添加青绿饲料对塔里木马鹿瘤胃原虫数量的影响

为了解塔里木马鹿瘤胃生理特点,研究其瘤胃内原虫的数量、频数等的变化规律,试验采用显微镜镜检的方法对塔里木马鹿瘤胃内原虫进行鉴定,并通过添加一定量的青绿饲料来替代粗饲料,采用血细胞计数板的方法对原虫数量进行统计。结果表明:塔里木马鹿瘤胃内主要的原虫为内毛属、双毛属、头毛属、等毛属、前毛属原虫;添加青绿饲料前后,瘤胃内原虫总数、原虫活力及原虫频数均未出现显著变化(P0.05)。     

不同添加剂和日粮类型对黄牛瘤胃原虫计数影响的研究

以莫能霉素、盐霉素添加剂和自制的复合营养舔(饲)块饲喂本地黄牛,研究其对瘤胃原虫(Rumen protozoa)计数厦瘤胃pH值的影响。试验结果表明,黄牛日粮添加莫能霉素、盐霉素使瘤胃原虫数减少,而补喂舔(饲)块使原虫数增加。同时发现,喂稻草型日粮时,黄牛瘤胃原虫计数较放牧时少。研究我国养牛条件下原虫的作用对牛饲料添加剂、瘤胃调控制的利用具有重要意义。     

反刍动物瘤胃原虫的化学感知及信号通路研究进展

瘤胃内原虫数量较少,但体积相对较大,在瘤胃发酵和营养利用中的作用也不容忽视。与传统的低质粗饲料饲养模式相比,瘤胃原虫在现有的高精料条件下对小肠蛋白质的贡献应当被提到相当重要的位置。原虫对不同营养底物有化学感知作用,其中PI3K/AKT/mTORC1通路在瘤胃原虫对营养的化学感知上发挥重要作用。了解瘤胃原虫化学感知作用信号传导规律,对于保障反刍动物瘤胃健康和提高日粮氮利用率具有重要意义。     

瘤胃原虫对瘤胃营养物质代谢的影响研究进展

本文在广泛查阅有关瘤胃原虫对糖代谢包括淀粉和可溶性糖代谢及纤维素代谢、对蛋白质代谢和对瘤胃内甲烷代谢等的影响研究资料的基础上，分析、总结了瘤胃原虫对反刍动物营养物质代谢的影响机理，指出瘤胃原虫对反刍动物的营养代谢既有有利的方面，也有不利的方面。实际应用时可根据不同的日粮类型、家畜的生理状况、当地的饲养管理条件等实际情况因时控制瘤胃纤毛虫区系及种群密度，创造适宜的瘤胃内环境，充分发挥原虫在特定条件下的有利生理功能，从而提高反刍动物饲料利用率和生产性能。     

去除瘤胃原虫对反刍动物的影响

对去除瘤胃原虫的几种方法做了述评,并讨论了去除原虫后瘤胃微生物区系的变化情况,以及对反刍动物N代谢、瘤胃发酵和一些养分利用的影响。     

荧光光度法在瘤胃原虫对细菌吞噬作用研究中的应用

本试验旨在探讨荧光光度法研究瘤胃原虫对细菌吞噬作用的可行性.试验选用4只装有永久性瘤胃瘘管的徐淮山羊采集瘤胃液以获取瘤胃原虫和细菌,以4,6-二脒基-2-苯基吲哚(DAPI)标记细菌的荧光强度为主要测定指标,研究山羊瘤胃中原虫对细菌的吞噬作用.结果表明,DAPI标记的瘤胃细菌浓度与其荧光强度之间存在线性相关(R2=0.995 2).在日粮粗精比为7:3的条件下,徐淮山羊瘤胃原虫对细菌的吞噬速率为370.89 cells/(cell·h),瘤胃内原虫对细菌的吞噬速率存在一定的波动性,吞噬量在60 min达到饱和,其最大吞噬量为370,89 ceIls/cell.结果提示,荧光光度法可应用于瘤胃原虫对细菌吞噬作用的研究.     

奶牛瘤胃pH、消化酶活性及原虫数量的日动态变化研究

本试验旨在研究奶牛瘤胃pH、消化酶活性、原虫数量的日动态变化规律及原虫种类。试验选择3头体重为(500±30) kg、泌乳胎次为2～4、健康状况良好、装永久性瘤胃瘘管的干乳期荷斯坦奶牛作为试验瘤胃液供体,分别采集采食前后各时间点瘤胃液。试验预试期14 d,正试期7 d。结果表明:1)随时间变化,瘤胃pH呈一定规律性变化,即在奶牛采食前瘤胃pH较高,采食后随饲粮进入瘤胃,瘤胃pH迅速降低,然后随瘤胃中饲粮的减少,瘤胃pH逐渐恢复至采食前水平,且采食前后瘤胃pH差异显著(P0.05)。2)5种瘤胃消化酶在采食前活性维持相对较低水平,采食后酶活性升高,其中蛋白酶、淀粉酶、木聚糖酶活性上升的速度快,果胶酶、纤维酶活性上升速度慢,最终这5种酶活性逐渐恢复至原来水平,后3种酶活性很长时间维持在较高水平;除淀粉酶、木聚糖酶活性在第2次采食前后差异不显著(P0.05)外,采食前后5种酶活性均差异显著(P0.05)。3)随时间变化,原虫数量整体呈凹凸形式变化,即原虫数量在采食前较高,采食后随着饲粮进入瘤胃,瘤胃内容物被稀释,导致原虫数量降低,然后随食糜的排空,逐渐恢复甚至高于原来水平,但采食前后原虫数量均差异不显著(P0.05)。4)对原虫进行形态学鉴别,观察到了厚毛属(Dasytricha)、内毛属(Entodinium)、鞘甲属(Elytroplastron)、单甲属(Eremplastron)、前毛属(Epidinium)、后毛属(Metadinium) 6个属12种原虫,其中内毛属的种类最多。本研究结果为进一步了解反刍动物在采食饲粮前后瘤胃消化酶活性、微生物菌群日变化规律及原虫种类提供调控瘤胃功能和原虫形态学鉴定的基础数据。     

18S rRNA／rDNA序列分析技术在瘤胃原虫分类学研究中的应用

作者指出了反刍动物瘤胃原虫分类学研究中的难点，介绍了原虫分类学中新兴的分子生物学指标(18S rRNA／rDNA序列同源性)，综述了18S rRNA／rDNA序列分析技术在瘤胃原虫分类研究中的应用。     

反刍动物瘤胃原虫自溶现象的初步研究

瘤胃原虫自溶的初步研究,可为反刍动物瘤胃内微生物蛋白循环的研究及相关调控技术的开展提供基础资料。本试验采用氧诱导自溶的方法,在体外培养条件下研究原虫形态、自溶度、蛋白酶活性及脂肪酶活性的变化。结果表明,氧诱导处理原虫形态变化明显,表现出自溶特征。前期(0~4 h)自溶速度较快,4 h胞内蛋白释放达50%。自溶组蛋白酶活性较对照组降低9.92%(P0.05);而脂肪酶活性较对照组提高41.01%(P0.01)。胞外蛋白酶活性与原虫自溶度相关性最高(r=0.703)。脂肪酶活性对于原虫自溶度的直接效应最强(P=0.586)。综上所述,氧诱导处理使瘤胃原虫发生自溶,据此建立自溶模型。脂肪酶的作用是瘤胃原虫自溶的关键因素。     

常用牧草对山羊瘤胃内环境参数的影响

为从微观角度评定山羊饲料营养价值,探讨6种山羊常用牧草对瘤胃内环境参数的影响。试验选用2只装有瘤胃瘘管山羊,采用瘤胃尼龙袋法,分别测定瘤胃液pH、氨态氮质量浓度及细菌和原虫数量。结果表明:加入装有牧草的尼龙袋后,瘤胃pH先降低后回升,变化趋势基本一致;瘤胃氨态氮质量浓度在4 h时达到峰值,随后降低至初始水平;牧草对瘤胃原虫数量的影响不一致,但均对瘤胃细菌数量有促进作用。由此可见,添加装有牧草的尼龙袋,可降低山羊瘤胃pH,对瘤胃氨态氮质量浓度及原虫和细菌数量有一定的影响,可能与饲料的品质和营养含量相关。     

Effects of protozoa on methane production in rumen and hindgut of calves around time of weaning.

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight (-23%, P < 0.01), and rumen pool size of N (-36%, P < 0.01) and crude fat (-37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N (+3%, P < 0.01) and D-alanine-N per 16 g N (+13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH4 production in the rumen increased exponentially with the increase in protozoa population size (R2 = 0.68). In presence of 46 x 10(4) protozoa per ml rumen fluid, the in vitro CH4 production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R2 = 0.55) to the CH4 production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate-propionate ratio and butyrate proportion of VFA were reduced. In vivo, in the absence of protozoa not only the whole animal CH4 production (-30%, P < 0.05) but also the digestibility of carbohydrates (-4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Comparisons of Salmonella conjugation and virulence gene hyperexpression mediated by rumen protozoa from domestic and exotic ruminants

Recent studies have identified a phenomenon in which ciliated protozoa engulf Salmonella and the intra-protozoal environment hyperactivates virulence gene expression and provides a venue for conjugal transfer of antibiotic resistance plasmids. The former observation is relegated to Salmonella bearing the SGI1 multiresistance integron while the latter phenomenon appears to be a more generalized event for recipient Salmonella. Our previous studies have assessed virulence gene hyperexpression only with protozoa from the bovine rumen while conjugal transfer has been demonstrated in rumen protozoa from cattle and goats. The present study examined virulence gene hyperexpression for Salmonella exposed to rumen protozoa obtained from cattle, sheep, goats, or two African ruminants (giraffe and bongo). Conjugal transfer was also assessed in these protozoa using Salmonella as the recipient. Virulence gene hyperexpression was only observed following exposure to the rumen protozoa from cattle and sheep while elevated virulence was also observed in these animals. Conjugal transfer events were, however, observed in all protozoa evaluated. It therefore appears that the protozoa-based hypervirulence is not universal to all ruminants while conjugal transfer is more ubiquitous.     

绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数值

本试验旨在研究绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数(Km)值,为解释绵羊瘤胃细菌、原虫蛋白质分解代谢特征提供酶学依据。选用6只1岁左右安装永久性瘤胃瘘管的中国美利奴(新疆型)绵羊[平均体重为(32.00±1.36)kg],饲喂精粗比为30∶70的饲粮,依次采集饲喂前(0 h)和饲喂后1.5、3.0、6.0、9.0、12.0 h 6个时间点的瘤胃液,重复采集3次。分离和制备细菌、原虫破碎液,分别测定相关酶活力及谷氨酸脱氢酶体系的Km值。结果显示:1)绵羊瘤胃细菌、原虫破碎液中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力随饲喂时间的延长均呈现先升高后降低的动态变化规律,总体在饲喂后1.5 h达到峰值;谷氨酸和氨含量也呈现相似的变化规律。原虫破碎液中参与蛋白质分解代谢的这4种酶的活力在各时间点均极显著高于细菌(P0.01)。2)原虫破碎液谷氨酸含量极显著高于细菌(P0.01);原虫破碎液氨含量在1.5、6.0、9.0和12.0 h显著或极显著高于细菌(P0.05或P0.01)。3)绵羊瘤胃细菌、原虫谷氨酸脱氢酶对烟酰胺腺嘌呤二核苷酸(NAD)的Km值分别为2.60×10~(-7)、1.48×10~(-7)mol/L;细菌、原虫谷氨酸脱氢酶对谷氨酸的Km值分别为8.41×10~(-6)、4.91×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对还原型烟酰胺腺嘌呤二核苷酸(NADH)的Km值分别为3.80×10~(-8)、2.70×10~(-8)mol/L;细菌、原虫谷氨酸脱氢酶对α-酮戊二酸的Km值分别为1.16×10~(-6)、2.07×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对氨的Km值分别为2.97×10~(-5)、1.40×10~(-5)mol/L。结果提示,总体上,绵羊瘤胃细菌、原虫中蛋白酶、谷氨酸脱氢酶、谷丙转氨酶、谷草转氨酶的活力在饲喂后1.5 h达到峰值,之后逐渐降低;绵羊瘤胃原虫中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力均极显著高于细菌,原虫中蛋白质分解代谢更旺盛;瘤胃原虫中不仅存在谷氨酸转氨机制,还可能存在利用氨重新合成氨基酸的机制。     

用荧光标记细菌法研究丝兰提取物对瘤胃原虫对细菌吞噬的影响

采用荧光染色标记细菌(fluorescence labeled bacteria,FLB)技术,研究山羊瘤胃中原虫对细菌的吞噬速率.试验选用3头装有永久瘘管的徐淮山羊作为瘤胃液供体,采用体外培养法研究在精粗比为1:9的情况下,探讨不同丝兰提取物水平(A组0 mg/L、B组35 mg/L、C组70 mg/L)对瘤胃原虫吞噬细菌速率的影响.荧光镜检与回归分析结果表明:原虫吞噬细菌的速率:A组:339.9 cells/(cell·h);B组:314.7 cells/(cell·h);C组:339.9 cells/(cell·h).结果表明荧光标记细菌技术能够应用于瘤胃原虫吞噬细菌速率的研究.[动物营养学报,2009,21(3):417-422][中文全文见<动物营养学报>网站(www.ChinaJAN.com)中文版2009年21卷3期]     

A quantitative study on arginine catabolism by mixed ruminal bacteria,protozoa and their mixture in vitro

The catabolism of arginine (Arg) by mixed rumen bacteria (B), mixed rumen protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system in order to confirm the metabolic pathway of Arg and provide basic information for enzymatic and molecular studies as well as an understanding of the quantitative distribution of metabolites. Rumen microbial suspensions (B, P, and BP) collected from fistulated goats were anaerobically incubated with or without 1 mmol/L Arg at 39°C for 12 h. Arg and other related compounds such as citrulline (Cit), ornithine (Orn), proline (Pro) and 5‐aminovaleric acid (5AV) in both supernatant and hydrolyzates of B, P, and BP suspensions were analyzed by HPLC. The metabolic pathways of Arg in mixed rumen bacteria and mixed rumen protozoa were considered to be as follows: rumen bacteria, Arg → Cit → Orn → Pro → 5AV → VFAs + NH₃; rumen protozoa, Arg → Cit → Orn → Pro → 5AV. The disappearance of Arg (1 mmol/L) was approximately 52.9 and 88.2% in B, 33.9 and 55.6% in P, and 52.8 and 85.2% in BP during 6 and 12 h incubations, respectively. When expressed in units of ‘per gram (g) of microbial nitrogen (MN)’, the net degradation rate of Arg in BP (50.3 µmol/g MN/h) was approximately 46% lower than that of B during a 12 h incubation period. The presence of protozoa tended to inhibit the production of Orn from Cit and the production of 5AV from Pro which were thought to be rate‐limiting steps of Arg metabolism in rumen microorganisms. As a result, protozoa appeared to have a saving effect on Arg metabolism, that is, protozoa protected Arg from wasteful exhaustion in the rumen.     

茶皂素对奶牛瘤胃原虫区系的影响

本研究旨在研究茶皂素对奶牛瘤胃原虫区系的影响。选取12头健康荷斯坦奶牛[体重(550±30)kg,产奶量35 kg/(头·d),胎次2~4],随机分为4组,每组3头,均饲喂基础饲粮,分别于晨饲前灌服0(对照)、20、30、40 g/(头·d)的茶皂素。试验期49 d,其中预试期14 d,正试期35 d,正试期内每7 d于晨饲前1 h用口腔采样器采集瘤胃液。采用聚丙烯酰氨凝胶电泳(DGGE)结合18S r DNA序列分析技术研究瘤胃原虫区系的变化。结果表明:1)添加茶皂素可以选择性地抑制瘤胃中原虫的生长。2)与对照组相比,30、40 g/(头·d)茶皂素组的丰富度指数和香农多样性指数都显著降低(P0.05),优势度指数显著增加(P0.05),但是均一性指数无显著变化(P0.05);茶皂素显著减少了瘤胃中前庭亚纲内毛目的原虫数量。综上所述,饲粮中添加30、40 g/(头·d)茶皂素均可抑制奶牛瘤胃原虫的生长,并且降低瘤胃原虫的多样性。     

不同粗精比日粮对绵羊小肠中瘤胃微生物蛋白质利用的影响

用4只装有瘤胃瘘管、十二肠瘘管和回肠瘘管的去势(公母各半)萨福克羊体重平均为(45.5±5.2)kg,在不同粗精比日粮条件下 ,研究瘤胃细菌、纤毛虫体蛋白质在小肠内的利用率。结果表明 :到达十二指肠的瘤胃细菌和纤毛虫的量 ,随日粮中精饲料水平的提高而显著减少(P<0.05)。与此相反 ,过瘤胃饲料蛋白质的量则显著增加(P<0.05)。小肠内的细菌利用率显著受日粮变化的影响 ,而纤毛虫利用率则不易受日粮影响且显著地高于细菌的利用率(P<0.05)。小肠内被吸收的细菌和纤毛虫对微生物氮的贡献率在全试验组中平均为50 %。     

Pipecolic acid in rumen fluid and plasma in ruminant animals

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen‐produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.