小鼠桑椹胚简易玻璃化冷冻技术再探讨

本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）     

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane‐bound G‐coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out‐of‐season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen‐2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out‐of‐season breeding. GG and CC genotypes were having a higher prevalence in the studied population.     

电脉冲与化学激活联合处理对猪卵母细胞孤雌发育的影响

探讨了猪卵母细胞电激活后间隔不同时间用化学药物处理对猪卵母细胞孤雌发育的影响。结果表明 :1)电激活后间隔 0 ,3,4 ,5和 6h再分别用 6 二甲基氨基嘌呤 ( 6 Dimethylaminopurine ,6 DMAP)、放线菌酮 (cyclohex imide ,CHX)处理 6h ,各组卵裂率、囊胚细胞数差异不显著 (P >0 0 5 ) ,但在 6 DMAP组中对照组囊胚率极显著高于其余各组 (P <0 0 1) ,在CHX组中对照组囊胚率极显著高于间隔 5和 6h组 (P <0 0 1) ,而其余各组之间差异不显著 (P >0 0 5 ) ;2 )电激活后间隔 4h再分别用 6 DMAP ,CHX和细胞松弛素B(CytochalasinB ,CB) + 6 DMAP处理 6h ,各组卵裂率差异不显著 (P >0 0 5 ) ,对照组囊胚率极显著高于其余各组 (P <0 0 1) ,其余各组囊胚率显著性均无差异 (P >0 0 5 )。对照组中无单倍体囊胚 ,二倍体囊胚为 88 2 4 % ,但 6 DMAP ,CHX ,CB + 6 DMAP中产生的囊胚大部分是单倍体 ,且三者之间无显著性差异 (P >0 0 5 )。以上结果说明 ,猪卵母细胞电激活后间隔一段时间再用DMAP和CHX激活 ,激活作用随时间的延长而减弱 ;猪卵母细胞电激活后 4h再用 6 DMAP ,CHX ,CB + 6 DMAP激活 ,产生的囊胚大部分为单倍体 ,该方法适于猪卵母细胞ICSI的激活。     

转基因兔胚胎玻璃化冷冻保存的研究

在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。     

Structural and functional homology among chicken, duck, goose, turkey and pigeon interleukin-8 proteins

Interleukin (IL)-8-encoding regions of five avian species were cloned, sequenced and characterized. Each IL-8-encoding region is 312 nucleotides long and encodes IL-8 which is 103 amino acids. Pairwise sequence analysis showed that sequence identities of IL-8-encoding regions ranged from 87% to 100%. The IL-8 protein identities varied from 84% to 100%. Phylogenetic analysis indicated that IL-8-encoding regions and encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from binding reactivities of antiserum against each recombinant IL-8 (rIL-8) protein to homologous or heterologous rIL-8 proteins, chemotactic activities of each rIL-8 protein or reduction levels of the chemotactic activity of rIL-8 protein which was pretreated with homologous or heterlogous antiserum have suggested that all five IL-8 proteins were functionally active, and shared structural and functional identity with each other.     

Effect of a Gonadotropin-releasing Hormone Vaccine (ImprovacTM) on Steroid Hormones, Boar Taint Compounds and Performance in Entire Male Pigs

The objective of this study, comprising two trials, was to evaluate the effect of a gonadotropin‐releasing hormone (GnRH‐vaccine Improvac^TM; Pfizer Ltd) in a sample of the Swedish pig population. The pigs (n = 120) were assigned to three groups: control (entire male pigs), surgical castration and immunization against GnRH. Surgically castrated pigs did not express detectable levels of either testosterone or estrone sulphate (E1S) in plasma, or androstenone in fat and had lower skatole and indole levels in fat than entire male pigs. Immunization significantly reduced testes weight and bulbourethral gland length, plasma levels of the testicular hormones testosterone and E1S, and fat levels of androstenone, skatole and indole. Skatole levels in plasma were significantly lower than in entire male pigs in the second trial, but not in the first due to overall low skatole levels. All immunized pigs and surgically castrated pigs expressed skatole concentrations in fat below the level of 0.2 μg/g, above which meat is regarded as tainted. In contrast, eight entire male pigs exceeded this level. Indole levels in plasma from immunized pigs were lower than those from entire male pigs. Surgical castration caused lower daily weight gain in the suckling period compared with piglets raised intact, whereas in the post‐weaning period no difference was observed. Immunization resulted in higher feed intake and daily weight gain after the second injection. The estimated lean meat content was improved in comparison with the castrated pigs, but was lower than for entire male pigs. Dressing percentage was lower in immunized pigs than in surgically castrated and entire male pigs. The frequency of skin damage did not differ between immunized and entire male pigs or between immunized and surgically castrated pigs.     

玻璃化冷冻牛胚胎的分割移植实验初报

1 材料 供体牛为河北省临漳县狄丘牛场的西杂母牛和河北省邯郸市牛奶公司的中国荷斯坦母牛.受体牛为中国北方黄牛和中国荷斯坦青年母牛.促卵泡生成素(FSH)由中国科学院动物研究所产纯化FSH(批号:980001).玻璃化溶液为含40 %乙二醇、18 %聚蔗糖和0.3 mol蔗糖的mPBS液,即EFS40. 冲卵液为mPBS液.胚胎分割仪为瑞士产Leiz分割仪.     

Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005

This article reports the genetic and pathogenic characteristics of 34 isolates of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Genetic analyses showed that a unique lineage of H6N1 viruses has been established in domestic chickens in Taiwan since 1997, and this lineage of viruses differs from the H6N1 viruses circulating in Hong Kong and Southeastern China. Pathogenicity tests showed that all Taiwanese H6N1 viruses were of low pathogenicity but might lead to economic loss when associated with other diseases. Hemagglutination inhibition tests showed that antigenic drift has occurred in Taiwanese H6N1 viruses, and sequence comparison has identified a total of five possible antigenic sites on the hemagglutinin molecule of the H6N1 viruses. Some Taiwanese H6N 1 viruses could replicate in mice without preadaptation, indicating that these viruses have the potential to cause cross-species infection into mammals.     

小鼠孵化囊胚细管法和OPS法玻璃化冷冻保存技术的研究

利用小鼠孵化囊胚为模型 ,为猪孵化囊胚的冷冻保存摸索适宜的方法和条件 :在 2 5和 37℃条件下 ,用不同含量的玻璃化溶液 (EFS和EDFS) ,对小鼠孵化囊胚实施细管法和OPS法冷冻保存。在 2 5℃条件下 ,细管一步法冷冻保存后的囊胚恢复率仅为 32 5 %～ 74 4 % ,与对照组 (10 0 % )差异极显著 (P <0 0 1) ;而细管二步法冷冻组用方法C解冻后 ,孵化囊胚恢复率达 87 0 % ,为细管法最佳组 ,但仍与对照组有显著差异 (P <0 0 5 )。在 37℃条件下 ,改用OPS法冷冻后 ,以方法A脱出抗冻保护剂 ,孵化囊胚恢复率最高值为 87 2 % (P <0 0 5 )。当采用EDFS30和EDFS4 0对孵化囊胚冷冻保存后 ,以方法C脱出抗冻保护剂 ,恢复率分别高达 97 8%和 93 3% (P >0 0 5 )。利用细管法和OPS法 2种最佳冷冻 -解冻组获得的 133和 15 4枚胚胎 ,分别移植给 12和 10只假妊娠 3～4d的受体母鼠。使 2种冷冻方法均有 6只妊娠 ,各产仔 2 2和 36只 ,产仔率分别为 35 5 %和 39 1% ,与对照组(39 8% )差异均不显著 (P >0 0 5 )。证明 2种方法对胚胎冷冻后均起到了保护作用。     

An efficient method for the sanitary vitrification of bovine oocytes in straws

Background

At present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study.

Results

When in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation.

Conclusions

These results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.