小鼠生精细胞的分离和体外培养

为了建立一种操作性强、稳定、高效的小鼠生精细胞体外培养体系,从10只8周龄成年雄性健康清洁级昆明小鼠睾丸组织分离出细胞得到混悬液,采用支持细胞和生精细胞共培养方式,通过添加多种细胞培养液,以维持细胞的生长和增殖。结果表明:采用睾丸生精细胞、支持细胞共同培养的方式可得到密度较大的生精细胞,且精原细胞、精母细胞的数量占绝大多数。     

山羊睾丸生精细胞体外培养和鉴定

试验采用两步酶消化法对2月龄公羔睾丸进行处理,得到了总数为7.44×109的生精细胞悬液,经台盼蓝染色,细胞活率在90％以上;支持细胞的贴壁性极好,生精细胞附着在支持细胞上,随着培养时间延长,生精细胞、支持细胞逐渐退化,呈现“集合”的趋势,细胞数量逐渐减少;经BrdU标记、H.E.染色,检测培养的生精细胞到分化到圆形精子细胞阶段,表明成功构建了山羊睾丸生精细胞共培养体系,可为后续深入研究山羊精子体外成熟培养提供一定的实验依据.     

CREM在体外共培养山羊睾丸生精细胞中的定位研究

为进一步深入研究CREM基因在雄性动物中的表达调控机制,试验成功建立体外生精细胞和支持细胞共培养体系,并应用免疫细胞化学研究CREM在体外培养山羊睾丸生精细胞的表达特点。结果显示:CREM仅表达于圆形精子细胞,其他生精细胞和支持细胞检测不到CREM的表达。表明CREM基因在圆形精子细胞发育过程中起重要调节作用,但其作用机制有待深入研究。     

鸡睾丸生精上皮细胞冷冻保存研究

以广西土鸡为试验材料,利用两步酶解法从出壳后20～30日龄公鸡睾丸中获取生精上皮细胞,以含20％胎牛血清的DMEM为基础培养液,比较了不同浓度DMSO和蔗糖对生精上皮细胞冷冻保存效果的影响。结果表明：DMSO的最佳浓度为10％,添加蔗糖对冻存有害。以10％DMSO为防冻剂,复苏后生精上皮细胞原代混合培养可形成典型的精原干细胞集落,集落呈碱性磷酸酶（AKP）阳性,说明以10％DMSO为防冻剂的冷冻体系适合鸡睾丸生精上皮细胞的冷冻保存。     

PHGPx在体外共培养山羊睾丸生精细胞中的定位研究

为探讨磷脂氢谷胱甘肽过氧化物酶(PHGPx)在雄性动物生殖机能中的作用及其在体外培养山羊睾丸生精细胞的定位,在已成功构建的山羊睾丸支持细胞-生精细胞共培养的基础上,制作细胞爬片,采用免疫细胞化学技术定位PHGPx的表达。结果显示,PHGPx在支持细胞、精原细胞中不表达,在圆形精子细胞细胞质检测到阳性表达产物。表明PHGPx在精子发生后期圆形精子的变态发育中起特定的调节作用,但作用机制有待深入研究。     

四种抗冻剂20℃时对小鼠生精上皮单细胞存活率的影响

将小鼠生精上皮单细胞置20℃含不同抗冻剂的冻存液中一定时间后，台盼蓝染色测定细胞存活率，以筛选冷冻保存小鼠生精上皮细胞的侯选抗冻剂及使用浓度。结果，20℃30min，10％浓度的二甲基亚砜（DMSO）、丙二醇（PG）及乙二醇（EG）对7日龄小鼠生精上皮单细胞存活率均无显著影响，而10％甘油（G）则使细胞存活率显著下降；20℃ 30min,10%浓度的DMSO、PG、EG及G对成年小鼠生精上皮单细胞存活率均无显著影响；20℃ 5min,25%浓度的DMSO、PG、EG及G均使7日龄及成年小鼠生精上皮单细胞存活率显著下降。实验结果表明，10％DMSO、PG及EG可作为7日龄小鼠生殖细胞慢速冷冻保存时的侯选抗冻剂，10％DMSO、PG、EG及G可作为成年小鼠生殖细胞慢速冷冻保存时的侯选抗冻剂；在高浓度抗冻剂超速冷冻保存小鼠生殖细胞时，平衡时间应短于5min，或在4℃进行。     

不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响

本试验旨在研究不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18(CK18)表达量的影响。采集42日龄山羊的瘤胃上皮组织,分别采用酶消化法和组织块法对其进行体外培养。通过光学显微镜观察原代培养和传代培养阶段的细胞形态,检测第5代山羊瘤胃上皮细胞的生长曲线,并采用细胞免疫荧光法对山羊瘤胃上皮细胞进行鉴定。结果显示:1)经0.25%胰蛋白酶+0.02%乙二胺四乙酸消化获得的原代培养山羊瘤胃上皮细胞于2 d开始贴壁生长,5 d细胞开始明显增多,10 d细胞数量达到最大。2)经组织块法获得的原代培养山羊瘤胃上皮细胞于4 d开始爬出组织块,8 d细胞开始明显增多,14 d细胞数量达到最大。3)经免疫荧光染色显示2种方法获得的细胞胞浆内CK18均呈阳性表达且细胞纯度后者明显高于前者。4)组织块法获得的细胞CK18表达量显著高于酶消化法(P0.05)。综合得出,与酶消化法相比,应用组织块法可成功获得纯度更高的山羊瘤胃上皮细胞。     

鸡原代输卵管上皮细胞体外分离培养与鉴定

为了探讨鸡原代输卵管上皮细胞分离培养方法,本研究分别对消化酶、消化时间、取材部位和表面包被物等条件进行比较和优化,筛选出鸡原代输卵管上皮细胞分离和纯化的最佳方法,并对培养细胞进行鉴定与传代。结果显示,取鸡输卵管漏斗部,0.25%胰酶+0.02%EDTA联合消化15min、低速离心去除单细胞、差速贴壁除去成纤维细胞,在20%FBS包被的细胞培养瓶中可获得满意的输卵管上皮细胞分离效果,细胞贴壁性良好。培养的细胞在24h时成团贴壁,48~60h明显增殖,呈圆形或多角形"铺路石样"的单层细胞生长,72h后细胞增殖速度减慢,可以维持至10d以上,且传代后细胞贴壁生长良好,经姬姆萨染色和透射电镜观察鉴定为鸡输卵管上皮细胞。本研究建立的鸡原代输卵管上皮细胞分离培养方法可获得纯度较高的目的细胞,为研究鸭源鸡杆菌对鸡输卵管细胞的侵袭特性提供了良好的体外研究模型。     

己烯雌酚对体外培养仓鼠生精细胞毒性作用的研究

为研究己烯雌酚(DES)对仓鼠生精细胞毒性作用机制,试验采用原代培养的仓鼠睾丸生精细胞,用10、30、90μmol/L DES处理,倒置显微镜观察生精细胞生长状况,MTT法测定处理后8 h生精细胞存活率,分光光度法检测细胞培养液中超氧化物歧化酶(SOD)、丙二醛(MDA)、细胞中谷胱甘肽过氧化物酶(GSH-Px)的含量。结果贴壁培养的生精细胞用DES处理后,细胞活力显著降低,与正常对照组比较差异极显著(P0.01),随着DES剂量的增加,细胞中SOD、GSH-Px含量显著下降,MDA显著上升。说明DES对体外培养生殖细胞毒性作用与ROS密切相关,DES通过降低抗氧化酶水平,增加ROS含量,干扰生精细胞正常功能,表明氧化损伤可能是环境雌激素生殖毒性的作用机制之一。     

大鼠心肌细胞分离纯化方法的改良

心肌细胞原代培养是心血管相关研究方面、以及细胞生理方面广泛应用的研究工具,目前已是多门学科研究的热点。心肌细胞培养的相关报道方法很多,由于各实验室所解决的问题、实验室具体条件的不同,所采用的培养方法也不尽相同。如何建立一种比较简单、快速,并且尽可能地获得较高的细胞存活率、较高的细胞活力,仍是心肌细胞培养模型制作中的关键问题。本研究借鉴前人的经验,采用比较完善的方法培养出纯度较高、活力较强的新生SD大鼠的原代心肌细胞,足以满足相关科研的要求。     

Reducing endogenous estrogen during development alters hormone production by porcine Leydig cells and seminiferous tubules

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5-4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E(2)) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E(2) and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E(2) and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E(2) but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E(2) production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.     

山羊痘病毒的分离与鉴定

对贵州省2002年以来发生的疑似山羊痘病例样品进行了病毒的分离与鉴定。取山羊痘病羊的皮肤痘疹和水疱作待检病料,接种BHK-21传代细胞盲传3代后,出现了明显的、规律的细胞病变(CPE),病毒细胞培养物在F4代以后,能与山羊痘标准阳性血清在琼脂扩散试验中出现白色沉淀线,而与正常细胞的培养物及PBS不出现沉淀线;参照GenBank上山羊痘病毒P32基因序列,设计了1对特异性引物,对现场分离毒株进行PCR扩增,可扩增出963bp特异性的DNA条带。用待检病料感染的BHK-21细胞培养物接种9日龄鸡胚绒毛尿囊膜,随着传代次数的增加,痘斑病变的出现率从13.3%～20%上升至33.3%～40%;用山羊痘病变皮肤、鸡胚绒毛尿囊膜和感染细胞进行超薄切片,在电子显微镜下可以观察到典型的山羊痘病毒粒子。     

山羊痘病毒的分离鉴定及生物学特性的研究

本研究对2003年广西部分地区山羊群发生的疑似山羊痘进行了病毒分离鉴定及生物特性的研究。取疑似山羊痘病羊的皮肤丘疹、水泡或脓泡组织的病毒悬液，接种初生羔羊睾丸细胞观察到明显的细胞病变，免疫荧光试验结果显示，病毒能与山羊痘标准阳性血清反应，在感染的细胞浆内发出特异性的黄绿色荧光。病毒悬液接种乳鼠、小鼠、豚鼠、兔子都未发病，而接种3月龄山羊则出现典型的山羊痘症状和病理变化，接种9～10日龄鸡胚绒毛尿囊膜，未见出现痘斑，连传3代，均无异常变化。通过病理组织学观察可以看到在细胞浆内有大小不一圆形或椭圆形的包涵体，在电子显微镜下可以观察到150nm～300nm大小，卵圆形、砖形，有囊膜的病毒颗粒。利用一对山羊痘病毒P32基因引物进行了PCR扩增，将所得序列与GenBank收录的5株山羊痘病毒P32基因的核苷酸及氨基酸序列比较分析。结果与疫苗株的同源性分别为99．8％和99．4％。与国外其它毒株的同源性为99．6％和98、8％～99．4％。研究结果表明，所分离的病毒为山羊痘病毒，在生物学特性上与资料记载存在一定的差异，P32基因与疫苗毒和国外毒株之间同源性非常高。将该毒株命名为山羊痘病毒LiuJiang／2003株。     

Isolation of Moellerella wisconsensis from the lung of a goat

This report describes the first isolation of Moellerella wisconsensis from the lung of a goat in Italy and represents the fourth isolation of this rare enterobacterial species from non-human sources reported in the literature. In fact, since its first isolation, M. wisconsensis has only been described in a few other occasions and often in clinical samples associated with cases of human enteritis.     

羊成纤维细胞建系研究进展

本文总结了羊成纤维细胞建系及生物学特性检测技术,包括羊成纤维细胞原代、传代培养与纯化、冷冻保存与复苏、细胞活力测定、生长曲线、染色体分析、同工酶分析、微生物检测等,并回顾了体细胞技术已经取得的成就,展望其在我国现代畜牧业中的应用前景。     

影响山羊类胚胎干细胞分离克隆的因素分析

将本地槐山羊胎儿的原始生殖细胞(PGCs)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类ES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更适宜于山羊类ES细胞的分离与克隆;类ES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,类ES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)能显著提高山羊类ES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,适合作山羊类ES细胞的分离培养。     

不同粪便处理模式对羊粪中捻转血矛线虫虫卵杀灭效果的影响

为了评价不同粪便处理模式下羊粪中捻转血矛线虫虫卵的杀灭状况,2018年8月至10月,对福州市某羊场的羊粪,分别采用堆积发酵(Ⅰ组)、塑料罐密闭储存(Ⅱ组)、舍内自然放置(Ⅲ组)等3种方式进行处理。第Ⅰ组堆积发酵30 d后可完全杀灭粪中虫卵;第Ⅱ组密闭储存20 d后可完全杀灭虫卵;第Ⅲ组自然放置50 d可杀灭虫卵97.9%。试验结果可为羊场的粪便处理提供参考依据。     

LncWNT3-IT affects the proliferation of Sertoli cells by regulating the expression of the WNT3 gene in goat testis

The proliferation and differentiation ability of testicular Sertoli cells directly affects spermatogenesis and male reproductive development. WNT proteins are involved in the regulation of cell proliferation, differentiation and spermatogenesis. Therefore, to study whether lncRNAs, which regulate the expression of WNT proteins during cell proliferation and differentiation, are worthwhile. In this study, testicular tissue from the Dazu black goat (Capra, goat, Chongqing, China) at neonatal time (less than 7 days old), early puberty time (45 days old) and sexual maturity time (90 days old) at three ages was subjected to high-throughput sequencing to predict testicular growth and development associated with WNT lncRNA. The final screening of lncWNT3-IT may be targeted to regulate the expression of WNT3. At the same time, the expression of WNT3 was verified by lncWNT3-IT by paraffin sectioning, fluorescence in situ hybridization, interference, overexpression, cytotoxicity assay, Western blotting and qPCR. The following results were obtained: lncWNT3-IT was expressed in the testicular Sertoli cells and played a role in the Sertoli cell cytoplasm. Fluorescence in situ hybridization localization analysis showed that lncWNT3-IT positively regulated the expression of WNT3, and through cell viability and cell proliferation experiments, it was found that the expression of lncWNT3-IT assisted in Sertoli cell proliferation. In summary, lncWNT3-IT can influence the proliferation of Sertoli cells by positively regulating the expression of WNT3.     

Housing of Norwegian goat herds and associations with milk yield and milk quality

Abstract

Data from a questionnaire regarding housing factors were merged with data from the milk recording databases (herd mean annual milk yield per goat, somatic cell count (SCC) and bulk milk bacterial count), and the material included 235 herds. Associations with housing factors were tested at herd level and at individual goat level. Housing in insulated buildings with no access to outdoor areas during the winter season, expanded metal grating and no use of bedding predominated. None of the housing factors evaluated were significantly associated with milk yield. In herds using water nipples, SCC was lower compared to herds using water bowls. SCC was also lower in herds with milking facilities in separate milking stalls as compared with combined feeding milking stalls in common pens. No significant associations were found between housing factors and bacterial counts, but the counts were lower in herds with high average milk yields per goat.     

山羊奇异变形杆菌的分离鉴定及16SrDNA和ZapA基因的PCR-RFLP分析

从发病山羊分离到5株病原菌,对分离株进行游散行为分析,生化试验、动物试验、药敏试验及16SrDNA和ZapA基因克隆测序,选用限制性内切酶对分离株16SrDNA和ZapA基因进行PCR-RFLP分析。结果表明分离株出现游散生长现象,对丁胺卡那霉素和复达欣敏感,小鼠LD5。为2．5000×10 ^7CFU／mL-4．4453×10^7CFU／mL;分离株16SrDNA与奇异变形杆菌的同源率为99．5％～99．8％,ZapA基N与奇异变形杆菌的同源率为99．5％;PCR—RFLP分析发现,分离株酶切片段数目和大小与标准株相同。结果表明分离株是奇异变形杆菌,多态性较为单一。