H9亚型禽流感病毒钦州株HA基因的克隆与遗传进化

采集钦州活禽交易市场的鸡气管和泄殖腔的棉拭予样品,用H9亚型分型引物进行PCR初步筛选,阳性样品经SPF鸡胚尿囊腔接种分离病毒,通过RT-PCR方法扩增HA基因,并将其克隆到pMD—18T载体后进行序列测定和分析。结果表明,获得1株H9亚型禽流感病毒命名为：A／Chicken／Guangxi／qz40／2009（简称qz40）。测序结果表明qz40的HA基因片段全长1683bp,编码560个氨基酸;序列同源性比较结果表明,该毒株与参考毒株的核苷酸序列同源性为82．8％．99．9％,推导氨基酸同源性为87．5％．99．6％;HA基因的裂解位点氨基酸顺序为RSSRIGLF,为低致病性毒株;含有7个潜在的N-糖基化位点,其中5个位于HAl部分、2个位于HA2部分;基于H9亚型HA基因的进化树分析表明,qz40株属于欧亚种系的A／Chicken／Beijing／1／94（Ck／Bei—like）群系。     

貉受精的细胞学和X射线微区分析研究

应用电子显微镜和Ｘ射线微分析技术，对貉受精过程和受精卵膜元素研究的结果表明，貉卵子的卵丘细胞具有吞噬和过滤功能；卵丘细胞间的精子顶体尚未发生囊泡化，而附着于透明带的精子发生了顶体反应，并以８０°角穿入透明带，在其穿入的前方打开一个通道，最后穿过。穿过透明带的精子以赤道段或顶体后区同卵膜融合，并激发皮质颗粒释放，接着穿入卵内，最终发育成雌、雄原核。原核期受精卵质膜上具有高含量的钙，并呈集团分布，它在皮质颗粒胞吐释放中起着重要作用。     

冬季种养结合对稻田土壤微生物量及有效碳氮库的影响

以双季稻冬闲田种植绿肥与养鸡结合的新型种养制度为平台,通过冬季绿肥和鸡粪还田,减少水稻生育期化肥用量,探讨“冬季绿肥-双季稻”轮作种植制度和“冬季种养结合-双季稻”种养制度下对稻田土壤微生物碳、氮和可溶性有机碳、氮的影响。试验包括5个处理,分别为冬闲(F)、冬季种植黑麦草(R)、冬季种植紫云英(M)、冬季种植黑麦草与养鸡结合(RC)以及冬季种植紫云英与养鸡结合(MC)。结果表明,在整个试验的动态过程中,微生物量碳、氮及可溶性有机碳、氮含量高低总体趋势表现为绿肥养鸡>绿肥>冬闲,各处理间差异显著(P<0.05);“冬季种养结合-双季稻”种养制度能显著提高土壤微生物量碳、氮和可溶性有机碳、氮。各处理微生物量碳、氮和可溶性有机氮在3月24日达到最大值,RC、MC、R和M处理微生物量碳最大值分别为492.22,464.91,432.34和435.48 mg/kg;微生物量氮的最大值分别为118.20,101.03,70.13和85.46 mg/kg;可溶性有机氮的最大值分别为1001.47,926.21,832.80和870.75 mg/kg;可溶性有机碳在早稻苗期达到最大值,RC、MC、R和M最大值分别为278.95,266.40,246.13和249.84 mg/kg。微生物量碳含量的高峰在早稻移栽初期、晚稻孕穗期和灌浆期,微生物量氮的高峰出现在早稻分蘖期、齐穗灌浆期和晚稻孕穗期,可溶性有机碳在稻田养鸡及成鸡出栏后较高。与冬闲田种植绿肥和休闲相比,冬闲稻田种植绿肥结合养鸡对增加微生物生物量的贡献更大,并显著提高土壤的可溶性有机碳氮,间接反映了种养制度能提高土壤有机碳、氮的矿化速率和土壤的活性有机碳氮以及满足水稻生长期间自身养分需求。     

ORIGINAL ARTICLE: The effect of dietary methionine levels on endogenous nitrogen and endogenous amino acids flows in growing goats

The effect of dietary methionine (Met) levels on endogenous N and amino acids (AA) flows at different part of the digestive tract of growing goats was determined using a ¹⁵N isotope dilution technique. Three goats (25 ± 2.5 kg) were fitted with the ruminal, duodenal and ileal cannulae and allocated to three dietary treatments in a 3 × 3 Latin square design. The dietary treatments consisted of a total mixed ration containing three levels of Met (0.15%, 0.25% and 0.35%) respectively. It was found that at 0.15% Met level, the lowest flow in endogenous N and total AA at the duodenum and ileum occurred. The endogenous N secretion contributed to 26% and 23% of the duodenal and ileal total N flows, respectively, and the proportions were not affected by the dietary Met levels. The duodenal and ileal flows of endogenous total AA were 11.1, 11.8, 11.3 g/d and 2.9, 3.9, 4.1 g/d respectively. The average real digestibility of N was 65%, 87% and 95% in the forestomach, intestine and whole digestive tract respectively.     

绵羊体内外受精胚胎玻璃化冷冻保存

用 EFS4 0溶液对绵羊体内外受精的早期囊胚进行了玻璃化冷冻保存的研究。经卵母细胞体外成熟、体外受精和体外培养获得的早期囊胚 ,分别作如下处理 :( A) 1 0 %EG中预处理 5min后 ,在 EFS4 0平衡 3 0 s(两步法 ) ;( B) EFS4 0中平衡 1 min(一步法 ) ;( C) EFS4 0中平衡 2 min(一步法 )。然后投入液氮中冷冻保存 ,解冻胚胎的继续发育率分别为 80 %、78%和 50 %,A、B2组的结果与对照组 ( 88%)无显著性差异 ( P >0 .0 5)。经超数排卵获得的体内受精早期囊胚用两步法冷冻保存 ,解冻后胚胎发育继续率为 89%,与对照组( 93 %)也无显著性差异 ( P >0 .0 5)     

Putative human and avian risk factors for avian influenza virus infections in backyard poultry in Egypt

Highly pathogenic influenza A virus subtype H5N1 causes significant poultry mortality in the six countries where it is endemic and can also infect humans. Egypt has reported the third highest number of poultry outbreaks (n = 1084) globally. The objective of this cross-sectional study was to identify putative risk factors for H5N1 infections in backyard poultry in 16 villages in Damietta, El Gharbia, Fayoum, and Menofia governorates from 2010–2012. Cloacal and tracheal swabs and serum samples from domestic (n = 1242) and wild birds (n = 807) were tested for H5N1 via RT-PCR and hemagglutination inhibition, respectively. We measured poultry rearing practices with questionnaires (n = 306 households) and contact rates among domestic and wild bird species with scan sampling. Domestic birds (chickens, ducks, and geese, n = 51) in three governorates tested positive for H5N1 by PCR or serology. A regression model identified a significant correlation between H5N1 in poultry and the practice of disposing of dead poultry and poultry feces in the garbage (F = 15.7, p < 0.0001). In addition, contact between domestic and wild birds was more frequent in villages where we detected H5N1 in backyard flocks (F = 29.5, p < 0.0001).     

Establishment of Digital RT-PCR Assay for Detection of Swine Influenza Virus H3N2 Subtype

A new assay for the detection of swine influenza virus (SIV) was developed with a novel nucleic acid probe——Molecular beacon in this study. The specific primers and molecular beacon probes were designed according to the conserved region of H3 and N2 genes of SIV H3N2 subtype. A digital RT-PCR assay was developed for detection of SIV H3N2 subtype. The results showed that SIV H3N2 subtype could be identified simultaneously on this microarry with high sensitivity and reproducibility,which could reach to 10⁶ dilute viruse. The conclusion was that the digital RT-PCR method could analyze quantitatively the RNA templates.On the identification of H3N2 SIV,the digital RT-PCR method was much more scientific than Real-time quantitative PCR method.     

中草药复方添加剂对繁殖后期种公鸡精液品质及种蛋受精率的影响

选用80只50周龄尼克T种公鸡随机分成Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组,每组16只。Ⅰ、Ⅱ组为对照组,分别饲喂基础日粮和基础日粮添加鸡蛋,Ⅲ、Ⅳ、Ⅴ组为试验组,分别饲喂基础日粮中添加1%、2%、3%中草药复方添加剂的日粮,研究中草药复方添加剂对繁殖后期种公鸡精液品质及种蛋受精率的影响。结果表明:日粮中添加中草药复方添加剂能显著提高种公鸡的精液品质和精子活力,降低精子畸形率,并且能提高种蛋受精率和健雏率,其中2%添加组效果最明显。     

甲型H1N1流感病毒NS1蛋白核仁定位的研究

为研究2009年甲型H1N1流感病毒的NS1蛋白的核仁定位情况,采用RT-PCR对其NS1基因进行了扩增,将其克隆至PEGX-KG载体,构建重组质粒KG-NS1,转化大肠杆菌BL21,IPTG诱导表达重组蛋白.然后采用GST柱亲和层析方法纯化NS1重组蛋白,免疫家兔来制备多抗,Western blot检测抗体.通过间接免疫荧光对表达不同长度NS1 (NS1-219、NS1-230、NS1-237)的3种重组流感病毒进行了核仁定位的研究,3种重组毒的NS1蛋白存在于细胞核和细胞质,但都不能定位于核仁,说明NS1蛋白的截短与否并不影响其核仁定位,其生物学意义有待于进一步研究.     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.