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Establishment and evaluation of an immortalized steroidogenic goat granulosa cell line as a model system to study the endoplasmic reticulum (ER)-stress response on steroidogenesis

Establishment and evaluation of an immortalized steroidogeni...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： Diqi Yang Lei Wang Pengfei Lin Tingting Jiang Nan Wang Fan Zhao Huatao Chen Keqiong Tang Dong Zhou Aihua Wang Yaping Jin Key Laboratory of Animal Biotechnology of the Ministry of Agriculture Northwest A&F UniversityYangl College of Veterinary Medicine Northwest A&F UniversityYangling712100ShaanxiChina

Under the support of granulosa cells and theca cells, the ovaries are responsible for producing oocytes and secreting sex steroids such as estrogen and *** reticulum stress (ERS) plays an important role in follicle atresia and embryo *** this study, goat granulosa cells were isolated from medium-sized (4-6 mm) healthy *** primary granulosa cells were immortalized by transfection with human telomerase reverse transcriptase (hTERT) to establish a goat granulosa cell line (hTERT-GGCs).Test results showed that hTERT-GGCs expressed hTERT, and a relatively long telomere existed at passage ***, the hTERT-GGCs expressed gonadotropin receptors CYP11A1, StAR and CYP19A1, which are involved in *** addition, the concentration of progesterone was detectable in *** the proliferation potential of hTERT-GGCs significantly improved,there was no evidence to suggest that the hTERT-GGCs are *** addition, thapsigargin (Tg)treatment led to a significant decrease in the progesterone concentration and the expression of steroidogenic enzyme in a dose-dependent *** summary, in this study we successfully generated a stable goat granulosa cell *** this cell line we found that Tg induced ERS, which reduced progesterone production and steroidogenic enzyme expression in *** studies may benefit from using this cell line as a model to explore the molecular mechanism regulating steroidogenesis and apoptosis in goat granulosa cells.

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Influence of Dietary Selenium on Fermentation Pattern and Mucosal Growth in Colon of Goat

Influence of Dietary Selenium on Fermentation Pattern and Mu...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： ABBASI Benazir MALHI Moolchand RUI Rong College of Veterinary Medicine Nanjing Agricultural University

Purpose:The present study aimed to investigate the impact of dietary selenium yeast（SY） on the fermentation pattern and mucosal growth in the colon of ***:Ten cross-bred goats at 4 month of age and 10.5 KgBW were fed basal diet consisting of guar hay and *** concentrate（150 g） was divided into two equal portions at 08:00 and 17:00 h *** experimental groups were consisting of basal diet（C）,or basal diet with SY at the dose of 0.3mg/kg feed,mixed in concentrate at morning ***:Results revealed that Se concentration（0.062 ± 0.005 vs 0.034 ± 0.003;P<0.05） and glutathione peroxidase（GSH-Px） activity（29.71 ± 1.09 vs 22.37 ± 4.26） increased in the serum of goats fed SY diet compared with *** molar concentrations of propionate increased（P<0.05） in the colon of SY（26.01 ± 1.04） than in C（20.69 ± 0.43） which was manifested by the increase（P<0.05） in total SCFA concentrations and the decrease（P<0.05） in pH of colonic digesta of SY goats compared to *** weight of colon increased（P>0.05） in SY goats than in control（135.50 ± 0.65 g vs 126.75 ± 0.85 g）.The weight of colonic mucosa increased（P<0.05） in SY goats（76.83 ± 0.18） compared with *** of colonic wall revealed that thickness of mucosa tunic and number of goblet cells per mm increased（P<0.05） in colon of SY fed goats than that of *** GSH-Px activity increased（P<0.05） in colonic epithelium of SY goats than that of *** summary,the dietary SY supplementation in diet of goats improved fermentation pattern,induced mucosal growth and increased GSH-Px activity in the colon of goats.

关键词： antioxidants colon fermentation goat mucosal growth selenium yeast

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Knock-down of apoptosis inducing factor gene protects endoplasmic reticulum stress-mediated goat granulosa cell apoptosis

Knock-down of apoptosis inducing factor gene protects endopl...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者：杨迪琦 Tingting Jiang Pengfei Lin Huatao Chen Lei Wang Nan Wang Fan Zhao Aihua Wang 靳亚平 Key Laboratory of Animal Biotechnology of the Ministry of Agriculture Northwest A&F University College of Veterinary Medicine Northwest A&F University

The apoptosis of granulosa cells is the main cause of follicular atresia,and endoplasmic reticulum（ER）stress is involved in the apoptosis of granulosa *** inducing factor（AIF） mediates caspase-independent apoptosis and causes chromatin condensation and DNA fragmentation,but its role in ER stress-mediated granulosa cell apoptosis during goat follicular atresia remains largely *** aim of this study was to investigate the function of AIF in the apoptosis of goat granulosa cells media ted by ER *** results of immunohistochemical and Western blot analyses demonstrated that AIF was mainly located in granulosa cells,and the expression of AIF significantly increased during follicular ***,AIF-shRNA recombinant lentiviral vectors were constructed successfully and transfected into hTERT-goat granulosa cells（hTERT-GGCs）.Real-time quantitative PCR and Western blot analysis confirmed that AIF was effectively knocked down in *** cytometry results showed that the knock-down of AIF in hTERT-GGCs reduced apoptosis due to serum starvation or thapsigargin（Tg）***,AIF depletion changed the expression of related molecular markers molecules of ER stress under Tg *** conclusion,AIF may serve as a key factor during follicular atresia and AIF depletion protects ER stress-mediated goat granulosa cell apoptosis.

关键词： Knock-down of apoptosis inducing factor gene protects endoplasmic reticulum stress-mediated goat granulosa cell apoptosis

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Expression and Function of MUC1 in Early Pregnant/Non-pregnant Sheep Uterus Manipulated by Artificially Induced Oestrous

Expression and Function of MUC1 in Early Pregnant/Non-pregna...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： Xiangguo Wang Boyang Zhu Shanhui Xiong Xihui Sheng Xiaolong Qi Yu Yang Yong Guo Hemin Ni Animal Science and Technology College Beijing University of Agriculture

Mucin 1(MUC-1) is a cell surface glycoprotein with a previously described anti-adhesive role involved in different aspects of reproductive *** this study,we investigated the expression and function of MUC1 in early pregnant/non-pregnant sheep uterus manipulated by artificially induced oestrous on days 10,12,14,16 and 18 by in situ hybridization,real-time PCR,immunohistochemical staining and western blot *** to our results,In the uterus of artificially induced oestrous sheep,MUC1 mRNA and protein expression increased first and then decreased from day 10 to 18,and reached the highest expression on day ***,MUC1 protein localisation was expressed in endometrial luminal epithelium(LE) on days 10,12 and 14 and in endometrial glandular epithelium(GE) on days 16 and *** contrast,in the uterus of early pregnancy sheep by artificially induced oestrous,the expression MUC1 mRNA and protein increased on days 10 and 12,decreased on day 14,but increased again on days 16 *** the same time,MUC1 mRNA and protein were expressed in endometrial LE and ***,the MUC 1 mRNA and protein expression levels in uterus of early pregnancy sheep by artificially induced oestrous was significantly lower than that in non-pregnant sheep uterus on days 10,14,and 18,except on day 16(P<0.01).Our data indicated that MUC1 might play an important role in the process of sheep embryo implantation,and MUC1 expression was mainly regulated by active embryos and steroid *** expression of MUC1 can be used as a judgment of a clear marker of uterine receptivity state of sheep embryo implantation.

关键词： sheep uterus MUC1 endometrium progesterone

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The effect of Sericin on mice oocyte maturation and embryonic development

The effect of Sericin on mice oocyte maturation and embryoni...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： Qipeng Jia Yong Zhang College of Veterinary Medicine Gansu Agricultural University

Purpose Sericin was a water-soluble component of silk protein,which has been identify with antibacterial,antioxidant,UV resistant properties,and absorbs and releases moisture,The present study was designed to investigate the effect of sericin supplementation during in vitro maturation(IVM) medium on oocyte maturation and synchronization of nuclear and cytoplasmic maturation by observing subsequent mice embryo development compared with oocyte maturational in *** female Kunming mice(day 21-30 of age) were randomly divided into two groups,the first group mice oocyte were collected from the ovaries and culturesd in TCM-199 with ricin at various concentrations of 0(control),0.1,0.25,0.5,1.0%and incubated at 37° in humidified 5%C02 *** group mice oocyte were collected from the oviduct,subsequently in vitro fertilization(IVF) and in vitro embryo culture(IVC).outcome evaluation were maturation,cleavage and blastocyst formation *** The rates of maturation to metaphase Ⅱ with 0.1%sericin had significantly higher than other groups(P<0.05),the addition of 1.0%sericin were similar maturation rates compared with control groups(P>0.05).After MII stage oocytes from mice ovaries and oviduct were coexistence with sperm,rates of cleavage and blastocyst from oviduct were significantly higher than other groups that from ovaries(P<0.05),And morula rates were similar(P>0.05) in oviduct group and 0.1%sericin group,As oocytes from ovaries,the addition of 0.1%sericin significantly improved the rates of cleavage,morula and blastocyst(P<0.05).when presumptive zygotes were cultured in the Sofa medium with 0%and 0.1%sericin concentration respectively,The present findings indicated that The maturation oocytes from the oviduct(in vivo) have a higher(P<0.05) cleavage,morula and blastocysts rates after IVF compared with that from the ovary(in vitro),and the maturation oocytes from the oviduct have similar(P>0.05)leavage,morula and blastocysts rates between 0%(control

关键词： mice sericin IVM in vitro in vivo embryonic development

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疫苗接种对靶动物免疫学功能的影响

疫苗接种对靶动物免疫学功能的影响

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者：田伟国纪世春朱根宝单鑫董森黄伟平刘亚安徽农业大学动物科技学院安徽地方畜禽遗传资源保护与生物育种省级实验室

促性腺激素释放激素(GnRH)和性激素在调节免疫系统和发展起重要作用。为探究免疫去势对大鼠免疫功能的影响,本实验采用流式细胞术和酶联免疫吸附(ELISA)检测方法对免疫去势的大鼠的T淋巴细胞分型,B淋巴细胞和T调节细胞变化和Th1、Th2和T... 详细信息

促性腺激素释放激素(GnRH)和性激素在调节免疫系统和发展起重要作用。为探究免疫去势对大鼠免疫功能的影响,本实验采用流式细胞术和酶联免疫吸附(ELISA)检测方法对免疫去势的大鼠的T淋巴细胞分型,B淋巴细胞和T调节细胞变化和Th1、Th2和Th17型细胞因子水平;目的是分析受免动物免疫细胞数量上的变化和受免动物免疫功能上的变化。试验中将45只18至21日龄的SD大鼠根据遗传背景平均分为三组:GnRH-MAP油佐剂组,GnRH油佐剂组和对照组。分组注射疫苗,每两周加强一次,共免疫三次,对照组不作处理。最后一次加强免疫后,每隔2周,称重后处死5只免疫组及对照组大鼠采集血液离心制备血清,取下胸腺组织进行B淋巴细胞、T调节细胞和T淋巴细胞分型检测。结果表明:(1)GnRH主动免疫的18～20d大鼠产生高滴度抗体时间较长,血清睾酮浓度一直处于极低水平(P<0.01),睾丸中曲细精管多数变形,管腔内细胞稀薄。免疫大鼠血清中Thymulin呈先升后降趋势,在免疫6周时显著高于对照组(P<0.05);在免疫4～6周时下丘脑内Thymulin显著高于对照组(P<0.05)。免疫大鼠下丘脑内GnRH浓度显著低于对照组(P<0.05)(2)在最后一次加强免疫1个月后血液中CD3+、CD4+细胞比例显著降低(P<0.05),2个月后与对照组无显著性差异(P>0.05);CD8+细胞和T调节细胞则呈先升后降的趋势,至免疫2个月后与对照组差异不显著(P>0.05)。GnRH主动免疫引起胸腺CD3+、CD4+CD8-、T调节细胞和双阳性细胞比例显著升高(P<0.05),CD4-CD8+和双阴性细胞比例显著降低(P<0.05)。总之,本研究发现,GnRH主动免疫对18～20d和8月龄大鼠的去势效果不同,对18～20d大鼠的睾丸造成不可逆损伤,睾酮水平处于极低状态;在最后一次加强免疫1月后,GnRH主动免疫负向调节血液T淋巴细胞,2月后又恢复至正常水平,而在此期间胸腺T淋巴细胞正向选择增强,T淋巴细胞向外周T淋巴细胞池的输送比例加大,为维持和促进机体T淋巴细胞的免疫功能奠定基础。

关键词：免疫去势 GnRH 雄性大鼠

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者：李谷月吕鹏郑鸿飞潘阳阳崔燕余四九甘肃农业大学动物医学院甘肃省牛羊胚胎工程技术研究中心

目的:为研究牦牛(Bos grunniens)热休克蛋白70-9B(heat shock 70 000protein-9B,HSPA9B)、热休克蛋白70-12B(heat shock 70 k D protein-12B,HSPA12B)和肿瘤抑制蛋白(tumor protein p53,Tp53)在牦牛早期体外受精胚胎中的表达差异及其与体外受精胚胎发育的关系。材料与方法:在正常生理条件下,采集青海高原牦牛卵巢,捡取A级和B级卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs)进行体外受精和体外培养,采用实时荧光定量(RT-qPCR)和间接免疫荧光技术,检测HSPA9B、HSPA12B和Tp53在不同时期体外受精胚胎中的表达差异及其蛋白分布。结果:RT-qPCR结果表明:HSPA9B、HSPA12B和Tp53在牦牛体外受精2-4细胞期胚胎、5-8细胞期胚胎、桑椹胚和囊胚中均有表达。其中HSPA9B在4～8细胞期胚胎中的相对表达量较高;HSPA12B在2-4细胞期胚胎中的相对表达量相对较高;Tp53在2～4细胞期和囊胚期表达量相对较低,Tp53在5～8细胞期和9～16期表达量相对较高。间接免疫荧光染色结果表明:在2～4细胞期胚胎、4～8细胞期胚胎和桑椹胚中,胞核HSPA9B的相对表达量较胞质弱,在囊胚中胞核HSPA9B的相对表达量较胞质强;Tp53蛋白主要存在于细胞核中,少量存在于细胞质中,其作用和DNA的损伤有关。结论:由此得出结论:HSPA9B可能在胚胎早期发育中发挥重要作用,通过蛋白水平研究发现HSPA9B在2～4细胞期胚胎、4～8细胞期胚胎和桑椹胚中,胞核HSPA9B的表达量较胞质的表达弱,在囊胚中胞核HSPA9B的表达量较胞质的表达强;在体外培养条件下,牦牛体外受精胚胎发育至囊胚时,HSPA9B蛋白的分布可能发生了移位。HSPA12B在牦牛体外受精胚胎的发育过程中非常重要,可能起着抑制细胞凋亡,延缓细胞衰老,调控胚胎早期发育的作用。Tp53在调节牦牛早期胚胎发育中也起着非常重要的作用,Tp53的表达量与胚胎发育阻断的发生几率成正相关。HSPA9B、HSPA12B和Tp53在参与牦牛体外早期受精胚胎发育和凋亡过程中起重要作用。

关键词：牦牛体外早期受精胚胎 HSPA9B HSPA12B Tp53

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Establishment and evaluation of an immortalized steroidogenic goat granulosa cell line as a model system to study the endoplasmic reticulum(ER)-stress response on steroidogenesis

Establishment and evaluation of an immortalized steroidogeni...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者：杨迪琦王磊 Pengfei Lin Tingting Jiang Nan Wang Fan Zhao Huatao Chen Keqiong Tang Dong Zhou Aihua Wang 靳亚平 Key Laboratory of Animal Biotechnology of the Ministry of Agriculture Northwest A&F University College of Veterinary Medicine Northwest A&F University

Under the support of granulosa cells and theca cells,the ovaries are responsible for producing oocytes and secreting sex steroids such as estrogen and *** reticulum stress(ERS) plays an important role in follicle atresia and embryo *** this study,goat granulosa cells were isolated from medium-sized(4-6 mm) healthy *** primary granulosa cells were immortalized by transfection with human telomerase reverse transcriptase(hTERT) to establish a goat granulosa cell line(hTERT-GGCs).Test results showed that hTERT-GGCs expressed hTERT,and a relatively long telomere existed at passage ***,the hTERT-GGCs expressed gonadotropin receptors CYP11A1,StAR and CYP19A1,which are involved in *** addition,the concentration of progesterone was detectable in *** the proliferation potential of hTERT-GGCs significantly improved,there was no evidence to suggest that the hTERT-GGCs are *** addition,thapsigargin(Tg)treatment led to a significant decrease in the progesterone concentration and the expression of steroidogenic enzyme in a dose-dependent *** summary,in this study we successfully generated a stable goat granulosa cell *** this cell line we found that Tg induced ERS,which reduced progesterone production and steroidogenic enzyme expression in *** studies may benefit from using this cell line as a model to explore the molecular mechanism regulating steroidogenesis and apoptosis in goat granulosa cells.

关键词： Establishment and evaluation of an immortalized steroidogenic goat granulosa cell line as a model system to study the endoplasmic reticulum stress response on steroidogenesis

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miR-101 Regulates Development of Bovine SCNT Embryos via Targeting ING3

miR-101 Regulates Development of Bovine SCNT Embryos via Tar...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： Rongxia Xie Fusheng Quan College of Veterinary Medicine Northwest A&F UniversityKey Laboratory of Animal Biotechnology of the Ministry of Agriculture

The SCNT(somatic cell nuclear transfer) technique is a key technology of modern biotechnology,has been widely used in livestock breeding of excellent individuals,transgenic technology and ***,due to incomplete reprogramming factors,the low birth rate of cloned animals and the high proportion of abnormal individuals affect the further development of SCNT *** evidence suggests that some microRNAs(miRNAs) are involved in the development potential of SCNT *** the abundant miRNA-101 in SCNT donor cells and bovine oocytes,numerous studies have demonstrated that miR-101 can regulate cell proliferation and ***,its roles in bovine SCNT donor cells and embryo development are not *** order to elucidate the roles of miR-101 in bovine SCNT donor cells and embryos,miR-101 was overexpressed in donor cells and the effects on the donor cell proliferation and embryo development were ***,luciferase reporter and western blot assays identified ING3 as a novel target of miR-101 in bovine *** results of Luciferase Reporter demonstrated that the 3'-UTR in ING3 mRNA contained a complementary site for the seed region of *** protein level of ING3 was detected by western blot and the result showed that miR-101 could down-regulate ING3 protein ***,real time PCR and flow cytometry methods were introduced to examine the effects of miR-101 on gene transcripts and cell *** results showed miR-101 reduced ING3 mRNA level,and also inhibited expression of P53,P21,UHRF1,BAX and DNMT3 *** addition,miR-101 promoted the growth of bovine fetal fibroblasts and decreased its ***,the effects of miR-101 overexpression on the development of SCNT embryos and blastocyst quality were *** utilizing donor cells with overexpressed miR-101,the blastocyst rate of bovine SCNT embryos in miR-101 group(35.75%) was significantly higher than the control group(26.06%)(P <0

关键词： miR-101 ING3 SCNT bovine embryo bovine fetal fibroblasts

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Prevalence of virulence and resistance to antibiotics in pathogenic enterococci isolated from mastitic cows

Prevalence of virulence and resistance to antibiotics in pat...

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中国畜牧兽医学会兽医产科学分会第十三次学术研讨会

作者： Xiaohu Wu Quanwei Zhang Youji Ma Yong Zhang Xingxu Zhao College of Veterinary Medicine Gansu Agricultural University College of Life Science & Technology Gansu Agricultural University College of Animal Science & Technology Gansu Agricultural University

Multidrug-resistant enterococci have become increasingly common in human and veterinary medicine and are causing a world wide health *** are commonly found in animal intestines and their feces and were considered commensal organisms until the late 1970s when antibiotics were widely used in both humans and *** prevalence and mechanisms of antibiotic resistance and virulence profiles on enterococcifirom human medicine have been widely examined, whereas reports on enterococci in animal disease arelimited, particularly reports concerning enterococcal dairycow mastitis in *** the present study, we investigated the virulence profile,antibiotic patterns, and pathogenicity of enterococciisolated from mastitic milk and evaluated their effects ondairy cows and public *** prevalence of enterococci was examined in 280 milk samples collected from dairy cattle diagnosed with mastitis in threeprovinces of western China. Sixty strains of enterococci were isolated and the species were determined based on their biochemical charactersand 16 S rRNA sequences. Resistance to seven antibiotic agents, frequency of seven virulence genes, and pathogenicity in Kunmingmice were tested to evaluate biological risks. The correlation between the number of virulence genes and pathogenicity in Kunming micewas also evaluated. The 60 isolates were allocated to Enterococcus hirae(68.3%), ***(25.0%), E. mundtii(3.3%), and E. durans(3.3%). A total of 83.3% of the isolates were resistant to penicillin, whereas 15.0% were resistant to ampicillin, 15.0% to vancomycin,6.7% to tetracycline, and 25.0% to ciprofloxacin. Moreover, isolates exhibited 50.0% and 21.7% resistance to high levels of gentamycinand streptomycin, respectively. The gene asal was detected in all enterococcal isolates, whereas 66.7% of strains harbored three or morevirulence factors and 56.7% were asal-ccf-gelE-positive. In pathogenicity tests, isolates harboring numerous virulence factors did not sh

关键词： antibiotic resistance pathogenic enterococci prevalence virulence

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