曲古抑菌素(Trichostatin)A对猪卵母细胞体外成熟及孤雌胚胎发育能力的影响

曲古抑菌素A（Trichostatin A,TSA）是一种组蛋白去乙酰化抑制剂,用TSA处理鼠核移植胚胎可显著提高胚胎的囊胚率。检验TSA对猪卵母细胞体外成熟以及孤雌胚胎发育的影响。在猪卵母细胞体外成熟液及胚胎培养液中添加TSA,比较不同浓度TSA对卵母细胞成熟的影响,不同浓度TSA对孤雌胚胎发育能力的影响以及TSA处理不同时间对孤雌胚胎发育能力的影响。结果发现：（1）5nmol／LTSA处理对卵母细胞体外核成熟无显著影响,却显著提高了卵母细胞孤雌胚胎的卵裂率和囊胚率（P〈0．05）;（2）50nmol／LTSA处理显著提高了孤雌胚胎的卵裂率及囊胚率（P〈0．05）;（3）50nmol／LTSA处理24h能显著提高胚胎的卵裂率及囊胚率（P〈0．05,82．1％±2．6％和37．4％±3．1％）。结果表明TSA对猪卵母细胞的体外成熟及孤雌胚胎发育具有显著的促进作用。5nmol／L的添加量对卵母细胞的体外胞质成熟具有促进作用;胚胎培养基中添加50nmol／LTSA处理24h能提高孤雌胚胎的发育能力。     

家兔胚胎细胞核移植与连续移植

将发育不同时期的兔胚和移核胚的卵裂球细胞核与去核的成熟卵母细胞共同组成移核胚,通过中间受体培养和移植实验检验胚胎的发育能力。结果表明,（１）兔囊胚之前各个时期的胚胎细胞核均可使移核胚发育到囊胚;（２）胚胎极化前后的卵裂球参与组成的移核胚发育到囊胚的比例无显著差异。但极化后 ６４细胞胚的卵裂球与去核卵母细胞的融合率低于极化前的 ８细胞胚胎卵裂球;（３）兔 １６细胞胚与去核的卵母细胞组成的移核胚可以发育到期,产仔率为 ３．１６％ ;（４）兔移核胚卵裂球用于连续核移植,其后 ２ 代均可发育成囊胚,其中第 １ 代移核胚与第 ２ 代移核胚发育率相似,但显著高于第 ３ 代移核胚;（５）兔移核胚和各代连续移核胚卵裂球与去核卵的融合率无显著差异。     

山羊毛囊干细胞分离、培养及鉴定

采用2．4U／mLDispase酶消化和机械切割法分离毛囊隆突部,经胰酶（0．5mg／mL胰酶＋0．2mg／mLEDTA）消化从山羊耳部皮肤分离得到毛囊干细胞,用自制无血清培养基培养,并用形态学观察、细胞生长曲线、克隆形成率及免疫组化染色检测,探讨毛囊干细胞体外分离培养方法及生物学特性。结果表明,所分离细胞具备毛囊干细胞特征：体积较小、表面光滑、立体感强,呈现未分化细胞特征;K19、integrin-β1以及p63免疫组化染色阳性;第3、5、7代干细胞克隆形成率分别为25．6％±2．6％、31．8％±1．9％和27．0％±3．9％,极显著高于对照组角质细胞克隆形成率（P〈0．01）。采用配制的无血清条件培养液可使山羊毛囊干细胞体外维持未分化状态并传代至19代。     

复方连翘有效成分在鸡体内的药代动力学研究

以岭南黄鸡为实验动物,利用RT-HPLC法为定量手段,研究中药复方连翘制剂以20g／kg（其中黄芩苷和绿原酸含量分别为158和53mg／kg）单剂量经胃灌服给药后在鸡体内的药物代谢动力学特征,用3p97药动学软件的非房室模型统计矩原理分析药物动力学参数,有效药物成分绿原酸、黄芩苷在鸡体内的主要药动学参数如下：AUC分别为（10．59±0．69）和（11．15±0．78）（mg／L）·h,AUMC分别是（106．06±3．11）和（138．44±2．86）（mg／L）·h^2,MRT分别是（10．01±0．68）和（12．42±0．43）h,t1／2分别为（6．93±0．18）和（8．61±0．36）h,Cmax分别为（0．98±0．25）和（1．04±0．25）mg／L,tmax分别为（1．93±0．15）和（2．45±0．19）h。结果表明2种有效成分在鸡体内血药浓度的变化表征了中药复方连翘在鸡体内代谢的变化规律,血药浓度一时问关系曲线均出现双峰现象,表示可能存在肝肠循环或胃肠循环,本研究为该复方制剂的临床应用提供了参考依据。     

发醇床猪含和传统猪含H2S和NH3浓度的比较研究

本试验分别对发酵床猪舍和传统猪舍的H2S和NH3浓度进行测定。上午8时发酵床猪舍H2S（mg/m3）浓度为：保育舍（3．14＋0．32）、母猪舍（2．25±0．47）、育肥合（2．31±0．21）,NH3（mg/3）浓度为：保育舍（2．54±0．62）、母猪舍（4．61±0．94）、育肥舍（1．24±0．75）;传统猪舍H2S（mg/m3）浓度为：保育舍（5．42±1．63）、母猪舍（6．41±1．73）、育肥舍（7．92±1．46）,NH3（mg／m3）浓度为：保育舍（4．84±0．87）、母猪舍（7．42±1．15）、育肥舍（8．26±1．35）。试验结果表明,发酵床猪舍H2S和NH3浓度显著低于传统猪舍。     

不同处理牛卵丘/颗粒细胞作为核供体对克隆胚发育的影响

本研究利用TSA（Trichostatin A）、ROS（Roscovitine）、血清饥饿和接触抑制方法处理牛卵丘/颗粒细胞,检测处理前后细胞乙酰化水平、周期分布及其对重组胚发育能力的影响,为提高核移植效率提供理论基础。分别采用上述方法处理牛卵丘/颗粒细胞,首先对处理的细胞形态及活率进行观察,并利用流式分选技术检测处理细胞的周期分布,再通过间接免疫荧光法检测经上述不同处理前后细胞乙酰化水平变化,最后以上述处理细胞作为核供体构建重组胚,比较重组胚发育水平的不同。试验结果显示,经上述处理的卵丘/颗粒细胞形态良好,活率均保持在94%以上;TSA处理卵丘/颗粒细胞后,其乙酰化水平较对照组和其他处理组明显增加（P〈0.05）,经ROS处理的卵丘/颗粒细胞乙酰化表达水平同样高于对照组（P〈0.05）,但仍低于TSA处理组（P〈0.05）,但经血清饥饿处理的卵丘/颗粒细胞乙酰化水平较对照组明显降低（P〈0.05）;利用TSA处理卵丘/颗粒细胞24 h后,细胞被明显抑制在G0/G1期,且较其他处理组的抑制现象更加明显（P〈0.05）;并且,经TSA处理后的卵丘/颗粒细胞充当供体细胞,其卵裂率和囊胚率较对照组明显增加（P〈0.01,（85.2±3.4）%vs（68.6±6.7）%;（30.2±5.7）%vs（10.4±8.3）%）。经TSA处理的牛卵丘/颗粒细胞,与其他处理组相比,乙酰化水平较高,细胞形态良好,细胞周期被明显抑制在G0/G1期,且获得了较高的卵裂率和囊胚率,作为供体细胞的处理方式较为适合。     

貉按摩法采精技术的研究

试验成功地进行了貉按摩法采精与精液评定,并初步建立了貉按摩法采精技术。采精成功率为27．9％,每次平均射精量为（0．29±0．18）mL,pH值为6．64±0．19,密度为（0．99±0．83）×10^8个／mL,活力为0．81±0．71,畸形率为16．41％±2．20％,活精子率为85．11％±8．30％。     

体细胞核移植生产转ω-3脂肪酸去饱和酶基因(sFat-1)的猪胚胎

本研究通过脂质体介导的方法将来源于线虫C.Briggsae的ω-3脂肪酸去饱和酶基因（sFat-1）转染至大白猪胎儿成纤维细胞。采用600μg·mL^-1G418药物浓度,经连续10 d筛选及PCR、RT-PCR鉴定,获得11个转基因阳性细胞克隆。以体外成熟42 h的猪卵母细胞与转基因细胞构建重构胚。经体外培养后观察,转基因克隆胚胎与非转基因胚胎的卵裂率（76.6%±4.1%vs.81.6%±3.1%）和囊胚率（10%±1.97%vs.9.7%±1.4%）均无显著差异（P〉0.05）。采用放线菌酮进行化学二次激活时,胚胎的囊胚率显著高于采用电二次激活胚胎（20.6%±0.89%vs.10%±1.97%,P〈0.05）,但二者的卵裂率并无显著差异（72.4%±4.96%vs.76.6%±4.1%,P〉0.05）。研究表明,通过脂质体介导的方法,可以获得转sFat-1基因大白猪胎儿成纤维细胞系;以该细胞系为核供体构建的转基因克隆胚胎与非转基因克隆胚胎的发育能力无显著差异;二次激活采用放线菌酮进行化学激活能够显著提高胚胎的囊胚发育率。     

细胞骨架抑制剂、电激活液对兔卵孤雌激活和发育的影响

应用细胞骨架抑制剂细胞松弛素B和秋水仙素处理兔成熟卵母细胞，然后采用电激活的方法激活兔卵母细胞，观察细胞骨架抑制剂对兔卵母细胞孤雌激活和孤雌发育的影响，结果表明：应用不同的电激活液，即甘露醇、Zimmerman氏和山梨醇对兔卵母细胞的孤雌激活效果无显著性差异（P〉0．05）；用7．5μg·mL^-1细胞松弛素B处理卵母细胞后孤雌激活，其2-细胞胚率（82．2％）和囊胚发育率（43．4％）与对照组（76．4％和51．5％）相比无显著性差异（P〉0．05）；用1μg·mL^-1秋水仙素处理兔卵母细胞，兔卵母细胞孤雌激活后的2-细胞胚率（64．9％）和囊胚发育率（23．8％）明显下降，与对照组相比存在显著性差异（P〈0．05）；用细胞松弛素B和秋水仙素共同处理的2-细胞胚率（62．5％）和囊胚发育率（30．9％）与秋水仙素单独处理的结果无显著性差异（P〉0．05）。因此，秋水仙素对兔卵母细胞的孤雌激活影响比CB更大。通过免疫荧光和激光扫描共聚焦显微镜观察发现，细胞骨架抑制剂处理后，经孤雌激活获得的囊胚中，微丝和微管结构未见异常。     

波兰猪IGF2基因内含子3上的已知突变A3072G与生长性能和胴体组成的相关性分析

胰岛素样生长因子2（IGF2）是控制肌肉发育的基因之一,属于父源表达的印迹基因,即仅来源于父系的等位基因可在后代中表达。本试验应用实时荧光定量PCR对IGF2进行了基因分型,评估了波兰地方猪（PL,n=271）和大白猪（LW,n=267）的IGF2基因A3072G位点的突变频率。试验结果显示,A等位基因常见于瘦肉率高的品种（LW、PL中等位基因频率分别为0.79、0.69）,这与先前的报道相一致。此外,本研究还对IGF2基因A/A和G/G基因型猪的体成分、生长性能和肉质性状进行了比较。结果表明,A等位基因能增加腰肌重（LW、PL的加性效应分别为450 g±50 g、213 g±64 g）、大腿重（LW、PL的加性效应分别为544 g±48 g、302 g±72 g）、眼肌面积（LW、PL的加性效应分别为4.9 cm2±0.46 cm2、2.1 cm2±0.95 cm2）和胴体产肉率（LW、PL的加性效应分别为3.12%±0.27%、1.89%±0.47%）;减少平均背膘厚（LW、PL的加性效应分别为-0.2 cm±0.048 cm、-0.2 cm±0.036 cm）。除此之外,在PL中,A等位基因还会增加里脊肉重（11g±0．01g）、日增重（30．7g±17．29g）；减少饲料摄入量（-121g±45g）和饲喂天数（-3．5d±2．08d），但对肉质性状无显著影响。因此，IGF2基因的突变在PL和LW中是非常有用的，其中G等位基因出现频率仍相对较高。     

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.     

Echinococcus ortleppi and E. granulosus G1, G2 and G3 genotypes in Italian bovines

To increase the knowledge on Echinococcus genotypes infesting cattle and water buffaloes (Bubalus bubalis) born and bred in Italy, the germinal layer of hydatid cysts was collected from the liver and the lungs of 80 animals slaughtered in 2007. Two mitochondrial genes (the cytochrome c oxidase subunit I and the NADH subunit I) were tested by PCR. Four genotypes were identified: G1 (sheep strain), G2 (Tasmanian sheep strain), G3 (buffalo strain), and G5 (cattle strain). Fertile cysts were detected only in the lungs of 4.5% of the total G1 lung cysts, of 9.4% of the total G3 lung cysts, and in the only G5 infected animal. This is the first report of Echinococcus ortleppi (genotype G5) in Italy.     

Full-length genome analysis of G2, G9 and G11 porcine group A rotaviruses

Group A rotaviruses with G2 and G9 VP7 specificity are common in humans, while G11 strains have been detected only sporadically. G2, G9 and G11 rotaviruses also circulate in pigs and swine rotaviruses have been suspected of interspecies and zoonotic transmissions in numerous studies. However, the complete gene constellation of G2 and G9 porcine rotaviruses has not yet been determined. In order to start filling this gap, the genomic make up of two G2, one G9 and one G11 porcine rotavirus strains, detected in Canada in 2005–2007, was determined. With the exception of a G2P[34] strain, with E9 NSP4 type and mixed I5 + I14 VP6 type, the constellation of genomic segments was rather conserved and were closely related to prototype porcine strains in the four viruses characterized (I5-R1-C1-M1-A8-N1-T7-E1-H1). Most notably, all the viruses displayed a rare NSP3 genotype, T7, which has also been identified in rare human reassortant strains and in the reference strain RVA/Cow-tc/GBR/UK/1973/G6P[5]. This study provides crucial genetic data on these complex viruses and will help understand the origin and ecological niche of gene segments and the role played by pigs in their evolution.     

Pharmacokinetics of penicillin G procaine versus penicillin G potassium and procaine hydrochloride in horses

OBJECTIVE: To compare the pharmacokinetics of penicillin G and procaine in racehorses following i.m. administration of penicillin G procaine (PGP) with pharmacokinetics following i.m. administration of penicillin G potassium and procaine hydrochloride (PH). ANIMALS: 6 healthy adult mares. PROCEDURE: Horses were treated with PGP (22,000 units of penicillin G/kg of body weight, i.m.) and with penicillin G potassium (22,000 U/kg, i.m.) and PH (1.55 mg/kg, i.m.). A minimum of 3 weeks was allowed to elapse between drug treatments. Plasma and urine penicillin G and procaine concentrations were measured by use of high-pressure liquid chromatography. RESULTS: Median elimination phase half-lives of penicillin G were 24.7 and 12.9 hours, respectively, after administration of PGP and penicillin G potassium. Plasma penicillin G concentration 24 hours after administration of penicillin G potassium and PH was not significantly different from concentration 24 hours after administration of PGP. Median elimination phase half-life of procaine following administration of PGP (15.6 hours) was significantly longer than value obtained after administration of penicillin G potassium and PH (1 hour). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that i.m. administration of penicillin G potassium will result in plasma penicillin G concentrations for 24 hours after drug administration comparable to those obtained with administration of PGP Clearance of procaine from plasma following administration of penicillin G potassium and PH was rapid, compared with clearance following administration of PGP.     

Peptide mapping of ovoglobulins G2A and G2B in the domestic fowl

1. The purification of the two principal genetic variants of ovoglobulin G2 from egg‐whites is described.

2. Both ovoglobulin G2A and G2B had molecular weights of 47 ±2 kDa. They differed in their mobilities in non‐denaturing gels and in their isoelectric points, which were 4.9 and 5.3 for G2A and G2B respectively.

3. Peptide mapping using either chymotrypsin or V8 digestion revealed additional proteinase sensitive sites in ovoglobulin G2A. The results are consistent with one of the differences between the two ovoglobulins being the replacement of an acidic amino acid residue in G2A by a neutral residue in G2B.     

Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec.

Fecal samples from diarrheic and nondiarrheic dairy calves (1 to 3 weeks old) from 12 regions of Quebec, collected between 1992 and 1994, were screened for group A bovine rotavirus (BRV) using a combination of 2 VP6-specific monoclonal antibodies (MAbs) in an enzyme-linked immunosorbent assay (ELISA). The overall prevalence of BRV infection was 26.4% (107/405). In diarrheic calves, BRV infection reached 74.3% (55/74), but only 15.7% (52/331) in nondiarrheic calves. BRV-positive samples were serotyped by enzyme-linked immunosorbent assay (ELISA) using G6 and G10 specific MAbs. The analysis of 107 field samples revealed that, in diarrheic calves, 34.5% (19/55) were G6, 27.2% (15/55) were G10, 9% (5/55) were G6 and G10 positive, and 29.9% (16/55) were G6 and G10 negative. In nondiarrheic calves, 19.2% (10/52) were G6, 19.2% (10/52) were G10, 7.6% (4/52) were G6 and G10 positive, and 53.6% (28/52) were G6 and G10 negative. Rotavirus dsRNA was extracted from BRV-positive samples and examined by polyacrilamide gel electrophoresis (PAGE). Of 107 samples tested, 74 (69.1%) were positive, and all the samples demonstrated a typical group A rotavirus migration pattern.     

Pharmacokinetics of penicillin G in the turkey

Parmacokinetics of penicillin G was determined for the turkey. The study was prompted by the isolation of a sulfonamide-resistant strain of Pasteurella multocida from tissues of turkeys involved in an outbreak of fowl cholera and the subsequent discovery that little pharmacologic information was available concerning other antimicrobial agents in that species. Penicillin G was chosen for study because P multocida is susceptible to this antibiotic. The elimination of the antibiotic followed first-order kinetics, and the half-life was found to be 0.5 hours. Parenteral administration of benzathine-procaine penicillin G resulted in higher concentrations, which persisted for longer periods than did procaine or potassium salts of the antibiotic.     

Benzathine Penicillin G and Procaine Penicillin G in Piglets: Comparison of Intramuscular and Subcutaneous Injection

The disposition of penicillin G in piglets is described after intramuscular or subcutaneous injection of depot preparations. The piglets were injected with 33 000 IU/kg or 100 000 IU/kg benzathine + procaine penicillin G intramuscularly or subcutaneously, or 100 000 IU/kg procaine penicillin G intramuscularly or subcutaneously. Intramuscular injection of benzathine + procaine penicillin resulted in higher maximum concentration in plasma (C_max) than did subcutaneous injection. The mean residence time (MRT) of penicillin G was longer when the drugs were injected subcutaneously rather than intramuscularly. The plasma concentration versus time profiles of the subcutaneous injections of benzathine + procaine penicillin revealed secondary peaks, possibly reflecting a certain degree of inflammation at the injection site.     

Clearance of penicillin G in the newborn calf

Sodium penicillin G was administered intravenously (4545 IU/kg) to calves on the day of birth (12-24 h old) and at 5, 10, and 15 days of age. Serum was collected at varying intervals for 120 min after injection and analysed for penicillin G. The mean total body clearance (ClB) of penicillin G on the day of birth was 2.98 ml/min/kg compared to 4.83 ml/min/kg at 5 days, 3.11 ml/min/kg at 10 days and 4.65 ml/min/kg at 15 days of age. Clearances at 5 and 15 days were significantly (P less than or equal to 0.05) higher than on the day of birth. The half-life (t1/2 beta), however, did not change significantly over the 15-day period of the study. These results indicate that the newborn calf has an appreciable ability to excrete penicillin G before it is 24 h old, and that total body clearance of the antibiotic increases rapidly in the immediate postnatal period.