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1.
采集25~28日龄的五指山小型猪(WZSP)胚胎原始生殖嵴细胞(PGCs),用于 WZSP生殖细胞(EG)建系培养技术研究。研究发现:以DMEM含15%胎牛血清、0 1 mol/L巯基乙醇等为基础培养液,分别添加生长因子20 ng/mL LIF、40 ng/mL hSCF、20 ng/mL hbFGF,以STO为饲养层,进行 EG培养建系,WZSP 3 d 可出现克隆,其克隆具有出现早,生长数量多,增殖快,成熟亦早,传代率高的特点。培养 7~10 d需传代或冷冻保存。EG类细胞冷冻、解冻后尚可复苏,并传至11代。若基础培养液不添加生长因子,培养 6~10 d后亦可出现 EG克隆细胞株,但其克隆具有出现晚、数量少、增殖慢等特点。研究中还发现25~26日龄的WZSP胚胎的原始生殖嵴小、不易观察、PGCs分离较困难; 27 ~ 28 日龄的 WZSP 胚胎的原始生殖嵴体积大、易观察、便于分离并可获得多量的PGCs。同时对PGCs进行了形态学、体外分化、AKP染色鉴定等研究。2002 年 9-12 月,在中国农业科学院畜牧研究所五指山猪保种场先后从22头超排处理的大白猪供体中,于配种后(当天为 0 d)5 5~7 d实施手术法获得252枚可用胚。将123枚胚胎显微注射WZSP的EG细胞,每枚注射5~15个细胞,与未注射的 103 枚混合分别移植给9头受体,平均每头受体移植胚胎25 11±5 11枚、含注射 EG细胞胚胎 13 67±3 57 枚,其中 7 头分别  相似文献   

2.
从本地槐山羊胎儿中,将原始生殖细胞与其生殖嵴周围组织细胞共同分离,经传代培养后获得了具有干细胞特征的山羊胚胎生殖(EG)细胞。结果表明:高糖DMEM培养基和低糖DMEM培养基相比较,低糖DMEM更适宜于山羊EG细胞的分离与克隆;EG细胞在山羊胎儿成纤维细胞饲养层上生长效果较好,可传4代或5代,而在小鼠成纤维细胞饲养层上EG细胞仅传3代;联合添加白血病抑制因子(LIF)、干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)能显著提高山羊EG细胞分离与克隆的效率;胎龄为30～45d的胎儿原代培养时可获得大量的细胞集落,克隆培养可传至5代,适合做山羊EG细胞的分离培养。  相似文献   

3.
昆明白小鼠胚胎生殖细胞的分离与培养   总被引：3，自引：0，他引：3  
以昆明白品系小鼠胎儿生殖嵴为材料，以不同的培养液分离培养胚胎生殖细胞（EG cells），发现小鼠胎儿肝细胞条件培养液的效果好于未添加白血病抑制因子（LIF）的基础培养液，而差于添加了1000IU／mL LIF的基础培养液。同时，试验对比了从不同胎龄胎儿分离培养原始生殖细胞（PGCs）的情况，鉴定了EG细胞的生物学特性；EG细胞经体外培养，可以分化为神经样细胞、上皮样细胞和简单类胚体。  相似文献   

4.
从五指山近交系 (ＷＺＳＰ)培育群中 ,先后选用自然或同期发情 1 1头 5 8月龄青年母猪 ,分别于授精后 2 5～ 2 9ｄ采集胎儿 75个 ,进行原始生殖嵴 (ＰＧＣｓ)细胞分离、培养等建系技术研究。培养液为ＤＭＥＭ Ｆ1 2 ( 1∶1 ) ,并含 1 5 %胎牛血清、0 1ｍｍｏｌ/Ｌ硫基乙醇、40ｎｇ/ｍｌｈＳＣＦ、2 0ｎｇ/ｍｌｈＬＩＦ、2 0ｍｍｏｌ/Ｌ谷胱甘肽 ,另添加或不添加 2 0ｎｇ/ｍｌｂＦＧＦ作为对比试验。用ＳＴＯ细胞作饲养层 ,在 37 5℃、5 0 %ＣＯ2和湿润的气相中进行培养。分别冷冻保存胚胎生殖嵴细胞 (ＥＧ)细胞系 1 1个 ,其中 2个ＥＧ…  相似文献   

5.
本研究对发育至第19期的鸡胚性腺原始生殖细胞(primordial germ cells，PGCs)分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖、PVP（聚乙烯吡咯烷酮）分别组成6种慢速冷冻保护液和6种玻璃化冷冻保护液，进行超低温冷冻保存。复苏后苔盼蓝染色测定细胞存活率，体外接种培养、传代。结果：①慢速冷冻保存中，PGCs在慢速冷冻液V（10％EG+10% FBS+0.1 mol/L蔗糖）条件下复苏后存活率最高（92.20%），且与慢速冷冻液I（10％ DMSO+10% FBS）存活率之间差异显著（P<0.05）。②玻璃化冷冻保存中，PGCs在玻璃化冷冻液I（10% DMSO+10% EG+20% FBS+10% PVP）条件下复苏后存活率最高（84.15%），且与其余5种玻璃化冷冻液下复苏后存活率之间差异均极显著（P<0.01）。③培养传至第3代的慢速冷冻复苏后PGCs细胞和培养传至第2代的玻璃化冷冻复苏后PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。  相似文献   

6.
研究采用 1 4～ 1 8d兔胎儿的生殖嵴及周围组织与其同源成纤维细胞共培养 ,低糖DMEM +1 0 %NBS +1 0 %FCS +1 0ng/mLLIF +1 0ng/mLSCF+0 1mol/Lβ 巯基乙醇 +1 0 0U/mL青霉素 +80U/mL链霉素作培养基 ,分离出兔原始生殖细胞 (PGC) ,克隆并多次传代。从原始生殖细胞 (PGC)中获得胚胎生殖细胞 (EG)细胞集落 ,1 4d胎儿原代观察到类EG细胞集落 ,传至 4代后丢失。 1 6d胎儿的类EG只传 2代 ,1 8d胎儿没有得到EG细胞集落。EG细胞具有干细胞的诸多特征 ,呈典型的团块状聚集生长 ,碱性磷酸酶 (AKP)染色呈阳性 ,在衰老饲养层的培养基中生长形成类胚体、上皮细胞、神经细胞和成纤维细胞等  相似文献   

7.
发育至第19期和第28期的鸡胚性腺原始生殖细胞（PGCs）分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖进行超低温冷冻保存，比较冷冻保护剂在单独或联合使用条件下对PGCs的冷冻保护效果，复苏后台盼蓝染色测定细胞存活率，体外接种培养、传代。结果表明：（1）第19期PGCs冷冻保存中，10％EG＋10％FBS＋0．1mol／L。蔗糖条件下细胞存活率最高为（92．20±2．18）％，且与10％DMSO＋10％FBS的存活率差异显著（P〈0．05），其余各冷冻保护液间存活率差异不显著；第28期PGCs冷冻保存中，5％DMsO＋5％EG＋10％FBS＋0．1mol／L。蔗糖条件下细胞存活率最高为（92．41±2．82）％，但各冷冻保护液间存活率差异均不显著（P〉0．05）。（2）解冻后体外培养的PGCs，在饲养层和3种细胞因子（I。IF、SCF、bFGF）的共同作用下，可持续增殖，传至第3代的PGCs，PAS染色、AKP染色均呈阳性并保持完整的二倍体核型，仍处于未分化状态。  相似文献   

8.
为了研究不同条件对ICR小鼠ES细胞的影响,试验以12.5~13.5 d ICR小鼠胚胎成纤维细胞(MEF)为饲养层,以3.5 d ICR小鼠胚胎为试验材料,探讨了血清、生长因子及传代方法对ICR小鼠ES细胞分离培养的影响。结果表明:采用含15%FBS的ES细胞培养液的囊胚贴壁率及ICM增殖率(79.3%,69.0%)均比含15%KSR、5%FBS+10%KSR的细胞培养液高(42.9%,28.6%;75.0%,54.2%),ES细胞最高传至6代;培养液中添加10 ng/mL LIF+10 ng/mL SCF的效果比单独添加1种因子的效果好,最高传至6代,高于单独添加1种因子的传代数(4代,2代);用3种传代方法进行传代时,采用差异贴壁法传代效果最佳,最高传至8代,酶消化法传至4代,机械加酶消化法传至6代。  相似文献   

9.
山羊PGCs用于分离与克隆类ES细胞   总被引：11，自引：1，他引：10  
选择健康成年本地白山羊，自然发情，配种后44d取胎儿，以传统的原始生殖细胞（PGCs)分离与克隆的方法和PGCs与其胎儿生殖嵴周围组织细胞共同培养的方法获得类胚胎干细胞（类ES细胞），并对山羊类ES细胞在不同饲养层上进行培养。结果表明，采用传统方法与共培养的方法并添加细胞因子均能分离获得类ES细胞。分离获得的类ES细胞在同源（山羊）胎儿细胞饲养层上生长效果较好，可传4代或5代，而在小鼠原代成纤维细胞饲养层上类ES细胞仅传3代。另外，共培养不添加细胞因子组仅获1个ES细胞集落，传代后丢失。  相似文献   

10.
为探讨水牛睾丸生精上皮细胞冷冻保存的适宜冷冻保护剂及其浓度,在DMEM中分别添加不同浓度的冷冻保护剂二甲基亚砜(DMSO)、甘油(G)、丙二醇(PG)和乙二醇(EG),以及10%的胎牛血清(FBS),对3～5月龄水牛生精上皮细胞进行冷冻保存,解冻后台盼蓝染色测定细胞活率.结果显示,当以0%,5%.10%,15%,20%DMSO添加时,10%DMSO组的细胞解冻后活率均显著高于其他各组(P<0.05);当以0%,20%,25%,30%,35%,40%G添加时,35%G组的细胞解冻后活率均显著高于其他各组(P<0.05);当以0%,5%,10%,15%,20%,25%PG添加时,15%～25%PG各组的细胞解冻后活率均显著高于其他各组(P<0.05),但以20%PG组为最高;当以0%,5%,10%,15%,20%EG添加时,5%～20%EG各组的细胞解冻后活率均显著高于0%EG组,但以10%EG组为最高;而对4种冷冻保护剂各最优浓度组的细胞解冻后活率比较,10%DMSO、35%G组的细胞活率均显著高于20%PG、10%EG组(P<0.05).结果表明,以添加10%DMSO或35%G于含10%FBS的DMEM冷冻液中,采用两步慢速冷冻,液氯保存,37℃水浴1 min解冻,是一种具有较高复苏率的冷冻保存水牛生精上皮细胞的方法.  相似文献   

11.
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。  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.  相似文献   

17.
The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.  相似文献   

18.
The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.  相似文献   

19.
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.  相似文献   

20.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
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