Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.     

基于ISSR分子标记的紫云英种质资源遗传多样性及结构分析

紫云英(Astragalus sinicus L.)是我国重要的稻田绿肥作物,为揭示不同地区种质资源遗传特征,本研究利用ISSR分子标记对77份紫云英种质资源进行了遗传多样性及结构分析。研究发现,77份供试紫云英种质资源遗传相似性系数为0.567 7～0.868 6,可划分为6个聚类。不同地区群体间分化系数为0.136 3,达到极显著水平(P<0.001),其中种群间变异占比13.9%,种群内变异占比86.1%。不同ISSR引物扩增信息鉴别紫云英资源的能力存在差异,构建了以P809第3,8,9,10,12,15扩增位点以及P886第1,3,5,9,10,11扩增位点为特征的指纹图谱,能有效区分77份供试紫云英资源。以资源所在省份为单元进行群体分析,供试紫云英资源遗传结构呈现分化特征,河南-安徽-江苏-上海资源遗传结构相似,构成北方亚群;四川-广西-湖南-江西资源相似,构成西南方亚群;福建种质资源则呈现混合型特征。本研究结果可为我国紫云英种质资源的科学利用及新品种选育提供依据。     

微贮牧草对山羊生产性能、饲粮养分消化率和消化道微生物数量的影响

试验使用黑曲霉、乳酸菌、酵母菌3种微生物的固体菌剂以及尿素作为微生态制剂青贮(微贮)高丹草和牛鞭草,并对64只黑山羊进行30 d饲养对比试验,研究高丹草和牛鞭草青贮对山羊生产性能、饲粮养分消化率和消化道微生物数量的影响。试验结果为,相比于普通青贮(普贮),微贮高丹草酵母菌、黑曲霉、乳酸菌数量分别增加了475.61%、290.65%和513.37%;微贮牛鞭草酵母菌、黑曲霉、乳酸菌数量分别增加了466.67%、762.07%和486.05%;微贮组羊瘤胃中的酵母菌、黑曲霉和乳酸菌数量均极显著增加(P0.01);微贮组羊粪中的酵母菌、黑曲霉和乳酸菌数量显著或极显著增加(P0.05),大肠杆菌数量极显著降低(P0.001);微贮组饲粮除中性洗涤纤维的表观消化率极显著高于普贮组(P0.01),其余均显著高于普贮组(P0.05);微贮组平均日增重和饲料转化率显著高于普贮组(P0.05)。综上所述,微生态制剂青贮不仅可增加瘤胃和粪中酵母菌、黑曲霉和乳酸菌数量,减少粪中大肠杆菌的数量,还能提高山羊饲粮表观消化率和生长性能。     

公司化运营的丹寨模式：新型基层兽医服务模式（三）

公司化运营丹寨模式是贵州省建立的一种新的基层兽医服务模式。为推广这一新模式,从模式的成立背景、项目营运、收入分配和运转成效等方面进行了系统介绍,从村级防检员收入、服务能力等方面剖析了该新型模式的先进性,并提出了推动该模式持续健康发展的相关建议。     

团头鲂幼鱼饲料中α-亚麻酸、亚油酸的适宜含量

在半纯化饲料配方的基础上,分别设计6个α-亚麻酸含量(0.02%、0.55%、1.08%、1.60%、2.13%、2.65%)、6个亚油酸含量(0.86%、1.29%、1.73%、2.16%、2.59%、3.03%),以亚麻籽油、玉米油、棕榈油调节饲料中α-亚麻酸、亚油酸的含量,配制等氮等能(粗蛋白质含量为30.09%,粗脂肪含量为6.87%)的12种半纯化试验饲料,探讨团头鲂幼鱼[初始均重为(59.5±0.5)g]饲料中α-亚麻酸、亚油酸的适宜含量。养殖试验分为α-亚麻酸和亚油酸试验2部分,均设6组,每组4个重复,每个重复20尾,养殖周期为85 d。结果表明:在α-亚麻酸试验中,依据回归方程计算得到,在饲料α-亚麻酸含量分别为1.32%、1.33%时,团头鲂幼鱼具有最大的特定生长率和最小的饲料系数;0.02%组的脏体指数显著低于除1.08%组外的其他各组(P0.05),肥满度、肝体指数各组间无显著差异(P0.05);0.02%组血清总胆固醇、甘油三酯含量显著高于0.55%、1.08%组(P0.05),1.60%组血清高密度脂蛋白含量显著高于0.55%、2.13%组(P0.05),0.02%组血清低密度脂蛋白含量显著高于其他各组(P0.05)。在亚油酸试验中,依据回归方程计算得到,在饲料亚油酸含量分别为2.02%、2.03%时,团头鲂幼鱼具有最大的特定生长率和最小的饲料系数;3.03%组的肝体指数显著低于其他各组(P0.05),肥满度、脏体指数各组间无显著差异(P0.05);1.29%组血清总胆固醇含量显著高于1.73%组(P0.05),0.86%组血清甘油三酯含量显著高于1.73%组(P0.05),1.29%组血清高密度脂蛋白含量显著高于0.86%、3.03%组(P0.05),各组血清低密度脂蛋白含量无显著差异(P0.05)。以特定生长率、饲料系数作为主要评价指标,结合部分血清生化指标和形体指标,得到适合团头鲂幼鱼快速生长、维持鱼体正常健康的饲料中适宜的亚麻酸、亚油酸含量分别为1.32%~1.33%、2.02%~2.03%。     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

Cloning,Expression of Clostridium perfringens α-toxin Gene and Preparation of its Antisera

One pair of primers had been designed and synthesized based on the α-toxin gene of Clostridium perfringens.The complete α-toxin gene fragment was amplified by polymerase chain reaction (PCR), and then was cloned into pGEM-T Easy vector to construct pGEM-T-α.Digested with EcoRⅠ and Hind Ⅲ, a fragment of 1125 bp was cloned into the expression plasmid vector pET-28a(+).The recombinant plasmid was transformed into the BL21(DE3)plys and induced by 1.0 mmol/L IPTG at 37 ℃.The expression product was found to be 46.1 ku as expected one identified by SDS-PAGE, and confirmed by Western blotting with Clostridium perfringens type A antisera, indicating similar reactivity with native α-toxin.Recombinant α-toxin protein was simultaneously found in culture supernatant, postsonic supertanant and inclusion bodies, most protein was expressed in inclusion bodies, which indicated recombinant α-toxin protein was expressed in the extracellular, periplasm and cytoplasm.Recombinant α-toxin protein in postsonic supertanant could not make mice die, indicating its non-toxicity.Toxin-antitoxin neutralization test showed that antisera of recombinant α-toxin protein were specific to α-toxin.Upon immunization of rabbit with the recombinant α-toxin protein, antisera with high antibody titer neutralizing 100 MLD toxin per 1 mL were prepared.     

No effect of exogenous melatonin on development of cryopreserved metaphase II oocytes in mouse

Background

This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation.

Results

First, mouse metaphase II (MII) oocytes were vitrified in the open-pulled straws (OPS). After warming, they were cultured for 1 h in M₂ medium containing melatonin at different concentrations (0, 10⁻⁹, 10⁻⁷, 10⁻⁵, 10⁻³ mol/L). Then the oocytes were used to detect reactive oxygen species (ROS) and glutathione (GSH) levels (fluorescence microscopy), and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10⁻³ mol/L melatonin group was significantly lower than that in melatonin-free group (control). The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp90aa1, Hsf1, Hspa1b, Nrf2 and Bcl-x1 with qRT-PCR in oocytes treated with 10⁻⁷, or 10⁻³ mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90aa1 expression in 10⁻⁷ mol/L melatonin-treated group than in the control (P < 0.05); the Hsf1, Hsp90aa1 and Bcl-x1 expression were significantly decreased in 10⁻³ mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10⁻⁷ or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them.

Conclusions

Our results indicate that the supplementation of melatonin (10⁻⁹ to 10⁻³ mol/L) in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.     

四川悬钩子属植物资源调查分析

研究了四川悬钩子属植物种类、分布、生态环境、生物学、繁殖特性等；已鉴定悬钩子属有28种7变种，其中光滑高梁泡、川莓、山莓、插田泡、红泡刺藤等分布广泛；繁殖方式有根蘖、种子、顶端生根和匍匐茎繁殖；并根据悬钩子的经济性状和用途进行了分类。     

在高寒地区樟子松育苗中应用外生菌根真菌的效应*

人工接种外生菌根真菌对高寒地区樟子松新播苗、移植苗的生长有明显的促进作用,苗高、地径、侧根数、干重和菌根化程度均比对照有较大提高。随着苗木生长期的延长,这种积极作用越显著。根据各项生长指标综合分析,筛选出的优良菌种为:臭红菇(Russula foetens)、牛肝菌(Boletus sp.)、血红铆钉菇(Gomphidius rutilus)。