The effect of some cryoprotectants on dromedary camel frozen‐thawed semen

The cryopreserved camel semen is often associated with poor quality and fertility. This study aimed to improve the dromedary frozen semen quality by comparing the efficiency of four cryoprotectant agents (CPAs) on sperm freezability. Semen samples were collected from seven male Maghrabi camels, diluted with Shotor diluent supplemented with glycerol (Sh‐G), dimethyl formamide (DMF, Sh‐DF), dimethyl sulfoxide (DMSO, Sh‐DS) or ethylene glycol (EG, Sh‐EG), all at 6% final concentration, and the samples were subjected to cryopreservation. The results revealed the superiority of Sh‐DF over Sh‐G and Sh‐DS in terms of post‐thaw motility (55.83 ± 2.20 vs. 47.50 ± 4.33 and 45.00 ± 2.89%, respectively), sperm membrane (49.00 ± 0.58, 39.33 ± 3.33 and 42.67 ± 1.45%, respectively) and acrosomal integrities (53.00 ± 0.58, 57.33 ± 0.88 and 52.33 ± 1.45%, respectively). Sh‐EG group showed the lowest post‐thaw motility, plasma membrane and acrosome integrities (12.50 ± 1.44, 22.67 ± 1.45 and 30.67 ± 1.45, respectively). In conclusion, the protocols of dromedary camel semen cryopreservation could be enhanced using 6% DMF as a cryoprotectant agent.     

牛朊蛋白27-30基因的克隆与表达

利用PCR技术，从含有牛朊蛋白（Prion protein，PrP）基因Prnp的开放阅读框的克隆质粒BoPrnp—T中扩增出约420bp的目的基因（PrP猢基因）。将PrP^27-30基因和载体pPIC9K分别用限制性核酸内切酶EcoRⅠ和NotⅠ进行双酶切，T4DNA连接酶作用后，转化至E．coli JM109中，构建重组表达载体pPIC9K-boPrP^27-30。pPIC9K-boPrP^27-30经限制性核酸内切酶SalⅠ线性化后电转至毕赤酵母GS115中，经G418筛选后得到高拷贝的重组菌株GS115／pPIC9K-boPrP^27-30。GS115／pPIC9K-boPrP^27-30经1．0％甲醇诱导后，表达产物用SDS-PAGE和Western blot分析，结果表明牛PrP^27-30基因在毕赤酵母细胞中获得表达，表达产物的分子量约为27Ku，能够被单克隆抗体SAF-70识别。     

日粮营养对动物脂肪组织基因表达的影响

脂肪组织是一个强大的分泌器官,它能分泌许多与能量、脂肪代谢相关的酶、激素等.调控能量代谢和脂肪组织生长.本文综述了日粮对动物脂肪组织中基因表达作用的研究状况.并阐述了营养成分对这些基因上下游表达的作用机制.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

牛传染性鼻气管炎病毒gE基因的截短克隆与表达

以牛传染性鼻气管炎病毒Baaha Nu/67株的DNA作为模板，用PCR扩增gE基N并克隆至pGEM-T Easy裁体，再以此质粒作为模板将gE基因分成6个片段，分别插入原核表达载体pET32a并在大肠杆菌中进行了表达。蛋白电泳结果表明6个片段中有2个片段以可溶形式表达，1个片段以包涵体形式表达，另外3个片段没有表达。采用固定化金属离子亲和层析法在非变性条件下对两个可溶性片段进行了纯化。经免疫印迹试验，间接ELISA和交叉试验证明，两个纯化的重组蛋白均与牛传染性鼻气管炎阳性血清样品发生反应，而与牛传染性鼻气管炎阴性血清无任何反应，显示其具有良好的抗原性和特异性，可用于牛传染性鼻气管炎gE-ELISA诊断方法的建立。     

The effects of vitrification after equilibration in different concentrations of cryoprotectants on the survival and quality of bovine blastocysts

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Re‐expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane‐damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN‐t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.     

牛副流感病毒3型核衣壳蛋白基因的真核表达

根据牛副流感病毒3型(BPIV-3)内蒙09株(NM09)全基因组序列(GenBank登录号:JQ063064)设计特异性引物,利用RT-PCR方法扩增出牛副流感病毒3型分离株的核衣壳蛋白(N)基因,通过NheI和NotI限制性内切酶位点亚克隆至真核表达载体pcDNA3.1/Zeo(+),获得真核重组质粒pcDNA3.1-N。采用Superfect转染试剂将重组质粒转染至BSR细胞中,转染后的BSR细胞经间接免疫荧光试验和RT-PCR方法检测,重组质粒pcDNA3.1-N在BSR细胞中能正确表达N蛋白。本研究结果为BPIV-3新型疫苗的研制奠定基础。     

牛传染性鼻气管炎病毒糖蛋白gC基因抗原活性区的克隆与表达

通过聚合酶链式反应从牛传染性鼻气管炎病毒Bartha Nu/67株中扩增得到病毒gC基因并克隆至T载体pMD20T,再以后者为模板扩增gC蛋白第15~177位氨基酸对应的抗原活性区片段gCd。将gCd插入原核表达载体pET32a构建重组表达质粒pET32a-bhv1gCd。限制性内切酶以及序列分析鉴定表明重组表达质粒克隆片段序列与阅读框正确。对重组质粒转化大肠杆菌BL21(DE3)的培养物进行蛋白电泳,可检测到分子量约45ku的目的产物,IPTG诱导后5h表达量最高。免疫印迹试验结果证实,gCd重组蛋白与牛传染性鼻气管炎标准阳性血清发生特异性反应,表明gC抗原活性区片段在原核获得表达并具有良好的抗原性。该重组蛋白可用于建立ELISA等免疫诊断方法。     

Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 μmol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 μmol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.     

不同冷冻方法对牛卵母细胞及孤雌胚发育的影响

研究了麦管和OPS管法冷冻以及OPS法中保护剂种类对牛卵母细胞体外成熟及孤雌胚发育的影响。结果发现,OPS管冷冻牛卵母细胞形态正常率、卵裂率、囊胚率极显著高于麦管法(P<0.05)。在OPS法中,应用两种保护剂冷冻后,卵母细胞形态正常率、卵裂率、囊胚率差异极显著(P<0.01);采取38℃与25℃温度平衡,冷冻后卵母细胞各发育指标差异不显著(P>0.05);采用4℃平衡,冷冻后的卵母细胞激活后没有出现囊胚,各发育指标极显著降低(P<0.01)。结果表明,OPS法可以有效地保护卵母细胞,保证其后孤雌激活;采用低温平衡会对孤雌发育的囊胚阶段有较大影响。     

In vitro development of OPU‐derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen‐thawed embryos after transfer

The optimization of single‐embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick‐up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen‐thawed embryos after a direct transfer. When two‐cell‐stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen‐thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen‐thawed embryos after transfer to recipients.     

鸭毛囊中ASIP基因片段的克隆及组织表达分析

根据GenBank发表的鸡刺鼠信号蛋白(ASIP)基因序列的同源保守区域,设计了1对特异性引物。以番鸭、高邮鸭和英系北京鸭(即樱桃谷鸭)毛囊RNA为模板,经扩增、测序及拼接得到的部分mRNA序列均为编码序列,共308bp,对应编码102个氨基酸。与已发表的原鸡和鹌鹑的ASIP对应的mRNA核苷酸序列进行比对,番鸭、高邮鸭和英系北京鸭的同源性均在92.53%以上,相应编码的氨基酸序列同源性均高于92.16%。高邮鸭与英系北京鸭核苷酸,氨基酸序列一致,同源性为100%。半定量PCR结果显示,番鸭ASIP基因表达具有较高的普遍性,在皮肤组织(毛囊和皮肤)和中枢神经系统(下丘脑)及其他非皮肤中均有表达。皮肤和毛囊的表达差异不显著(P>0.05),下丘脑中的ASIP基因相对表达量最高,极显著高于皮肤中ASIP基因的表达量(P<0.01),而与毛囊中的差异不显著(P>0.05)。     

猪ERK6基因编码区的克隆及组织表达谱分析

利用RT-PCR方法扩增了猪ERK6基因编码区的序列，将扩增产物与pMD18-T载体连接，构建了重组质粒；重组质粒经PCR、酶切鉴定后进行测序；采用Northern杂交和半定量RT-PCR方法在猪不同部位骨骼肌中分析了ERK6基因转录本的个数、大小及组织表达谱。结果显示，所克隆的猪ERK6基因片段长1113bp，含有1个1104bp的开放阅读框（ORF），该ORF编码367个氨基酸；该基因与已报道的人ERK6基因核苷酸序列相似性为90％；猪ERK6基因在肌肉组织中只有一个转录本，大小约1．5kb。此基因在骨骼肌中的表达量最高，心、子宫次之，卵巢最低；而在脂肪、胃、肝、脾、肺、肾、十二指肠和胰腺中未见表达。结果表明，猪ERK6基因与已报道的小鼠和人ERK6基因一样，主要在骨骼中特异表达。     

贵州山羊品种RERG基因的克隆及不同组织表达

选择与羊同源性较高的牛ras相关的雌激素调节的生长抑制因子(ras-related estrogen-regulated growth inhibitor,RERG)基因组序列设计特异性引物,通过RT-PCR技术对RERG基因进行克隆测序及生物信息学分析.结果显示,克隆了羊RERG基因cDNA序列629 bp,完整的开放阅读框(ORF)为20～620 bp,其编码199个氨基酸.GenBank登录号分别为JN672576、JQ917222和JN580309.通过实时荧光定量RT-PCR技术分析RERG基因在贵州三大地方品种同一年龄段不同组织中的表达情况.结果表明,成年羊肺脏和脾脏表达量是最高,胸腺表达量最低,肌肉表达量为相对中度表达.为进一步研究地方品种羊生长性状的改善提供科学依据.     

生糖底物和神经内分泌因子对新生犊牛肝细胞PEPCK-C mRNA表达水平的影响

在原代单层培养的新生犊牛肝细胞培养液中分别加入不同浓度丙酸钠、丙酮酸钠、胰岛素、胰高血糖素和瘦蛋白，培养12h后，应用半定量RT-PCR方法检测体外培养的肝细胞PEPCK—C mRNA的丰度。结果显示，随着丙酸钠、丙酮酸钠浓度的升高，肝细胞PEPCK-C mRNA的丰度均先升高后下降（P〈0．01）；随胰岛素、胰高血糖素和瘦蛋白浓度的升高，肝细胞PEPCK-C mRNA的丰度分别剂量依赖性地降低、升高（P〈0．01）和无显著变化。表明，丙酸钠、丙酮酸钠能通过上调体外培养的新生犊牛肝细胞PEPCK—C mRNA的表达而促进肝糖异生代谢，但上调作用是有限的；胰岛素能通过下调体外培养的新生犊牛肝细胞PEPCK—C mRNA的表达而抑制肝糖异生代谢，且下调作用呈剂量依赖性；胰高血糖素与胰岛素作用刚好相反；瘦蛋白未起直接的调节作用。     

牛源犬新孢子虫NcGRA7基因的克隆及原核表达分析

为了解牛源犬新孢子虫NcGRA7基因的生物学特性,本实验采用PCR技术扩增牛源犬新孢子虫NcGRA7基因,并连接至pMD-18T载体中,构建重组质粒pMD 18-NcGRA7,经PCR、酶切鉴定及测序分析,将NcGRA7基因连接到原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-NcGRA7,转化大肠杆菌BL21,筛选阳性克隆,将PCR和酶切鉴定正确的重组质粒进行IPTG诱导表达,SDS-PAGE和westem blot分析表达产物.结果表明,扩增的NcGRA7基因片段大小为701 bp,编码233个氨基酸,与GenBank中登录的NcGRA7(AF176649)核苷酸序列同源性为98.7％;western blot分析表达蛋白的分子量为52 ku,具有较好的反应原性.本实验为犬新孢子虫NcGRA7基因疫苗研究奠定了基础.