鸡精原细胞分离纯化与体外培养初探

本研究以组合酶分步消化法并利用Percoll梯度分离、贴壁纯化的方法，从不同时期的鸡睾丸组织中分离精原细胞。结果发现：①Percoll分离后，出壳6d雏鸡获得的细胞总数、精原细胞总数、精原细胞纯度分别为282．1、174．6、61．9％，其中精原细胞纯度比孵化15d、19d鸡胚和出壳后13d雏鸡分别高10．2％、5．7％和2．5％；②贴壁纯化后精原细胞纯度达到82％，比未经贴壁纯化的精原细胞纯度高出15．6％；③在无饲养层培养条件下，比较Percoll梯度分离前、后鸡精原细胞体外培养的情况，Percoll梯度分离前精原细胞贴壁在时间上比分离后较快，且分离前精原细胞体外存活时间亦比分离后长。     

产角蛋白酶菌株D5培养条件的研究

为确定产角蛋白酶菌株D5的最佳培养条件，试验通过测定不同影响因素条件下角蛋白酶的酶活，确定产角蛋白酶菌株D5的最佳培养基为：新鲜鸡羽毛粉2．00％，酵母膏2．00％，磷酸二氢钾0．05％，硫酸铵0．05％，蛋白胨2．00％，硫酸镁0．04％，氯化锌0．01％；最适发酵条件为：温度37℃，pH6．5，装量80mL／250mL锥形瓶，转速150r／min，培养时间3d。     

鸡胚胎干细胞分离培养和单细胞克隆的制备

从鸡第X期胚胎中分离胚盘,以鸡成纤维细胞为饲养层,用添加了10%的胎牛血清、2％的鸡血清、2mmol／L L-谷氨酰胺、1mmol／L丙酮酸钠、5．5×10^-5mmol／L β-巯基乙醇、10μL／mL非必需氨基酸、以及含1000U／mL LIF、10ng／mL bFGF和5ng／mL SCF的高糖DMEM对细胞进行培养和传代。利用鸡第X期的胚胎干细胞,采用口吸管法、细胞稀释法和克隆环法3种方法,制备单细胞克隆,采用细胞化学法和免疫荧光法检测细胞表面标志物。结果显示,可获得传至5～6代的鸡胚胎干细胞。通过对传代培养后的鸡ES细胞进行AKP染色鉴定和SSEA-1的鉴定,证实细胞未发生分化,具有胚胎干细胞的特征。通过不同分离胚盘方法和不同消化时间的比较得出药勺法提取胚盘简单易行,原代消化5～8min的ES细胞适合于传代培养。口吸管法、细胞稀释法和克隆环法单细胞克隆形成率分别为0、4.2％和1．0％。经AKP活性和SSEA-1免疫荧光鉴定均呈阳性,扩增出的克隆能稳定增殖不分化。结果表明,细胞稀释法操作简单易行,试验时间短,对细胞伤害小,为鸡ES单细胞克隆进一步建系提供了可行的方法。     

机械破碎法分离奶牛乳腺上皮细胞的体外培养研究

采用机械破碎法分离奶牛乳腺上皮细胞 ,并对分离细胞 (试验组 )和未分离的乳腺组织块 (对照组 )进行体外培养 ,研究乳腺上皮细胞体外培养的效果。培养液由DMEM /F1 2 加 2 0 %小牛血清组成 ,培养条件为 5 %CO2 、 37℃。机械破碎法分离的细胞在培养 2d开始生长增殖 ,7d基本长满培养板底部 ;而培养的乳腺组织块细胞到 5d后才呈现旺盛生长 ,长满培养板底部需 1 4～ 1 5d。结果表明 ,机械破碎法是分离乳腺上皮细胞的一种有效方法     

转化型人参皂甙的生物活性:体外实验研究

将人参总皂甙经化学转化成转化型人参皂甙后分离得8个组分，在体外细胞上进行了转化型人参皂甙8个组分对CEF细胞毒性、生长活性、诱导肿瘤细胞凋亡、诱导单克隆抗体生成以及抗IBDV、AIV（H9）、NDV、MDV病毒的活性研究。结果发现，转化型人参皂甙各组分的细胞毒性作用在23．26～39．53mg／L之间；对鸡胚成纤维细胞（CEF）的生长和活性呈促进作用的工作浓度在10～20mg／L之间；转化型人参皂甙8号组分12．5mg／L诱导48h后，肝癌细胞7402株有60％的细胞凋亡，大鼠脑胶质瘤细胞C6株有35％的细胞凋亡；3、4、7号组分12．5mg／L和6．25mg／L对单克隆抗体6E6株杂交瘤细胞和单克隆抗体10B11株杂交瘤细胞的单抗生成具有明显促进作用，其HI效价为2^6～7与对照组的2^5相比有明显差异，IFA效价为10^4～5与对照组的10^2相比有极显著差异；4、7、8号组分15mg／L阻斯IBDV、AIV（H9）、NDV感染CEF细胞的作用都达到了51．1％以上，抑制IBDV、AIV（H9）、NDV在CEF上增殖的作用都在30％以上；6、7号组分12．5mg／L在阻断感染、抑制增殖、直接杀灭3种方式的抗MDV—RB1B强毒株在SPF鸡CEF细胞上的空斑减数率都达了到ACV抗MDV空斑减数率的80％，其中4、6号组分对MDV—RBIB株的直接杀灭作用与ACV的杀灭作用相等。表明人参总皂甙经化学处理转变成转化型人参皂甙，可明显改变其生物学活性。     

HPLC法测定饲料中喹乙醇的研究

建立了饲料中喹乙醇含量的HPLC测定法，流动相为15％甲醇，流速0．9mL／min，进样量5μL，检测波长260nm。在10～100μg／mL范围内，喹乙醇浓度与峰面积呈良好的线性关系，r为0．99998，回收率85．8％～99．4％，批内变异系数≤5．7％，批间变异系数≤1．1％。24批鸡配合料中未检出喹乙醇，62批鱼饲料中喹乙醇的检出率为8．1％，含量范围为19．7～42．0mg／kg。该方法具有很好的分离度，方法简便、准确、重复性好。     

高寒牧草内生细菌分离培养条件的优化

通过对高寒牧草内生细菌的分离培养方法研究,得出了优化的分离培养条件,即牧草不同组织器官用0．19／6SDS浸泡15min、3％NaClO浸泡3min、0．1％升汞浸泡10min、75％酒精1～2min处理后（各步间均用无菌水冲洗3～4次）,研磨并稀释至10^-3,涂布于TSA培养基上,置28℃恒温条件下培养5—7d,可从高寒牧草组织中分离获得数量、种类较多的内生细菌。用此方法,已从5种牧草不同组织器官（根、茎、叶、花）中分离获得大小、形态、颜色各异的内生细菌315株。     

HPLC法测定饲料中喹乙醇残留及应用HPLC法测定饲料中喹乙醇残留及应用

HPLC法测定饲料中喹乙醇的残留，具有很好的分离度，浓度10～100μg／mL范围呈良好的线性关系，R=0．99998，回收率范围为85．8％～99．4％，批内变异系数≤5．7％，批间变异系数≤1．1％。实践中检测24批鸡配合饲料，喹乙醇残留未检出，检测62批鱼饲料，喹乙醇残留检出率为8．1％，检出的样品中，残留量范围为19．7～42．0mg／k。表明鱼饲料中存在喹乙醇残留隐患。     

鲁西黄牛体细胞克隆保种技术的研究

以体外培养的鲁西黄牛耳皮肤成纤维细胞作核供体，研究利用体细胞克隆技术保存我国地方黄牛优良品种的技术方法。通过组织块培养法建立的鲁西黄牛（1♂，4♀）成纤维细胞系，作为核移植供体细胞，利用屠宰场母牛卵巢卵母细胞经体外培养成熟后作为核受体进行核移植，试验结果表明：重构胚的融合率为62．5％（242／387），分裂率为63．6％（154／242），体外培养第7天囊胚发育率为42．9％（66／154）。体外培养第7天的囊胚的内细胞团细胞（ICM）与滋养层细胞数平均为37和47，ICM占44．2％。体外发育到7d的囊胚新鲜胚胎的移植妊娠率（新鲜胚胎）移植受体10头，60d妊娠率为20％（2／10）。     

鸡肠上皮细胞原代培养方法的改进研究

鸡肠上皮细胞(Intestinal epithelial cell,IEC)既是消化吸收的功能性细胞又是肠道免疫的天然屏障,本研究旨在探讨IEC的分离方法和改进其体外培养条件.试验选取20日龄鸡胚,分离肠组织后利用刮取法获得了肠绒毛及隐窝单位,在体外与成纤维细胞共同培养最终获得了活性较高的肠上皮细胞.对所分离细胞进行形态学观察和生化指标的检测,鉴定为IEC.结果表明,共培养方法可以使体外IEC的培养时间延长,可有效满足实验需要,为长时间培养IEC提供了新的方法.     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

A brief history of the prevention of infectious diseases by immunisations

Infectious diseases have always been a terrible scourge for humans. The appearance of these plagues, as they were called without distinction, was generally connected to various conditions: asters, climatic changes or religious reasons. The concept of contagious, and then infectious, diseases came slowly. Variolation, i.e. transmission of ‘virulent’ matter to induce a natural disease and the immunity against it, was brought from Constantinople to England by Lady Montague, in 1721. This ‘variolation’ technique was also often performed in veterinary medicine against diseases like sheep-pox or pleuropneumonia. As ‘vaccination’ is the term generally accepted for ‘immunisation’, variolation can be the word designating such a technique. The second period of the history of immunisation began, in 1880, with the studies of Pasteur and his collaborators. A great number of bacterial vaccines were developed: dead, live but attenuated or only parts of pathogens. The viruses were produced in animals, then in eggs and at last, in tissue cultures. Second generation vaccines appeared with genetic engineering: recombinant vaccines, vector vaccines, nucleic acids vaccines, and markers vaccines, among others. These novel technologies can permit the development of new ones and improve the quality of the vaccines already existing.     

Micronutrient Content of Certain Tropical Conventional and Unconventional Feed Resources of Southern India

The ash, silica and certain important micronutrients were estimated in conventional and unconventional feed and fodder resources available in Southern India. Commonly used dry roughages, such as paddy straw, ragi straw, maize kadbi/stalk, jowar kadbi/stalk, bajra stalk and wheat straw, were high in ash (9.9% +/- 0.77%) and silica (6.4% +/- 0.65%) and low in most of the other micronutrients, except iron, with paddy straw containing most silica (>9%). Cultivated non-leguminous (maize, jowar) and improved green crops (hybrid napier, guinea, green panic, NB-21, CO-1) were also high in ash (10.5% +/- 0.60% and 12.5% +/- 0.51%) but were moderate sources of P, Mg and Cu and good sources of Zn (98 +/- 13.8 ppm and 55 +/- 6.7 ppm). Leguminous green fodders (stylosanthus, lucerne, cow pea, soyabean) were excellent sources of Ca (1.9% +/- 0.16%), Mg (0.40% +/- 0.05%), Cu (30 +/- 5.2 ppm), Zn (121 +/- 14.7 ppm) and Fe (1234 +/- 166 ppm) and moderate sources of P. Mixed local grasses and weeds were high in silica (6.9% +/- 1.00%) but were good sources of Cu, Zn and Fe. Cereal grains (maize, wheat, rice, ragi) were low in ash (2.9% +/- 0.33%) and were relatively poor sources of Ca (0.22% +/- 0.03%), Mg (0.19% +/- 0.03%) and Cu (13 +/- 3.1 ppm). Pulses were low to medium sources of most minerals and good sources of Fe (1230 +/- 293 ppm). Oil seed cake/extractions (groundnut cake, cotton seed cake, soyabean meal, sunflower cake, safflower cake) and cereal by-products (rice polish, rice bran, wheat bran) were excellent sources of P (1.1% +/- 0.47% and 2.3% +/- 0.19%) and good sources of Zn (65 +/- 3.9 ppm and 66 +/- 10.7 ppm) and Fe (938 +/- 130 ppm and 662 +/- 126 ppm). Among the unconventional feeds screened, orange peel, sunflower heads, meat meal, rubber seed cake, spirulina algae and sea weeds contained plentiful Ca, Zn and Fe: tree leaves/top feeds (mulberry, erythrina, glyricidia, banana, subabul, groundnut haulms) were excellent sources of Ca (1.5% +/- 0.13%), Zn (120 +/- 22.9 ppm) and Fe (1033 +/- 133 ppm) but relatively poor sources of P. Soyabean husk, cocoa seed husk, rubber seed cake and meat meal were moderate to good sources of P (1.0% and 0.33%). The high Zn and Fe values of most feeds/fodders were probably due to soil contamination. This account of the micronutrient content of feed/fodder resources should help in strategic supplementation intended to alleviate local deficiencies.     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Bovine Mastitis in Selected Areas of Southern Ethiopia

A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia. A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian, were examined by clinical examination and the California mastitis (CMT) test. Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively. Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.2%) clinically and 129 (60.8%) subclinically. The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season. Mastitis increased with parity number (R = 0.9). Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative. Of the 199 positive samples, Staphylococcus accounted for 39.2%, Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0%. It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.     

不同日粮氮水平对山羊氮代谢和微生物蛋白质合成的影响

本试验旨在探讨不同日粮氮水平对内蒙古白绒山羊氮代谢和微生物蛋白质合成(MCP)的影响,以达到提高氮利用率,减少环境污染和饲料资源的浪费。试验选用9只体况良好、体重为(43.83±2.95)kg,装有瘤胃瘘管的内蒙古白绒山羊,按体重随机分为3组,每组3只。日粮分为低氮7.5%、中氮10.5%、高氮13.5%3个氮水平。试验使用常规试验方法分析测定粪尿血液等生化指标,通过全收尿法和尿嘌呤衍生物法估测MCP。结果表明:随着氮水平增加,氨氮(NH3-N)浓度、氮摄入量、尿氮排出、尿中尿素氮(UUN)和MCP显著增加(P0.05),干物质采食量(DMI)显著降低(P0.01);沉积氮占总摄入氮的比例在中氮组最高,MCP在高氮组最高。综上所述,适当降低日粮氮水平可减少粪尿氮排放,提高反刍动物氮利用率。     

Microsporidial keratopathy in two dogs

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin‐fixed, paraffin‐embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow‐up are discussed.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.