生长抑素和皮质抑素双表达DNA疫苗免疫杂交水牛育成牛的促生长效果研究

试验旨在探究生长抑素（SS）和皮质抑素（CST）双表达DNA疫苗免疫杂交水牛育成牛的促生长效果,并对其安全性进行验证。将鉴定正确的pVGS/2SS-2A-S/CST-asd质粒电转入减毒猪霍乱沙门氏菌C500构建生长抑素和皮质抑素双表达活载体疫苗。将16头6~12月龄杂交水牛育成牛随机分为低（TL）、中（TM）、高（TH）剂量组及PBS对照组（NC）,每组4头,试验组和对照组分别用双表达疫苗和PBS进行鼻腔免疫,初免时每天09：00和15：00免疫2次,连续免疫3 d,2周后以相同程序加强免疫1次,对免疫后不同时期的增重效果和免疫应答水平等进行监测。免疫应答结果显示,各剂量组免疫后均引起了免疫应答,TL组在初免后2周产生的SS抗体水平高于TM和TH组,而TM组SS抗体阳性率最高,为100%,优于TL （75%）和TH组（50%）;初免后2周产生的CST抗体水平则呈现完全相反的趋势,抗体水平随免疫剂量下降呈下降的趋势;初免后7周SS抗体和CST抗体水平和阳性率整体呈下降趋势,但也有部分牛出现抗体升高趋势;免疫因子检测结果显示,初免后2周TL和TM组IL-4水平极显著高于对照组（P<0.01）,而免疫后7周对照组的IL-4水平显著高于TL组（P<0.05）,与其他各组之间差异均不显著（P>0.05）;对照组INF-γ水平在初免后2周显著高于TL和TM组（P<0.05）,免疫后7周对照组显著高于TL组（P<0.05）,与其他各组之间差异均不显著（P>0.05）;对比抗体阳性组和阴性组发现,初免后2周抗体阳性组的IL-4水平极显著高于阴性组（P<0.01）,初免后7周则显著低于对照组（P<0.05）,而抗体阳性组INF-γ在初免后2和7周均显著低于阴性组（P<0.05）。增重效果检测显示,初免后2周各试验组平均日增重均显著高于对照组（P<0.05）,而在其他各时期各组之间差异均不显著（P>0.05）,抗体阳性组仅在初免后2周显著高于阴性组（P<0.05）。激素检测结果显示,各试验组生长激素（GH）和胰岛素样生长因子-1（IGF-1）在初免后2周均极显著高于对照组（P<0.01）,而初免后7周TL组GH和IGF-1水平显著低于对照组（P<0.05）,其他各组之间均差异不显著（P>0.05）;抗体阳性组的GH和IGF-1水平在初免后2周极显著高于阴性组（P<0.01）,而初免后7周则极显著低于阴性组（P<0.01）。安全性检测结果显示,各时期采集的水样、土样、粪样中均未检测到目的片段,证实了疫苗的安全性。本试验结果表明,双表达疫苗免疫后机体能产生良好的免疫应答,能提高育成牛的平均日增重,且对环境未产生安全性问题。     

Influence of varying levels of dietary cation anion difference on ruminal characteristics,nitrogen metabolism and in situ digestion kinetics in buffalo bulls

This study was conducted to examine the influence of varying dietary cation anion difference (DCAD) on nutrient intake, digestibility, ruminal characteristics, blood acid base status and in situ digestion kinetics in Nili Ravi buffalo bulls. Four iso‐nitrogenous and iso‐caloric diets having ?110, +110, +220 and +330 mEq/kg dry matter (DM) DCAD were formulated which were represented by A (anionic), LC (low cationic), MC (medium cationic) and HC (high cationic), respectively. These diets were used in four ruminally cannulated Nili Ravi buffalo bulls in a 4 × 4 Latin Square Design. Improved nutrient intake was recorded at high DCAD levels while digestibility remained unaffected. Ruminal ammonia nitrogen, rumen pH, acetate and acetate : propionate ratio were higher in buffalo bulls fed MC and HC diets than those fed A and LC diets. Blood pH and HCO₃^‐ also tended to increase as DCAD level was increased in the diet. Serum Ca and Cl concentrations were higher in bulls fed A and LC diets whereas serum Mg, P and S remained unaffected. Urine pH increased with increasing DCAD level. Nitrogen intake and blood urea nitrogen concentrations were also higher in bulls fed MC and HC diets. There was a consistent increase in ruminal DM and neutral detergent fiber degradability, rate of disappearance and extent of digestion at high DCAD levels in the diet. However, lag time decreased at high DCAD level. This study indicated that buffalo bulls fed MC and HC diets improved feed intake, ruminal characteristics and digestion kinetics.     

Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China

Astroviruses are the principal causative agents of gastroenteritis in humans and have been associated with diarrhea in other mammals as well as birds. However, astroviral infection of animals had been poorly studied. In the present study, 211 rectal swabs collected from cattle and water buffalo calves with mild to severe diarrhea were tested for bovine astrovirus (BAstV) by RT-PCR. Results: 92/211 (43.6%) samples were positive for BAstV, at a rate of 46.10% (71/154) in cattle and 36.84% (21/57) in water buffalo. Phylogenetic analysis based on the partial and full-length of 25 ORF2 amino acid sequences obtained in this study classified the Guangxi BAstVs isolates into five subgroups under the genus of Mamastrovirus, genotype MAstV33, which suggested that the water buffalo was a new host of this genogroup that previously included only cattle and roe deer. Despite the origin of the host, the Guangxi BAstV isolates were closely related to the BAstV Hong Kong isolates (B18/HK and B76-2/HK), but highly divergent from the BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the U.S.A. Nucleotide sequence-based characterization of the ORF1b/ORF2 junction and corresponding overlapping regions showed distinctive properties, which may be common to BAstVs. Our results suggested that cattle and water buffalo are prone to infection of closely related astroviruses, which probably evolved from the same ancestor. The current study described astroviruses in water buffalo for the first time and is thus far among the largest epidemiological investigations of BAstV infection in cattle conducted in China.     

水牛瘤胃氮及矿物质代谢若干规律的研究

试验采用４头装有永久性瘤胃瘘的本地生长雄性水牛。在秋季自由采食稻草或青干草日粮条件下，采用Ｃｒ－ＥＤＴＡ连续灌流标记法，测定瘤胃液的体积、周转率和停留时间，观察瘤胃氮、矿物质代谢水平和昼夜动态。结果表明，在这两种日粮条件下，瘤胃液的体积、周转率和停留时间差异不显著。但瘤胃乙酸、丙酸、丁酸、氨氮（ＮＨ３－Ｎ）、细菌蛋白氮、以及Ｎａ＾＋、Ｃａ＾＋＋、Ｍｇ＾＋＋、Ｐ的浓度青干草期均显著高于稻草期（Ｐ＜０     

RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P<0.01);细胞增殖受到显著抑制(P<0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P<0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P<0.05),Caspase9、Fas基因表达量极显著上升(P<0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P<0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。     

水牛发情周期生殖激素变化规律及唾液结晶的分析

试验对水牛发情周期血清和唾液中雌二醇(E_2)和孕酮(P_4)的浓度变化规律、水牛唾液结晶与卵泡发育变化分别进行了分析研究,为进一步探讨水牛发情规律、指导生产提供依据。采用酶联免疫分析法(ELISA)测定发情母水牛血清和唾液中E_2和P_4的浓度变化,并对血清和唾液的激素变化规律进行相关性分析。结果表明,水牛血清和唾液中的E_2和P_4呈波动性变化。发情前期,唾液中P_4浓度一直维持在6.50～7.10 ng/mL,发情第13天达到11.09 ng/mL,随后快速下降。唾液中E_2浓度在发情第3～5天出现一个峰值178.53 pg/mL,在第14～17天唾液中E_2浓度显著升高,出现第二个峰值179.10 pg/mL。母水牛唾液中E_2和P_4浓度的变化趋势与其在血清中的变化趋势基本一致,均呈显著相关(P<0.05);唾液中E_2与P_4浓度呈极显著相关(P<0.01)。水牛发情当天唾液结晶呈现明显的蕨类作物形状且分维值显著低于其他时间点(P<0.05)。水牛发情周期唾液结晶图形的变化与卵巢卵泡发育基本同步,可作为监测水牛发情及预测排卵的可靠指标之一。     

水牛骨骼标本的串制工艺新探

选择体格健康的摩拉杂交水牛,参照家畜解剖学图谱和X光片,遵循以牢固和保持其固有形态为主,兼顾关节运动方式的原则,串制了摩拉杂交水牛骨骼标本。串制顺序为：先串制四肢骨骼,然后串制躯干骨骼,最后将前、后肢骨骼分别连接于肩带和盆带部。通过反复试验,认为聚氨酯填缝剂可作为较理想的修补剂;针对骨块特别是小骨块容易弄乱甚至丢失,去肉时可在较易弄乱的骨块上（如肋骨）刮去骨膜,标上记号,而较小的骨块（如籽骨）则应用纱布打包后再进行处理。在串连四肢时,每一肢均用2根铁丝由远端穿至近端,然后合为1根,可使骨骼标本更加美观、自然、牢固;串制中使用螺丝杆,既可稳固整个骨架,又可根据需要灵活调整使其保持自然形态。