永生化绵羊瘤胃成纤维细胞系的建立

[目的]建立永生化绵羊瘤胃成纤维细胞系,为基础研究和动物病毒的研究提供稳定的体外细胞模型。[方法]采用组织块贴壁法培养绵羊瘤胃成纤维细胞（ovine ruminal fibroblasts cells,ORFCs）,利用PEI试剂将带有hTERT基因的pCI-neo-hTERT质粒转染ORFCs,经G418筛选得到阳性克隆细胞。采用RT-PCR、Western Blot检测F₁₅和F₃₅代阳性克隆细胞的hTERT基因的转录和表达情况;利用血球计数法绘制原代ORFCs和F₃₅代阳性克隆细胞的生长曲线比较其增殖能力。[结果]将hTERT基因成功转入ORFCs并稳定表达;阳性克隆细胞的增殖能力显著高于原代ORFCs。[结论]该试验成功建立了永生化绵羊瘤胃成纤维细胞系。     

亚急性瘤胃酸中毒奶山羊瘤胃上皮形态结构和通透性的变化

本试验旨在研究亚急性瘤胃酸中毒(SARA)对瘤胃上皮形态结构和通透性的影响。选用体况良好、体重相近的泌乳期萨能奶山羊9只,随机分为3组(对照组、SARA组、恢复组,n=3),对照组饲喂基础饲粮[非纤维性碳水化合物与中性洗涤纤维比(NFC/NDF)=1.40],SARA组和恢复组先后饲喂NFC/NDF为1.40、1.79、2.31、3.23的4种试验饲粮诱导SARA发生,每种饲喂15 d,恢复组奶山羊待SARA诱导成功后自由采食青干草30 d。对照组奶山羊分别在饲养30、60(与SARA组3只同时)和90 d(与恢复组3只同时)各屠宰1只。采集瘤胃腹囊部上皮组织用于石蜡切片、透射电子显微镜观察及尤斯灌流系统(Ussing chamber)研究。结果表明:1)组织切片结果显示,瘤胃上皮角质层厚度SARA组显著高于对照组和恢复组(P0.05),恢复组显著低于对照组(P0.05);颗粒层厚度对照组显著高于SARA组和恢复组(P0.05),但SARA组和恢复组之间无显著差异(P0.05);棘突层厚度3组之间无显著差异(P0.05);上皮总厚度恢复组显著低于对照组和SARA组(P0.05),但对照组与SARA组之间无显著差异(P0.05)。透射电子显微镜结果显示,SARA组瘤胃上皮紧密连接被破坏,细胞间隙增大,棘状层细胞线粒体出现降解并出现空泡。2)与对照组相比,SARA组和恢复组瘤胃上皮短路电流(Isc)、组织导电性(Gt)和辣根过氧化物酶(HRP)流速显著升高(P0.05),跨膜电位差(PD)显著降低(P0.05)。综合得出,SARA破坏了奶山羊瘤胃上皮形态结构的完整性,使瘤胃上皮通透性增加,导致瘤胃上皮屏障功能长期受损。     

Number of nitrate- and nitrite-reducing Selenomonas ruminantium in the rumen, and possible factors affecting its growth

The cell number of Selenomonas ruminantium (S. ruminantium) that reduces nitrate and nitrite in the rumen was usually 8–10% of the total number of S. ruminantium (an order of 10⁶/mL). The percentage was not affected by the roughage/concentrate ratio or nitrate content of the diet in 2 weeks. However, feeding a high‐nitrate diet for 12 weeks increased the percentage. The percentage of lactate‐using S. ruminantium, such as the ssp. lactilytica, was less than 1% of the total number of S. ruminantium. No S. ruminantium was found that used formate as an electron donor for nitrate and nitrite reduction. Lactate and H₂ appeared to be important for nitrate and nitrite reduction by S. ruminantium. Nitrate reduction by S. ruminantium was enhanced by the coexistence of amylolytic bacteria in a medium containing starch, and as a result, nitrite accumulation increased. Coexistence of cellulolytic bacteria facilitated the growth of S. ruminantium in a medium containing cellulose, and consequently increased nitrite reduction. In order to suppress nitrite accumulation in the rumen, it may be important to enhance fiber digestion.     

估测饲料蛋白质瘤胃降解率活体外新方法的研究

研究了一种新的根据活体外人工瘤胃产气量与游离 NH_3-N 量估测反刍动物饲料蛋白质瘤胃降解率的方法。饲料样本与瘤胃液在活体外培养6 h,其产气量与组入瘤胃微生物蛋白质中的NH_3-N 量呈高度正相关(r>0.99),故产气量可用于准确度量组入微生物蛋白质中的 NH_3-N量。以饲料样本的游离 NH_3-N 量与根据产气量估测的组入 NH_3-N 量之和占其总 N 量的比例,作为其瘤胃蛋白质降解率的度量。本研究方法简单、快速、成本低,所测结果与体内法结果具有高度相关性(r>0.97)。     

醋酸盐缓冲合剂对碱过多性胃肠弛缓的疗效

对６９例前胃弛缓和瘤胃积食等胃肠弛缓自然病牛的瘤胃内环境参数和植物神经调控指标进行了检测。前胃弛缓、瘤胃积食病牛的瘤胃内环境参数基本一样,特征是ｐＨ值明显升高,分别为（７．９３±０．４７）和（８．０４±０．５９）,漂浮沉降时间明显缩短,次甲蓝反应时间明显延长,总酸度降低至（１３．１０±４．７５）Ｕ和（１１．２３±４．５５）Ｕ,纤毛虫数量减少、活力减退,细菌以Ｇ－者为主,乳酸增多,乙酸、丙酸和ＶＦＡ总量减少。前胃弛缓、瘤胃积食病牛血清肾上腺素（Ｅ）、去甲肾上腺素（ＮＥ）、多巴胺（ＤＡ）和儿茶酚胺（ＣＡ）等第一信使指标,ｃＡＭＰ、ｃＧＭＰ等第二信使指标改变不大。根据瘤胃内环境参数检测结果,结合临床表现,确认６９例胃肠弛缓病牛均为碱过多性胃肠弛缓,对其中５９例投服醋酸盐缓冲合剂（无水醋酸钠１３１ｇ,冰醋酸２２．９２ｍＬ,常水１０Ｌ）进行治疗,治愈率在９０％以上,平均投用方剂数仅为１．３付。     

Pathophysiological evaluation of subacute ruminal acidosis (SARA) by continuous ruminal pH monitoring

Evaluation of the radio‐transmission pH‐measurement system for monitoring the ruminal pH and subacute ruminal acidosis (SARA) in cattle is described. This is done in order to reveal the possible application of this system for detection and pathophysiological research of SARA by continuous ruminal pH measurement. The possibility of using this system for assessment of the ruminal pH in SARA cattle, and the presence of negative correlation between the ruminal pH and ruminal temperature in heathy and SARA cattle were determined. In addition, the 16S rRNA gene pyrosequencing analysis showed that the ruminal microbial community was simpler in SARA cattle, and the bacterial numbers in SARA cattle were lower than those in healthy hay‐fed cattle. Concentrate feeding might have reduced the diversity of the ruminal microbial community. Changes in the ruminal microbial community of SARA cattle might be related to the changes in ruminal pH followed by the decrease in the number of some bacteria. Continuous monitoring of the ruminal pH using the radio‐transmission pH‐measurement system would be applied for detection and prevention of SARA in the field and pathophysiological research of SARA, including ruminal zymology and bacteriology, which have been determined previously by sampling of the ruminal fluid and measuring of ruminal pH.     

高精料饲粮条件下反刍动物瘤胃适应机制的解析

饲喂高能、高淀粉饲粮是集约化生产中提高反刍动物生产性能的常用策略,但高精料饲粮易引起一系列的营养代谢疾病,其中以瘤胃酸中毒最为常见。反刍动物瘤胃不仅具有消化、吸收营养物质的功能,瘤胃上皮亦是重要的免疫屏障,故瘤胃健康对反刍动物至关重要。本文主要从反刍动物采食高精料饲粮时其瘤胃组织形态、瘤胃上皮适应分子机制和瘤胃微生物区系3个方面的变化进行阐述,以期为高精料饲粮条件下瘤胃适应机制的研究提供参考。     

同/异型发酵乳酸菌对全株玉米青贮营养成分和瘤胃降解特征的影响

以新饲玉10号全株青贮玉米为研究材料,旨在探究同/异型发酵乳酸菌对其主要营养成分和瘤胃降解率特征的影响。采用真空袋法调制青贮,共设计4个发酵处理,分别为不添加任何菌剂(CK);复合同型发酵乳酸菌植物乳杆菌+戊糖片球菌(T),添加量为1∶1,1×10^5 cfu·g^-1;异型发酵乳酸菌布氏乳杆菌(Y),添加量为1×10^5 cfu·g^-1;复合同、异型发酵乳酸菌(TY),添加量为1∶1∶1,1×10^5 cfu·g^-1。通过对模拟开窖60 d主要营养成分和绵羊瘤胃降解特征参数的分析。结果表明:干物质(DM)、中/酸性洗涤纤维(N/ADF)、可溶性碳水化合物和粗脂肪含量最优均为Y处理,粗蛋白和淀粉含量各试验处理均极显著高于CK处理(P<0.01),粗灰分含量最高为CK处理。DM和NDF有效降解率按高低排序均为T和Y处理>CK和TY处理(P<0.01)。有机物(OM)有效降解率最高为Y处理(P=0.003)。各处理ADF有效降解率间差异不显著(P>0.05)。说明玉米青贮中Y处理可显著增加开窖60 d时各营养成分含量(粗灰分除外),且显著提高DM和OM有效降解率,TY处理可显著提高NDF有效降解率。综合营养成分和有效降解率12项指标进行隶属函数评价,各处理按优劣排序为:Y处理>T处理>TY处理>CK处理。     

Effects of substituting kraft pulp with corn silage on dry matter intake,ruminal mat formation,rumen fermentation,and rumination activity in non‐lactating cows

The present study examined the effects of substituting kraft pulp (KP) with corn silage (CS) on dry matter intake (DMI), ruminal mat and rumen fermentation characteristics, and rumination. Four non‐lactating, rumen‐cannulated Holstein cows were fed a CS diet comprising 36% grass silage (GS) and 64% CS or a KP diet comprising 36% GS, 57% KP, and 7% soybean meal. DMI was significantly lower in cows fed the KP diet than in those fed the CS diet (p < 0.05), whereas rumination time did not significantly differ between the treatments. Dry matter content in the rumen immediately before and 3 h after feeding was significantly higher in cows fed the KP diet than in those fed the CS diet (p < 0.05). The consistency and thickness of the ruminal mat did not significantly differ between the treatments. The ruminal mean retention time of feed particles tended to be longer in cows fed the KP diet than in those fed the CS diet (p < 0.10). The ruminal digestion rate of KP was comparable to that of GS and CS. Because ruminal mat was formed and rumination was stimulated, KP was considered to have the equivalent physical effectiveness as CS.     

Effects of supplementary mother liquor,by‐product of monosodium glutamate,on in vitro ruminal fermentation characteristics

Mother liquor (ML) is monosodium glutamate by‐product and contains much crude protein (CP). The objective of this study was to investigate the effect of supplementation of two types of MLs having different chemical compositions on in vitro ruminal fermentation characteristics. ML1 had 74% organic matter (OM) and 70% CP and ML2 had 85% OM and 83% CP on a dry matter (DM) basis. The experiments were conducted using 0.5 gDM rolled barley or ryegrass straw as substrates. Urea and freeze‐dried MLs were added at three levels of nitrogen to be isonitrogenous. The ML1 treatment increased gas production compared with the ML2 treatment in barley substrate condition (p < 0.05). The supplementary ML2 increased gas production compared with the supplementary urea, and the digestibilities of DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) were higher for the supplementary ML2 than for the supplementary ML1 in straw substrate condition (p < 0.05). In both the conditions, ammonia nitrogen concentrations of the ML1 and ML2 treatments were lower than that of the urea treatment (p < 0.05). The results indicated that the supplementary ML1 and ML2 activated in vitro ruminal fermentation particularly in concentrate and roughage substrate conditions respectively.