)、B(玉米蛋白粉)、C(豆粕)和D(鱼粉)。结果表明:pH值在5.80-6.80之间变化,均值以C组最低为6.19,A组最高为6.61;氨氮浓度变动范围为3.68-12.01 mg/100ml,均值以A组最低为5.49 mg/100ml,C组最高为9.95 mg/100ml;微生物蛋白产量在各组间差异显著或极显著(P<0.05、P<0.01),以D组最高(0.6513 mg/ml),A组最低(0.5289 mg/ml),C组细菌蛋白量(0.3309 mg/ml)为四组之最高。底物对微生物区系有选择作用。原虫与细菌比值以A组最高(107.00 %),C组最低(84.30 %)。SSCP图谱分析表明微生物区系内的类群结构也因底物发生了改变。另外底物还引起了微生物AA-N比例的变化,以D组最高(17.75),C组最低(16.48)(P<0.01)。综合以上试验结果认为不同蛋白质补充料体外培养条件下其瘤胃微生物发酵和微生物区系特征有明显不同。
试验8、9混合日粮蛋白质对徐淮山羊瘤胃微生物结构及AA组成的影响
试验8以4只瘘管山羊作为试验动物,采用4×4拉丁方设计进行试验。研究由羽毛粉(A)、玉米蛋白粉(B)、豆粕(C)和鱼粉(D)等不同蛋白质饲料配合的混合日粮对瘤胃发酵、微生物群体结构、微生物蛋白(MCP)产量及其AA组成模式的影响规律。在第一期正试期开始时分别从四只羊瘤胃中采集瘤胃液,用于相应日粮作为培养底物的预先体外培养试验,以确保复杂与昂贵的体内试验的有效性和必要性。结果表明,由不同蛋白补充料所配制混合饲料底物对瘤胃发酵、微生物类群结构及AA-N影响与单纯的蛋白补充料相比,在底物样本间差异程度上虽然有所降低,但影响规律影响基本一致,并且微生物类群结构及AA-N仍有明显或显著变化,表明进一步开展体内试验是必要和有意义的。
试验9继而开展的104天的4×4拉丁方体内试验结果表明:瘤胃液pH值在5.60-6.80之间变化,均值以A和C组较高,B和D组较低(P<0.05),A和C、B和D的pH均值虽相近,但随时间的动态变化模式相差较大;氨氮浓度变动范围为6.77-21.67 mg/100ml,均值以A组最低为11.08 mg/100ml,C组最高为15.04 mg/100ml,各组随时间动态变化模式也有所不同;日粮蛋白显著影响MCP产量,以C、D组微生物蛋白较高,A、B组较低(P<0.05);虽然C、D组微生物蛋白相当,但C组细菌蛋白产量却显著高于D组(P<0.05)。原虫与细菌区系比例在组间差异显著,豆粕组(81.27 %)显著低于其他三组(P<0.05)。进一步克隆测序分析表明细菌中的类R.flavefaciens、R.bromii、Roseburia faecalis等8群系在组间差异显著;原虫中除Diplodinium外其他4类群都有显著差异。微生物的AA-N比例在各组间也有显著差异,并以C组最低,D组最高,并且与细菌(原虫)蛋白质呈负(正)相关关系。研究同时发现部分种类AA含量在微生物区系间和同一区系内不同组间差异显著。原虫蛋白的Val比例高于细菌,而细菌蛋白的Lys则高于原虫。细菌蛋白的Arg;原虫蛋白的Met、Leu和His在各组间差异显著。差异性AA的变化与微生物类群的变化有一定的关联。综上可见混合日粮蛋白质对瘤胃发酵、微生物类群结构和微生物AA组成都有一定的影响。
The most important sources of AA at the duodenum of ruminants are the microbial crude protein (MCP) synthesized in the rumen, any change in microbes or MCP, such as, microbial flora, microbial AA-N ratio, and AA profile of microbes etc., is bound to affect AA supply to small intestine, and consequently influences animal performance. For a long time, it is well accepted that ruminal microbial AA is constant; moreover, it is common to use certain AA to direct ruminant practice. However, later studies reveal that, microbial AA is not constant. A review by Clark et al. (1992) indicated great differences in the AA composition of rumen bacteria, and followed by a continuous debate about this subject. Unfortunately, whether the microbial AA change or not, are still controversial issues till now. Therefore, in vitro and in vivo experiments were conducted using varied sources in this study, and aimed to illustrate whether microbial AA change, and how microbial AA change with diets. The Fluorescence-Labeled Rumen Bacteria Techniqe (FLRB) was developed to determine grazing rate of protozoa on bacteria in rumen, and the molecular technique including SSCP, cloning and sequencing etc., were also introduced into this paper. This dissertation was described in the following nine sections.
Trail 1 Establishment of Fluorescence-labeled Rumen Bacteria Technique and Study on the Grazing Rate of Protozoa
Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled rumen bacteria. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows: one group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid (WFLB); the other group was bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid (FLB). The result showed that, the bacterial predation rates of rumen protozoa were 398.40 cells/(cell h) for the group WFLB, 230.40 cells/(cell h) for the group FLB; When the corresponding values expressed as bacteria-N were: 2.15 pg N/(cell h) for the group WFLB and 1.24 pg N/(cell h) for the group FLB respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.20 mg N/(d head) for the group WFLB, 59.50 mg N/(d head) for the group FLB, respectively. It was estimated that protein recycling were 0.645 g Pr/(d head) for the group WFLB and 0.372 g Pr/(d head) for the group FLB, respectively. And finally, the fluorescence-labeled rumen bacteria technique (FLRB) would be a potential assay for determination of bacterial predation rate by rumen protozoa.
Trail 2 Effects of Dietary Concentrate to Forage Ratio on Microbial Protein Recycling in the Rumen of Goats
In this study, the main aims were to investigate effects of dietary concentrate to forage ratio on microbial protein recycling in the rumen of goats using a technique of FLRB, developed in trail 1. 4×4 Latin squares were conducted by using 4 Xuhuai goats with permanent cannulas, and diets were divided into A (10:90), B (30:70), C (50:50), and D (70:30) by varying concentrate to forage ratios, which concentrated food were corn-soybean meal, while forage were straw in this experiment. The result showed that, rumen micro-ecosystem was shifted heavily by concentrate to forage ratio. The highest protozoal density were recorded by group C which dietary concentrate to forage ratio was set as 50:50, whereas densities of both protozoa and bacteria were lowest for the group A; grazing rates of rumen protozoa on bacteria were, respectively: 429.50 cells/(cell h), 366.74 cells/(cell h), 389.48 cells/(cell h), and 402.20 cells/(cell h) for group A, B, C, and D. When the corresponding values expressed as bacteria-N were: 2.319 pg N/(cell h), 1.98 pg N/(cell h), 2.103 pg N/(cell h), and 2.172 pg N/(cell h) respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat with different diets, there were 136.49 mg N/(d head), 369.02 mg N/(d head), 485.99 mg N/(d head), and 440.56 mg N/(d head) for group A, B, C, and D respectively. It was estimated that, protein recycling of rumen bacteria were 0.853 g Pr/(d head) , 2.306 g Pr/(d head), 3.370 g Pr/(d head), and 2.754 g Pr/(d head) respectively, with group C recording the highest protein recycling of rumen bacteria and turnover rates (3.07 %).
Trail 3 Effects of Dietary Concentrate Levels on Rumen Fermentation, Protozoal Profiles and Grazing Rates
The objectives of present studies were to demonstrate the effects of dietary concentrate to forage rate on rumen fermentation, protozoal structure and grazing rates in goats’rumen. The experimental animal and desigh were the same as trial 2. The result showed that, rumen fermentation was shifted heavily by concentrate to forage ratio. High microbial activity and high NDF degradability were observed in group B, and pH was stable in this group, comparatively. It was also observed that, protozoal dynamics was modulated by dietary structure. Percentages of Entodiniinae and Isotrichidae were higher for diets which concentrate level were high, whereas percentages of Diplodiniinae and Ophryoscolecinae were higher for diets which forage level were high. The predation rates were 361.90, 606.30, and 607.50 cells/(cell h) for Entodiniinae, Diplodiniinae, and Ophryoscolecinae respectively, and marked difference was found among genus (P=0.000), however, the change rule of grazing rate with dietary structure seemed to be similar across genus. Regression analysis revealed that, there were strong cubic relationship between predation rates and dietary structure (Y=-724.53X+563.15X2-124.666X3+646.833, R2=0.97864). Additionally, multivariate regression analysis revealed the existence of linear correlations between predation rates and cell densities (protozoa and bacteria) (R2=0.839). The equation was presented bellow: Y=445.514-3.078X1+1.864X2 (Y- predation rate; X1 - bacteria density; X2 - protozoa density).
Trail 4 Effects of Different Structure of Carbhyborate on Rumen Fermentation and Microbes characteristics in vitro
Three goats fisted with cannulas were used to investigate the effects of rations in different starch to cellulose ratio on rumen fermentation and microbial characteristics in vitro. Substrates were designed by varying the level of starch/cellulose ratio as follows: 100:0, 70:30, 50:50, 30:70, 0:100. The results showed that: Cellulose degradability and microbial biomass were highest when starch/cellulose ratio in the culture was set to 30:70; The regression analysis between microbial protein and substrate were: Bacteria: Y=0.2410+0.0855X-0.0371X2 +0.0029X3 (R=0.7397); Protozoa: Y=0.2276+0.0853X-0.0380X2+0.0030X3 (R=0.7370) (Y: microbial protein, mg/ml; X: Starch/Cellulose), respectively. Significant differences were found in microbial AA-N ratio between groups (P<0.05); Furthermore, protozoa to bacteria ratio had a tendency to increase firstly, followed by decline when starch/cellulose ratio decreased, and the highest record falling in the group provided ration containing starch to cellulose ratio of 50:50; SSCP analysis further revealed that microbial structure was shifted by substrates; and also, cell-counting of protozoa showed that, Entodinium and Isotricha decreased, whereas Diplodinium and Ophryoscolecinae increased according to the decrease of starch/cellulose ratio, and revealed that the profile of protozoa was subjected to substrates, which agreed with SSCP. Additionally, the average recovery rate of microbes after detaching was 53.29 %; the DNA extraction ratio from bacteria (45.40 %) were significantly less than protozoa (56.10 %); the size of DNA fragment extracted from all the samples were larger than 20 kb; and fit for advanced research. It was therefore concluded that carbohydrate structure influenced both rumen fermentation and microbial characteristics. And finally SSCP has a potential for research on characterizing rumen mixed microbes, ITS1 is also good at research on protozoal diversity. So far, a measurement system was developed for the research on microbial characteristics in this trail.
Trail 5 Effects of Different Nitrogen Compounds MCP Yields and Microbial Diversity of Rumen
In this study, the main aims were to investigate effects of different N compounds on rumen fermentation, MCP yields and microbial diversity in vitro, using rumen liquor provided by 3 goats. Four treatments were NH4Cl, mixed oligo-peptide (<3, 000 Da), mixed oligo-peptide (<500 Da), and free amino acid respectively. Results showed that, the recorded pH-value ranged between 6.40 and 6.90, the average pH-value was lowest for the group with free amino acid, and highest for the group with NH4Cl in the culture. The varied range of NH3-N concentration was from 11.60 to 29.45 mg/100ml. The mean concentration of NH3-N was lowest in mixed oligo-peptide, and the highest was found in free amino acid. It was also observed that, yields of microbial protein varied with substrates, with lowest record dropping in NH4Cl, and microbial yields of both two peptides groups were comparatively higher. The protozoa to bacteria ratio was also shifted by substrates, and oligo-peptide recorded the highest peak, while the opposite was found for NH4Cl (P<0.01). Additionally, significant differences were found in microbial AA-N ratio between groups, with the lowest record falling in the group whose substrate was free amino acid (P<0.05). Furthermore, microbial diversity was demonstrated in SSCP fingerprint clearly, revealed that the structure of bacteria or protozoa community was altered by substrates, and the diversity in NH4Cl was much low. It could be concluded that, both rumen MCP yields and microbial structure were modified by different N compounds.
Trail 6 Study on Effects of Certain Amino Acids-Removal on the Growth of Rumen Microbe in vitro
The objectives of this study were to determine the effects of certain amino acids on rumen fermentation and microbial growth in vitro. Three goats fitted with cannula were used to provide rumen liquor, and the removal method was introduced into current experiment. Treatments were, respectively, total essential amino acid (A), His-removal (B), Lys-removal (C), Met-removal (D), branch chain amino acid (BCAA)-removal (E). Results showed that, the pH-value ranged between 5.90 and 6.80, and the highest mean value was observed in the group E (6.54). Concentration of NH3-N ranged between 10.99 to 30.51 mg/100ml, with the highest mean value dropping in the group A (17.85 mg/100ml). It was also observed that, yields of microbial protein and limiting degree on microbial growth were varied with treatments, with the group E demonstrating the lowest record (0.1389, 0.1772 and 0.3161 mg/ml for bacteria, protozoa, and microbes respectively) (P<0.01). The microbial yield of this group, comparing with the group A, decreased by 44.52 %. And also, the response to the substrate of protozoa differed from bacteria, and significant differences were found in the protozoa to bacteria ratio between groups. The group C interpreted the lowest data (89.12 %), and the reverse was true for the group E, displayed the highest value (127.60 %) (P<0.01). Additionally, microbial AA-N ratio was shaped by substrates, and lowest in the group A (16.89), while highest in the group E (18.76) (P<0.01). Further genetic fingerprint analysis revealed that, microbial profile was modified by substrates within bacteria or protozoa community. In conclusion, the rumen fermentation and microbial growth responded differently to certain amino acid, based on current in vitro trail.
Trail 7 Effects of Different Protein Supplement on Rumen Fermentation and Microbial Community in vitro
Certain protein supplement were used as substrates in this trail, and aimed to detect the characteristics of rumen fermentation and microbial community in vitro, by three goats fitted with cannula. Treatments: A (Plume meal), B (Corn gluten meal), C (Soybean meal), D (Fish meal). Results showed that, the pH-value ranged between 5.80 and 6.80, and the lowest mean value was found in the group C (6.19), while the highest occurred in the group A (6.60). NH3-N concentration ranged between 3.68 to 12.01 mg/100ml, with the group A recording the lowest average NH3-N concentration (5.49 mg/100ml), while the group C (9.95 mg/100ml) writing the highest value. Also, yields of microbial protein were varied with treatments (P<0.05, P<0.01), and the lowest record was observed in the group of A (0.5289 mg/ml), the other way round, the highest was found in the group of D (0.6513 mg/ml), it should be noted that the group C showed the highest bacteria yield (0.3309 mg/ml). It was further observed that, the protozoa to bacteria ratio was lowest in group C (84.30 %), the opposite was found for group A (107.00 %), interpreting the highest peak. Further SSCP analysis revealed that, profiles of both bacteria and protozoa subjected to substrates. Additionally, significant differences were found in microbial AA-N ratio between groups, and AA-N ratio was lowest (or highest) in the group of C (or D) (P<0.01). In a word, protein stuff brought out the changes, which were not just in microbial fermentation and flora, but in microbial AA-N ratio.
Trail 8, 9 Effects of Dietary Protein on Microbial Community and AA Composition of Rumen
Trail 8 The objectives of these parts were to investigate how rumen fermentation, microbial community, microbial protein (MCP) yields, and AA composition of MCP changed with dietary protein. Four goats fitted with rumen cannula, were used in a 4×4 Latin square design. And formula diets were divided into 4 groups according to their nitrogen source, which was, respectively, plume meal (A), corn gluten meal (B), soybean meal (C), and fish meal (D). At the beginning of first experimental period, rumen liquor was collected from each goat, and used for in vitro culture with the corresponding mixed diet, before the animal experiment. This pre-experiment aimed to ensure the necessity and validity of the complex and expensive animal experiment. The results showed that, marked differences were found in fermentation parameters, microbial diversity, and AA-N ratio etc. across groups, although the degree of variation had a somewhat decline, compared with the results of sole culture of protein supplement (trail 7). And these results indicated that, it was necessary and significant to carry out animal experiment using these mixed diet.
Trail 9 The results of the following 104-day in vivo experiment were described here. The recorded pH-value ranged between 5.60 and 6.80, and mean pH value of group A and C were high, the reverse was true for group B and D (P<0.05), the most interesting thing was that, although the mean pH value of group A and C (B and D) seemed to be similar, the change patterns of pH with time differed from each other; Concentration of NH3-N ranged between 6.77 to 21.67 mg/100ml, with the group of A interpreting the lowest average NH3-N concentration (11.08 mg/100ml), while highest peak droping in the group of C (15.04 mg/100ml). Yields of microbial protein were also varied with diets; microbial protein of the group C and D were much higher than that of the group A and B comparatively (P<0.05). And surprisingly, the microbial protein yield of group C and D were similar, but the bacterial protein yield of group C was significantly higher than that of group D. As for microbial community, protozoa to bacteria ratio had notable differences across groups, with group C recording the lowest value of 81.27 %, through a comparison with 3 other groups (P<0.05). Further cloning and sequence analysis revealed that, significant differences were found in 8 kinds of bacteria, such as: R.flavefaciens, R.bromii, and Roseburia faecalis etc., and also, there were marked differences in 4 protozoal genuses except Diplodinium. The microbial AA-N ratio was also alerted by dietary protein. Group C recorded the lowest valve, and the opposite was found for group D, recorded the highest peak. Furthermore, multiple regression analysis showed that AA-N ratio had a negative (positive) correlation with bacteria (protozoa) protein. It was also observed that, significant differences were found between protozoa and bacteria in AA content for some, but not all, amino acids. Within each microbial fraction, additionally, significant differences in the content of AA were found between rations. Protozoal Valine content was higher than bacterial, while Lysine content was high in bacteria. For bacteria, significant difference was detected in Arginine only; for protozoa, Methionine, Leusine, and Histidine showed significant differences. And the variations of AA were related to the changes of microbial fractions in some degree. All in all, rumen fermentation, microbial profile, AA-N content, and AA composition of ruminal MCP were all modified by dietary protein.
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