拟茎点霉B3与有机肥配施对连作草莓生长的影响

通过盆栽试验探讨了内生拟茎点霉B3与有机肥配施对连作草莓土壤的改善及对草莓生长的影响。试验共设5个处理,分别为对照(CK)、施有机肥与灭菌固体培养基(A)、施有机肥与内生拟茎点霉B3固体菌种(B)、施有机肥与绿色木霉、黑曲霉、枯草芽孢杆菌固体菌种(C)、施加有机肥与绿色木霉、黑曲霉、枯草芽孢杆菌和内生拟茎点霉B3固体菌种(D)。结果表明:A、B、C和D处理平均单果鲜重分别为对照(CK)的1.1、1.4、0.9和1.1倍。B处理比对照增产19.7%,A处理增产8.2%,C和D处理产量均比CK低。B处理草莓生长最好,植株株高及叶面积均值比其它4个处理大。发病率及病情指数结果表明B处理抗病效果最明显,推断内生拟茎点霉B3可以用作生防菌剂。进一步的研究表明土壤真菌和细菌数量在整个生育期先上升后下降,在花期达到最大。成熟期A、B、C、D处理的土壤放线菌数量分别比CK增加7.2%、160.3%、124.5%及82.6%。在花期,B处理及D处理蔗糖酶酶活达到最大,其中A、B、C及D处理的蔗糖酶酶活分别比CK高11.1%、69.4%、50.3%及77.2%。B处理整个生育期都保持较高的土壤蔗糖酶活性。花期是草莓生长的关键期,需氮量较高。A、B、C及D处理脲酶酶活分别比CK处理高250.0%、700.0%、250.0%及175.0%,B处理花期土壤脲酶酶活性显著高于其它4个处理,促进了有机氮向速效氮的转化。花期A、C处理磷酸酶酶活比对照低67.0%、46.7%,B、D处理比对照高122.5%,227.5%。B处理在整个生育期都有较高的土壤磷酸酶酶活, D处理组在花期土壤磷酸酶酶活较高。可见含内生拟茎点霉B3菌的B及D处理组能增加土壤磷酸酶酶活。B处理在苗期和花期土壤纤维素酶活较低,而结果期和成熟期较高。说明内生拟茎点霉B3菌剂与有机肥配施可以改善连作草莓土壤微生物区系,提高土壤酶活性,增强草莓抗病能力,增加草莓产量,是一种有效缓解草莓连作障碍的方法。     

水蒸气蒸馏法提取大蒜挥发油的工艺研究

以挥发油提取率为指标,通过单因素和L9(34)正交试验设计确定了水蒸气蒸馏法提取大蒜挥发油的最佳提取工艺及条件。结果表明,各因素作用主次顺序为蒸馏时间(D)>料液比(C)>发酵温度(A)>浸泡时间(B),各因子的最优组合为A2B2C2D3,即以1∶3.5的料液比(m∶V),在35℃时浸泡3h后蒸馏2h为大蒜挥发油的最佳提取工艺,此时挥发油的提取率达到0.32%,证明此方法是大蒜挥发油提取的可靠、易行的科学方法。     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

急性心肌梗死患者肠道菌群多样性分析

目的初步探究急性心肌梗死患者肠道菌群的多样性。方法选择2015年6月至2016年3月于大连大学附属中山医院住院治疗的急性心肌梗死患者作为观察组,年龄51~70岁。选择同期经冠脉造影排除冠心病的住院患者为对照组,年龄51~70岁。排除近1个月内发生感染、炎性肠病及应用抗生素的患者。所有患者按性别与年龄分为A、B、C、D组。直接从患者粪便标本中提取细菌总DNA,PCR扩增后进行梯度凝胶电泳(DGGE)分析。结果 DGGE分析显示急性心梗患者肠道菌群丰度均较对照组下降。A组中观察组与对照组患者电泳条带数为(33.71±4.39)vs(38.71±2.56),t=-2.058,P=0.040;C组患者为(31.14±2.67)vs(35.29±3.55),t=-2.005,P=0.045;差异均具有统计学意义。B与D组中心梗患者肠道菌群丰度亦有下降趋势,但与对照组比差异无统计学意义。UPGAM法聚类分析显示除D组外,各组中观察组与对照组患者肠道菌群呈现分离现象;急性心梗患者肠道菌群有较高相似性,与非冠心病患者肠道菌群差异明显。结论急性心梗与非冠心病患者肠道菌群存在差异,急性心梗患者肠道菌群多样性较低。肠道菌群改变可能与急性心肌梗死存在一定相关性。     

小地老虎食诱剂糖醋酒液配方筛选及发酵增效作用

【目的】为筛选和规范诱集小地老虎Agrotis ipsilon成虫的最佳糖醋酒液配方及最佳发酵时间,确定发酵液中的挥发物成分。【方法】优选以单一纯物质糖、醋、酒和水进行混配的配方A, B, C和D, 其配比分别为蔗糖(g)∶乙酸(mL)∶无水乙醇(mL)∶纯水(mL)(m/v/v/v)=3∶1∶3∶80, 3∶1∶3∶160, 3∶1∶6∶80和1∶1∶3∶80及常见配方E［白糖(g)∶白醋(mL)∶白酒(mL)∶自来水(mL)= 6∶3∶1∶10(m/v/v/v)］,分别并采用Y型嗅觉仪及GC-MS测试和分析小地老虎成虫对发酵1~15 d后的糖醋酒液的趋性及发酵液中的挥发物成分。【结果】趋性试验结果表明,糖醋酒液B配方对小地老虎诱集效果显著优于A, C, D和E配方,C配方次之。小地老虎成虫对B和C这两种配方发酵8 d发酵液的选择反应率均显著高于对未发酵及发酵5 d和7 d外的其他发酵时间发酵液的选择反应率。挥发物成分组成分析结果显示,糖醋酒液B配方发酵8 d发酵液的挥发物共有41种化合物,主要包括17种烃类化合物、8种醛类化合物、2种酮类化合物、4种醇类化合物、5种酯类化合物和5种醚类化合物。而且发酵8 d的糖醋酒液B配方与其未发酵以及发酵4 d和发酵12 d的糖醋酒液B配方之间的挥发物成分差异较大。【结论】优选并明确单一物质糖、醋、酒和水进行混配的小地老虎食诱剂配方(3∶1∶3∶160, m/v/v/v),其糖醋酒液通过发酵可以产生增效作用,最佳发酵时间为8 d。     

接种AM真菌对PAEs污染土壤中微生物和酶活性的影响

土壤灭菌条件下 ,添加 5 0mg·kg-1DEHP和 5 0mg·kg-1DBP ,在温室进行盆栽试验 ,观察土壤施加DEHP与DBP和接种AM真菌 (Acaulosporalavis,光壁无梗球囊霉 ,菌号 :34)后菌根际 (简称A)、菌丝际 (简称B)和常规土 (简称C)中土壤微生物和部分土壤酶活性的变化。结果表明 ,土壤施加DEHP和DBP后 ,A、B和C层土中土壤微生物数量和土壤酶含量下降 ;接种AM真菌后 ,受AM直接影响的A和B层土中细菌、放线菌和真菌数量比不接种低 ,而C层土中三菌数量比不接种高 ;A和B层土中中性磷酸酶和脲酶含量下降 ,脱氢酶含量在A、C层土中下降 ,在B层土中稍有增加 ,AM对土壤脱氢酶活性影响不大。接种AM真菌没有降低DEHP和DBP对土壤微生物生长和土壤酶活性不利影响的程度     

日舒安洗液联合克霉唑治疗小鼠外阴阴道念珠菌病的疗效观察

目的观察日舒安洗液联合克霉唑治疗小鼠外阴阴道念珠菌病的疗效。方法将95只BALB/c雌鼠(8周龄)随机分为A组(5只,空白对照组),B组(30只,模型对照组),C组(30只,克霉唑组)和D组(30只,日舒安洗液联合克霉唑组)。对B组、C组和D组小鼠阴道接种白念珠菌。对A组和B组予等量的生理盐水冲洗阴道,C组单用克霉唑治疗,对D组进行日舒安洗液联合克霉唑治疗。在接种前及治疗过程中观察各组小鼠体重变化。B组、C组和D组在治疗后的第1、2、3、6、8、10、14和16天取阴道灌洗液进行菌落培养,采用涂布法检测小鼠的阴道真菌载荷量,并通过ELISA检测IL-2和IL-8的水平;qRT-PCR法检测小鼠阴道组织中NLRP3、ASC、caspase-1的mRNA表达。结果在前8 d,4组小鼠的体重均保持稳定,接种后B、C和D组小鼠体重下降,随后体重逐渐上升,接近接种前水平。与空白对照组A相比较,B、C和D组阴道灌洗液中阴道真菌载荷量增加,但B、C和D组之间存在统计学差异,D组阴道真菌载荷量少于B组和C组,C组阴道真菌载荷量少于B组;与空白对照组相比较,B、C和D组阴道灌洗液中IL-2和IL-8增加,但治疗后B、C和D组之间IL-2和IL-8水平存在统计学差异,D组IL-2和IL-8水平显著小于C组, C组IL-2和IL-8水平显著小于B组(P0.01)。与空白对照组相比较,B、C和D组阴道组织中NLRP3、ASC、caspase-1 mRNA表达上调,但B、C和D组之间存在统计学差异,B组mRNA上调水平大于C组和D组,C组mRNA上调水平大于D组(P0.01)。结论日舒安洗液联合克霉唑对小鼠外阴阴道念珠菌病治疗作用显著。     

口服特比萘芬联合外用联苯苄唑乳膏治疗足癣长期临床研究

目的 为足癣治疗提供较理想的方案.方法 盐酸特比萘芬250 mg,口服1次/d;1%联苯苄唑乳膏,外用1次/d.250例足癣患者随机入组.A组口服1周加外用1周,B组口服1周加外用2周,C组口服2周加外用1周,D组口服2周加外用2周,E组单独外用4周.在治疗结束时、治疗结束后4周、24周、40周、56周、72周时对各组的疗效、复发率进行评价.结果 在停药后第24周时,A、B、C、D组与E组疗效比较差异有统计学意义.在停药后第40周、56周、72周时,D组与A、B组疗效比较差异也出现统计学意义.在停药后24周、40周时,A、B、C、D组真菌学疗效与E组比较差异有统计学意义.在停药56周、72周时,D组与A、B组真菌学疗效比较差异也出现统计学意义.在停药40周时,A、B、C、D组与E组复发率比较差异有统计学意义.在停药56周、72周时,D组与A、B组复发率比较差异也出现统计学意义.结论 口服特比萘芬2周联合外用1%联苯苄唑乳膏2周治疗足癣的有效率和真菌学疗效最高,复发率最低.     

左旋布比卡因浓度和容量选择对小儿骶管阻滞效果影响

为探究不同浓度和容量左旋布比卡因对小儿骶管阻滞效果的影响,研究人员对我院2013年至2015年收治的90例小儿手术患者病例进行了研究。所有患儿采用左旋布比卡因行骶管阻滞麻醉,按照随机数表法平均分至A~F 6组,分别给予不同浓度和容量的左旋布比卡因:A组(0.25%,1.0 m L/kg)、B组(0.25%,0.7 m L/kg)、C组(0.20%,1.0 m L/kg)、D组(0.20%,0.7 m L/kg)、E组(0.15%,1.0 m L/kg)、F组(0.15%,0.7 m L/kg)。平均动脉压,A组明显低于C组和E组,B组显著低于D组和F组。E组麻醉时间、下肢肌力恢复时间及肛门排气时间明显低于A、C两组,而补充氯胺酮次数及疼痛评分显著高于A、C两组。F组麻醉时间、下肢肌力恢复时间及肛门排气时间明显低于B、D两组,而补充氯胺酮次数及疼痛评分显著高于B、D两组。E、F两组不良反应发生率显著高于其余4组。C组疼痛评分显著低于D组。0.20%的左旋布比卡因1.0 m L/kg用于小儿骶管阻滞既可达到满意麻醉效果,且并发症少,可作为最佳有效浓度。     

巴戟天水提液有效萃取成分促进微波致雄性大鼠生殖损伤的修复

目的探讨巴戟天(Morinda officinalis F.C.How)水提液对微波辐射致生殖系统损伤的修复作用。方法以900MHz频率、218μm/cm2辐射密度的低功率微波持续辐射10天,建立雄性SD大鼠生殖损伤模型。66只健康成年雄性SD大鼠随机分为11组。A组为空白对照组,B组为辐射未给药组,C组为辐射+生理盐水组,D组为巴戟天水提液乙酸乙酯萃取水层治疗组,E组为巴戟天水提液乙酸乙酯萃取酯层治疗组(其中D、E组各有10g、20g、30g、40g生药量/kg浓度梯度组)。每天灌胃1.5ml,持续14d。观察大鼠性行为学指标差异,同时检测血清睾酮变化;比较睾丸、附睾指数以及睾丸组织形态学的改变。结果巴戟天水提液乙酸乙酯萃取各层浓度梯度组分别给药后,D3、D4组扑捉潜伏期与B、C组相比缩短,D4组扑捉次数较之B、C组增加,余治疗组性行为学指标与B、C组无明显差别。D3、D4组睾酮水平较之B、C组均显著升高。HE染色和PCNA/SCP3双重免疫荧光染色显示,随着给药剂量增大,D组大鼠生精细胞数量逐渐增多,以A型精原细胞和粗线期初级精母细胞修复最为明显。结论巴戟天水提液乙酸乙酯萃取水层可改善大鼠性欲、修复生精上皮生精细胞和精子生成,最佳治疗剂量为40g/kg,其机制可能与促进睾酮分泌增加有关。     

Effects of soybean oil and dietary copper levels on nutrient digestion,ruminal fermentation,enzyme activity,microflora and microbial protein synthesis in dairy bulls

ABSTRACT

The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.     

Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows

The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108 v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104 v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108 v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.     

Volatile fatty acids and anaerobic fermentation in temperate piscivorous and omnivorous freshwater fish

Endproducts of anaerobic fermentation, volatile fatty acids (VFAs), occur in the intestines of fish but no direct comparisons exist between VFA levels in fish with differing feeding ecologies during different seasons. We measured intestinal concentrations of six types of VFA in the upper and lower intestines of two freshwater omnivores ( Cyprinus carpioi and Dorosoma cepedianum ), and one piscivore ( Micropterus salmoides ) during the spring, summer and autumn. Acetate occurred in all species, and was highest in M. salmoides . In all species, concentrations were similar between upper and lower guts and higher during the summer. All three species contained anerobic bacteria and C. carpio and D. cepedianum contained cellulolytic types. Scanning electron microscopy revealed extensive colonization and suggested microbial breakdown of digesta in M. salmoides . In radio-tracer experiments, C. carpio dosed orally with [2–¹⁴C] acetate contained label in liver, muscle and blood tissues. Amounts of intestinal VFA did not appear to increase in species with refractile diets, and low VFA in D. cepedianum suggests fermentation plays a minimal role in the nutrition of this species. Low levels of intestinal VFA during cool seasons are consistent with the hypothesis that temperature limits fermentation in these Species.     

Protein enrichment of sweet potato residue with co-culture of amylolytic fungi by solid-state fermentation

Protein enrichment of sweet potato residue with amylolytic moulds by solid-state fermentation was higher than that obtained with amylolytic yeasts. The optimum initial moisture content for protein enrichment was 66% to 75%. Incrementally added nitrogen sources to the culture at zero time and at 24 h considerably improved the final protein content. During the cultivation, the moisture, ash and ATP contents increased, while the pH value decreased. A 1:1 co-culture of amylolytic mycelial fungi yielded a product with 32.4% crude protein after 4 days incubation at 30 degrees C.     

Increasing sodium bicarbonate level in high-concentrate diets for heifers. I. Effects on intake, water consumption and ruminal fermentation

Four ruminally fistulated Holstein heifers (BW = 264 ± 12 kg) were used in a 4 × 4 Latin square design experiment to determine the effect of increasing levels of sodium bicarbonate (BICARB; 0%, 1.25%, 2.50% and 5%, on concentrate dry matter (DM) basis) on DM intake (DMI), water consumption and ruminal fermentation. Sampling was carried out in the last week of each four 21-day experimental periods. Heifers were offered concentrate (13.4 ± 0.04% crude protein (CP), 13.3 ± 0.44% NDF, 51.7 ± 0.97% starch) and barley straw once daily at 0830 h ad libitum. There was a linear decrease in concentrate DMI and a linear increase in straw DMI with increasing buffer level in the diet, resulting in a tendency towards a linear decrease in total DMI. Intake of concentrate was 6.89, 7.66, 6.72 and 5.72 ± 0.83 kg/day, whereas straw intakes were 0.73, 0.84, 0.94 and 1.06 ± 0.14 kg/day, for the 0%, 1.25%, 2.5% and 5% BICARB, respectively. Water consumption was not affected by treatments when expressed as l/day or percentage of BW, but increased linearly when expressed as l/kg of DMI. The percentage of total daily water drunk in the morning (from 0830 to 1230 h) increased linearly with the level of buffer. Mean ruminal pH and total area under the pH curve were not affected with increasing buffer level. The lowest daily pH (5.65 ± 0.09) was not affected by treatments. A quadratic tendency (P 0.10) was observed in the number of hours and the area under the pH curve in which ruminal pH was below 5.8, with high values only at the 0% BICARB. Additionally, increasing bicarbonate level caused a linear increase in the ruminal pH at 2 and 4 h after feeding. Daily average NH3 N (2.4 ± 0.9 mg N/100 ml) and total volatile fatty acids (VFA) (143 ± 12 mM) concentrations were not affected by treatments. Daily average molar proportion of propionate decreased linearly, and acetate proportion and the acetate-to-propionate ratio were increased with increasing buffer level in the diet. Molar percentage of butyrate, isobutyrate and isovalerate, and branched-chain VFA concentration increased linearly as the level of bicarbonate increased in the diet. Results indicate that high levels of BICARB to finishing heifers fed high-concentrate diets may result in a decreased DMI without significant effects on mean ruminal pH, which may affect animal performance. All individual VFA proportions, except valerate, were changed by the addition of bicarbonate.     

Effects of dietary supplementation of rumen-protected folic acid on rumen fermentation,degradability and excretion of urinary purine derivatives in growing steers

The present experiment was undertaken to determine the effects of dietary addition of rumen-protected folic acid (RPFA) on ruminal fermentation, nutrient degradability, enzyme activity and the relative quantity of ruminal cellulolytic bacteria in growing beef steers. Eight rumen-cannulated Jinnan beef steers averaging 2.5 years of age and 419 ± 1.9 kg body weight were used in a replicated 4 × 4 Latin square design. The four treatments comprised supplementation levels of 0 (Control), 70, 140 and 210 mg RPFA/kg dietary dry matter (DM). On DM basis, the ration consisted of 50% corn silage, 47% concentrate and 3% soybean oil. The DM intake (averaged 8.5 kg/d) was restricted to 95% of ad libitum intake. The intake of DM, crude protein (CP) and net energy for growth was not affected by treatments. In contrast, increasing RPFA supplementation increased average daily gain and the concentration of total volatile fatty acid and reduced ruminal pH linearly. Furthermore, increasing RPFA supplementation enhanced the acetate to propionate ratio and reduced the ruminal ammonia N content linearly. The ruminal effective degradability of neutral detergent fibre from corn silage and CP from concentrate improved linearly and was highest for the highest supplementation levels. The activities of cellobiase, xylanase, pectinase and α-amylase linearly increased, but carboxymethyl-cellulase and protease were not affected by the addition of RPFA. The relative quantities of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes increased linearly. With increasing RPFA supplementation levels, the excretion of urinary purine derivatives was also increased linearly. The present results indicated that the supplementation of RPFA improved ruminal fermentation, nutrient degradability, activities of microbial enzymes and the relative quantity of the ruminal cellulolytic bacteria in a dose-dependent manner. According to the conditions of this experiment, the optimum supplementation level of RPFA was 140 mg/kg DM.     

内生真菌角担子菌B6对连作西瓜土壤尖孢镰刀菌的影响

建立了快速定量检测土壤中角担子菌(Ceratobasidum stevensii)B6的实时定量PCR方法,同时跟踪了土壤中尖孢镰刀菌数量的动态变化,以及不同的发酵组分对开花期西瓜土壤微生物区系的影响。PCR扩增分析表明引物Cf1/Cr1有很好的特异性,能对角担子菌B6特异性扩增得到371bp的条带,对其它10株真菌不能有效扩增。使用荧光定量PCR对施加角担子菌B6的土壤总DNA扩增,结果表明将活菌B6固体发酵物施加到土壤1周后,B6的数量有一定的增殖,达到7.4 log(pg DNA/g干土),随着时间推移,数量逐渐减少,在第5周的时候低于检测限;而液体发酵液处理从一开始施加到土壤后,B6的数量就开始逐渐减少,在第4周的时候就低于检测限。施加活的B6菌4周内能够有效地控制尖孢镰刀菌的数量(尖孢镰刀菌数量维持在5×103 CFU/g干土左右),之后随着B6数量的减少尖孢镰刀菌数量大量增加。活菌B6在土壤中能够存活1个月左右,不会过度影响土著微生物区系,是一株环境友好型菌株,对土壤微环境的干扰较小。     

Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1). Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)(-1)] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)(-1)]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1). Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.     

Effect of different levels of concentrate on ruminal microorganisms and rumen fermentation in Nellore steers

The aim of this study was to investigate the effect of different dietary levels of concentrate on feed intake, digestibility, ruminal fermentation and microbial population in steers. Eight Nellore steers fitted with ruminal cannulas were used in a double 4 × 4 Latin square design experiment. The dietary treatments consist of four different proportions of concentrate to roughage: 30:70, 40:60, 60:40 and 80:20% in the dry matter, resulting in Diets 30, 40, 60 and 80, respectively. The roughage was corn silage, and the concentrate was composed of corn, soybean meal and urea. Apparent digestibility of organic matter and crude protein showed a linear association with concentrate proportion (p = 0.01), but the increased concentrate levels did not affect the digestibility of fibre. The lowest ruminal pH-values were observed in animals fed with Diet 80, remaining below pH 6.0 from 6 h after feeding, while in the other diets, the ruminal pH was below 6.0 not before 12 h after feeding. After feeding Diet 80, the ammonia concentration in the rumen was significantly the highest. Higher dietary concentrate levels resulted in a linear increase of propionic acid concentrations, a linear reduction of the ratio acetic acid to propionic acid (p < 0.01) and a linear increased synthesis of microbial nitrogen (p < 0.001). The predicted production of methane was lower in diets with greater amounts of concentrate (p = 0.032). The population of methanogens, R. flavefaciens and R. albus decreased with higher concentrate levels, while the population of S. ruminantium increased (p < 0.05). The results indicate that greater amounts of concentrate do not decrease ruminal pH-values as much as expected and inhibit some cellulolytic bacteria without impairing the dry matter intake and fibre digestibility in Nellore steers.