Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone-induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

In the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.     

猪蛋白酪氨酸磷酸酶-1B基因第7内含子多态性与猪脂肪沉积性状的关联性

本研究旨在分析蛋白酪氨酸磷酸酶-1B(PTP-1B)基因部分SNP与猪脂肪沉积性状的关联性。选取国外瘦肉型猪种大约克夏16头、长白猪21头和中国脂肪型地方猪种梅山猪22头以及"大白×梅山"F2代资源家系213头为研究材料,利用PCR-Eco88I-RFLP方法检测PTP-1B基因第7内含子序列存在G/A突变的多态性,并进行方差分析。结果表明,大白和长白猪种为单一的A等位基因,而梅山猪种以杂合子GA基因型为主,对213头"大白×梅山"F2代资源家系进行性状关联分析,该位点与猪板油质量、内脂率、胸腰椎间背膘厚等脂肪沉积性状显著相关(P<0.05),且杂合子GA基因型个体的板油质量和内酯率都最低(GA基因型个体相似文献   

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

Phosphorylation of histone H3 on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes

Background

The p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; however, little is known about its role in porcine oocytes.

Result

Total p21-activated kinase 1 (PAK1) and phosphorylated PAK1 at Thr423 (PAK1^Thr423) were consistently expressed in porcine oocytes from the germinal vesicle (GV) to the second metaphase (MII) stages, but phosphorylation of histone H3 at Ser10 (H3^Ser10) was only expressed after the GV stage. Immunofluorescence analysis revealed that PAK1^Thr423 and H3^Ser10 colocalized on chromosomes after the GV stage. Blocking of endogenous PAK1^Thr423 by injecting a specific antibody decreased the phosphorylation level of H3^Ser10; however, it had no impact on chromatin condensation, meiotic progression, cleavage rate of blastomeres or the rate of blastocyst formation.

Conclusion

Phosphorylation of PAK1^Thr423 is a spontaneous activation process and the activated PAK1^Thr423 can promote the phosphorylation of H3^Ser10; however, this pathway is not required for meiotic maturation of porcine oocytes or early embryonic development.     

Effect of sex steroids on expression of sulfotransferase 2B1 immunoreactive protein in primary cultured porcine hepatocytes

An excessive accumulation of androstenone (5α-androst-16-en-3-one) in pig adipose tissue is one of the two major contributors to the phenomenon of boar taint. High levels of adipose tissue androstenone have been related to a low rate of hepatic androstenone metabolism, which includes two stages: oxidative and conjugative. Sulfotransferases (SULTs), alongside with other specific enzymes, play the key role in the conjugative stage of androstenone metabolism. The present study investigated the mechanism regulating expression of sulfotransferase 2B1 (SULT2B1) immunoreactive protein using primary cultured pig hepatocytes as a model system. A specific objective was to determine whether the expression of pig hepatic SULT2B1 is regulated by the sex steroids; androstenone, testosterone and estrone sulphate. The study was performed on entire male pigs of a Large White (40%) × Landrace (40%) × Duroc (20%) cross-breed, average carcass weight 72.2 kg. The study shows that SULT2B1 immunoreactive protein expression can be induced by testosterone (final concentrations, 10 and 500 nM) and repressed by estrone sulphate (final concentration, 100 nM). Androstenone had no significant effect on SULT2B1 immunoreactive protein expression in the range of concentration, 10 nM to 1 μM. Time-courses (0 to 48 h) of steroid effects were investigated. The maximum effects of testosterone and estrone sulphate were observed in 24 h after the steroid treatments. This study provides direct evidence for involvement of sex steroids in the regulation of porcine hepatic SULTs.     

融合表达猪圆环病毒2型ORF1和ORF2基因真核质粒的构建及对小鼠的免疫原性

用PCR法扩增出猪圆环病毒2型的Rep蛋白基因（933bp）和Cap蛋白基因（705bp）,将其定向克隆于真核表达载体pcDNA3.1（＋）的多克隆位点上,构建真核表达质粒pcDNA-ORF1-ORF2。将构建好的重组质粒pcD-NA-ORF1-ORF2按100μg/只腿部肌肉注射BALB/c小白鼠,同时设pcDNA-ORF1、pcDNA-ORF2、pcDNA3.1-（＋）、PCV2全毒疫苗和PBS为对照,共免疫2次,间隔2周。分别于首免后第0、7、14、21、28、42天用MTT法检测小鼠脾淋巴细胞的增殖效应,用ELISA法检测小鼠的抗体水平;并于首免后第0、7、14、21、28天测定脾淋巴细胞中各细胞亚群的比例,对该核酸疫苗的免疫原性进行初步评价。结果显示,重组质粒能诱导鼠体产生较强的细胞免疫和体液免疫,并从免疫后第7天起所测各组数据均显著高于（P〈0.05）或极显著高于（P〈0.01）其他试验组。结果表明,将ORF1和ORF2基因共同用于PCV2核酸疫苗的研发具有很好的前景,为研究新型猪圆环病毒疫苗奠定了基础。     

猪HSD11B1和MyoG基因多态性与妊娠期长短的关系

选择HSD11B1和MyoG2个基因作为影响妊娠期长短的候选基因。在7个中外猪群体中采用PCR-RFLP技术研究了HSD11B1和MyoG2个基因多态性与妊娠期长短的关系。HSD11B1基因的PCR-Bsh1236Ⅰ-RFLP分析结果表明：清平猪、新清平猪母系、大白猪、长白猪、杜洛克猪、中国瘦肉猪新品系DIV1和DIV2系猪群中A等位基因的频率分别为0.538、0.752、0.522、0.941、1.000、0.889和0.594;除DIV2外,HSD11B1不同基因型与妊娠期长短无显著关系（P〉0.05）。MyoG基因的PCR-MspⅠ-RFLP分析结果表明：清平猪、新清平猪母系、大白猪、长白猪、杜洛克猪、中国瘦肉猪新品系DIV1和DIV2系猪群中M等位基因的频率分别为1.000、0.534、0.370、0.115、0.094、0.382和0.243;清平猪中只存在MM基因型,其他群体中3种基因型皆有分布;新清平猪母系MM比NN基因型母猪妊娠期要短,且在经产长白猪中差异极显著（P〈0.01）。     

Prevalence of serum neutralising antibody to equine rhinitis A virus (ERAV), equine rhinitis B virus 1 (ERBV1) and ERBV2

The objective of this study was to determine the incidence of serum neutralising (SN) antibody to ERAV, ERBV1 and ERBV2 in a population of horses from birth to 22 years of age. The prevalences of ERAV, ERBV1 and ERBV2 SN antibodies in 381 sera obtained from 291 horses were 37%, 83% and 66%, respectively. ERAV, ERBV1 and ERBV2 maternal antibody was present in foals 12 h postsuckling but by 10-12 months, ERAV SN antibody was not detected in any of the horses, while ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively). Sera were obtained from 44 Thoroughbred horses when they were newly introduced into a training centre when their average age was 23 months and a second sample was obtained approximately 7 months later. ERAV SN antibody was present in 8 (18%) when first bled and in 27 (61%) when tested 7 months later. Accordingly 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. Among all the horses the average ERAV SN antibody titre was relatively high (3796) and in contrast, ERBV1 and ERBV2 titres were relatively low (average 84 and 45, respectively) and often fell to below detectable levels over time and at a rate comparable to new seroconversions in the same group of horses.     

几种脱毒剂对黄曲霉毒素B_1和玉米赤霉烯酮体外吸附脱毒效果的比较研究

文章采用酶联免疫法(ELISA),分析比较了市场上10种常见脱毒剂对黄曲霉毒素B1和玉米赤霉烯酮的体外脱毒能力。结果显示,不同的脱毒剂脱毒效果不同,且经过特殊方式处理后的蒙脱石不仅对黄曲霉度B1和玉米赤霉烯酮具有较好的吸附脱毒能力,其复合物稳定性也较好。     

文昌鸡MTNR1B基因的多态性及其与产蛋性状的关系

本研究旨在了解褪黑激素受体基因MTNR1B的多态性及其与文昌鸡产蛋性状的关系。用直接测序的方法检测MTNR1B5′调控区、外显子1和外显子2的单核苷酸多态性,并将其与文昌鸡的产蛋性状进行关联分析。结果表明,在外显子1和2的编码区未发现碱基突变或缺失,而在5′调控区发现15个单核苷酸多态位点。其中-778位点突变(A→G)对文昌鸡中期(22～31周龄)产蛋数和46周龄总产蛋数的显性效应分别达到了2.3和6.2枚(P<0.05)。对后期(32～46周龄)产蛋数和46周龄总产蛋数而言,-216位点C对A为优势等位基因,加性效应值分别为2.5和3.2枚,显性效应分别为3.5和6.8枚,且表现为超显性。在4种单倍型中,AA单倍型个体的后期产蛋数和46周龄总产蛋数最低,GC单倍型个体的开产日龄最早。AC/GA联合基因型母鸡拥有最高的中期产蛋数、后期产蛋数和46周龄总产蛋数。综上所述,MTNR1B基因的外显子高度保守,而5′调控区的SNPs有望成为影响产蛋性状潜在的遗传标记。     

Growth performance and puberty attainment in growing pigs fed dietary fumonisin B1

Twenty‐four male weaned piglets assigned to four diets containing 5.0, 10.0, 15.0 and 0.2 mg fumonisin B₁ (FB₁) /kg as diets 1, 2, 3 and control diet, respectively, were used to study the effect of dietary FB₁ on growth and puberty attainment in pigs in a 6‐month feeding trial. Lower feed intake during 0–4 months and a non‐significant (p > 0.05) but FB₁ concentration‐dependent decrease in live and DWGs in animals fed FB₁‐contaminated diets was observed at the end of the pubertal phase. The daily and the final live weight gains of animals fed diet 3 were 75.8% and 90.6%, respectively, of the control values. The mean ages at puberty by boars on diets 2 and 3 were significantly (p < 0.05) higher than those for animals on the control and diet 1. The animals on diet 3 attained puberty when mean live weight was 60.1 kg, some 30.3 days after the controls attained puberty, at 156.3 days, when the mean live weight was 46.9 kg. This study revealed that dietary FB₁ delays sexual maturity in growing pigs. Male weanling pigs for breeding should not be exposed to dietary FB₁ higher than 5 mg/kg for optimum growth and reproductive performance.     

猪繁殖与呼吸综合征病毒GP5蛋白-重组犬1型腺病毒构建及在猪体内免疫特性

本试验根据猪繁殖与呼吸综合征病毒(PRRSV)基因组GP5蛋白的免疫原性,将PRRSV河北分离株GP5基因的表达盒克隆到犬1型腺病毒的感染性基因组的复制非必需区内,转染MDCK细胞,获得了重组病毒,免疫新生仔猪,分别在免疫后0～12周采集血清,通过ELISA检测证明,猪体同时产生了针对犬1型腺病毒和PRRSVGP5的抗体.说明GP5-重组犬1型腺病毒具有作为蓝耳病疫苗的潜力.     

贵州黑山羊独立生长因子1B基因cDNA克隆与生物信息学分析

选择与羊同源性较高的牛独立生长因子1B(GFI1B)基因序列设计特异性引物,提取贵州黑山羊脾脏总RNA,通过RT-PCR技术对GFI1B基因进行克隆测序及序列分析。结果表明:首次克隆了贵州黑山羊GFI1B基因cDNA序列996 bp,GenBank登录号为JN662390,编码331个氨基酸。贵州黑山羊GFI1B基因与牛的同源性高达97.0%。聚类分析显示:哺乳动物、禽类和两栖类各为一类。生物信息学分析表明:山羊GFI1B分子包括1个低组分复杂性区域、6个锌指结构域ZnF-C4。同时,预测结果还表明GFI1B分子不包含信号肽序列且没有跨膜螺旋结构域,26个磷酸化位点,蛋白糖基化位点有7个。     

Aflatoxin B1 decreased flesh flavor and inhibited muscle development in grass carp(Ctenopharyngodon idella)

In nature,aflatoxins,especially aflatoxin B1(AFB1),are the common mycotoxins,which cause serious health problems for humans and animals.This paper aimed to study the effects of AFB1 on flesh flavor and muscle development of grass carp(Ctenopharyngodon idella)and its mechanism.There were 1440 individual fish in total,with 6 treatments and each treatment replicated 3 times.The 6 treatments were fed a control diet with different doses of AFB1(0.04,29.48,58.66,85.94,110.43 and 146.92 μg/kg diet)for 60 d.AFB1 increased myofiber diameter,as well as decreased myofiber density of grass carp muscle(P＜0.05).The contents of free amino acid decreased gradually(P＜0.05)as dietary AFB1 increased in the muscle of grass carp.The levels of reactive oxygen species,malonaldehyde and protein carbonyl(PC)were increased(P＜0.05)with the dietary AFB1 increased.The levels of antioxidant enzyme(glutathione peroxidase,glutathione,glutathione reductase,total antioxidant capacity,anti-superoxide anion,and anti-hydroxyl radical)were decreased(P＜0.05)with the dietary AFB1 increased.In addition,dietary AFB1 decreased the content of collagen,and downregulated the mRNA and protein levels of transforming growth factor-β(TGF-β)/Smads signaling pathway in grass carp muscle(P＜0.05).The mRNA and protein levels of myogenic regulatory factors were downregulated in grass carp muscle(P＜0.05).Furthermore,the activities of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9)were increased(P＜0.05),and the protein levels of phosphorylate-38 mitogen-activated protein kinase(p-p38MAPK),phosphorylate-c-Jun N-terminal kinase,urokinase-type plasminogen activator(uPA),MMP-2 and MMP-9 were upregulated(P＜0.05),but collagen I,laminin β1 and fibronectin were downregulated(P＜0.05)with the dietary AFB1 increased in the muscle of grass carp.Based on the results of this study,we can draw the following conclusion:dietary AFB1 might damage flesh flavor and inhibit the muscle development through MAPK/uPA/MMP/extracellular matrix(ECM)signaling pathway in grass carp.Moreover,the recommended safe limit of AFB1 in feed is no more than 26.77 μg/kg diet according to the PC levels in grass carp muscle.     

共表达与牛疱疹病毒1型VP22融合的猪繁殖与呼吸综合征病毒E及M蛋白的重组伪狂犬病毒的构建及其免疫原性观察

为了研制猪繁殖与呼吸综合征病毒（PRRSV）基因工程疫苗，以伪狂犬病毒（PRV）gE基因缺失标志疫苗株TK^-／gE^-／LacZ^＋为病毒载体，通过同源重组，构建了共表达与牛疱疹病毒1型VP22（BHV-1 VP22）融合的PRRSV E及M蛋白的重组伪狂犬病毒（rPRV）TK^-／gE^-／VP22E^＋／VP22M^＋。经PCR、Southern blot、Western blot证实rPRV构建正确，并能表达与BHV-1 VP22融合的PRRSV E及M蛋白。rPRV在IBRS-2、PK-15细胞中的增殖滴度与PRV亲本株相比无显著差异，表明外源基因的插入不影响rPRV增殖。用该rPRV免疫BALB／c小鼠,检测免疫小鼠抗PRRSV中和抗体及脾淋巴细胞增殖反应，并与未融合VP22的单表达PRRSV E蛋白及共表达E及M蛋白的rPRV TK^-／gE^-／E^＋与TK^-／gE^-／E^＋／M^＋进行比较。结果显示TK^-／gE^-／VP22E^＋／VP22M^＋可诱导小鼠产生更好的体液与细胞免疫反应，BHV-1 VP22发挥了佐剂效应。本研究为研制安全、有效的猪繁殖与呼吸综合征-伪狂犬病二价基因工程疫苗奠定了基础。     

黄曲霉毒素B1、复方中药和硒对肉鸡肝组织匀浆中自由基代谢的影响

为观察黄曲霉毒素B1(AFB1)对肉鸡肝组织匀浆自由基代谢的变化,并研究复方中药与硒对AFB1的拮抗效应,将10日龄120只试验肉鸡随机分成4组,每组30只,分别为Ⅰ组(对照组)、Ⅱ组(AFB1组:AFB10.2mg/kgBW)、Ⅲ组(AFB10.2mg/kgBW+亚硒酸钠2mgSe/kgDM)和Ⅳ组(AFB10.2mg/kgBW+复方中药20g/kgDM)。分别处理后于17、24、31、38日龄宰杀5只肉鸡,提取肝组织,检测肝组织中的总抗氧化能力(T-AOC)、总超氧化物歧化酶(T-SOD)活性和过氧化氢酶(CAT)及丙二醛(MDA)含量的变化。结果显示,口服AFB1(0.2mg/kgBW)后T-SOD、T-AOC和CAT活性降低(P<0.05),MDA含量明显升高(P<0.05),而复方中药与硒治疗组T-SOD、T-AOC和CAT活性升高(P>0.05或P<0.05),MDA含量明显下降(P>0.05或P<0.05)。证实,由自由基增多而引起的脂质过氧化损伤在肉鸡AFB1慢性中毒发病机理中起着重要的作用,复方中药与硒均可有效降低脂质过氧化造成的损伤。     

连续低剂量黄曲霉毒素B1对大鼠肝肾损伤作用

用含有不同质量浓度黄曲霉素B1（AFB1）（0、4、20、25、50μg／kg）的污染饲料连续饲喂各试验组大鼠60d,于7、15、30、60d静脉取血,测定各组大鼠肝功能与肾功能生化指标。每周检测大鼠体质量,试验第60天断头处死大鼠,检测肝肾组织病理学变化并计算各组大鼠的脏体系数。试验期间备组受试动物未发现明显的中毒症状和异常情况,60d内无受试动物死亡。与对照相比,不同剂量组AFB1对大鼠体质量无显著性影响;连续喂饲7d,大鼠血清AST和BuN含量显著升高（P〈0．05）;连续喂饲15d,大鼠血清AsT、BUN和ALB含量显著升高（P〈0．05）,连续喂饲30d,大鼠血清BUN含量显著升高（P〈0．05）,连续喂饲60d,大鼠血清CR和BUN含量显著升高（P〈0．05）。在各测试质量浓度条件下,大鼠虽未发现明显的中毒症状和异常情况,试验期间无动物死亡,但与空白对照相比,各剂量组AFBl对大鼠均产生了显著的肝肾组织病理学损伤,中毒程度与给毒剂量成正相关性。随着毒素暴露时间延长,大鼠肝脏对低剂量AFBl表现出一定的耐受性,而大鼠的肾脏损伤与毒素暴露时间表现出显著的积累效应。     

血管紧张素转化酶2和中性氨基酸转运载体BoAT1在不同日龄猪肠道中的表达差异

研究了肠道血管紧张素转化酶2(ACE2)和中性氨基酸转运载体B0AT1在不同生长阶段猪小肠各段中的分布表达,探讨其相互关系。研究选取3日龄仔猪、90日龄生长猪和120日龄成年猪十二指肠、空肠和回肠组织,采用体外扩增和荧光定量技术对各肠段组织中ACE2和B0AT1基因进行了定性和定量分析。结果显示ACE2基因在不同日龄猪小肠各段均有表达,相对表达量随日龄增长显著升高,120日龄猪表达量极显著上调。而肠道中B0AT1相对表达量逐渐下降,120日龄时显著下调。结果提示:ACE2在成年猪肠道组织中有较高表达,B0AT1在仔猪肠道组织中表达较高。     

福建省羊传染性脓疱病毒F1L、B2L和VIR基因的遗传变异分析

为探明2014年-2015年在福建省流行的羊传染性脓疱病毒(ORFV)遗传变异情况,对10株ORFV流行毒株的F1L、B2L和VIR基因进行克隆、测序及分析。结果表明,10株ORFV F1L基因之间的核苷酸序列同源性为97.6%~100%,与国内株的核苷酸序列同源性为96.8%~99.7%,与NZ2参考株的核苷酸序列同源性为96.3%~97.1%;同NZ2参考株比较,FJ-YT2014缺失2个氨基酸;10株ORFV B2L基因之间核苷酸序列同源性为97.5%~99.9%,与国内株的核苷酸序列同源性为96.7%~99.5%,与NZ2参考株的核苷酸序列同源性为96.7%~97.7%;10株ORFV VIR基因之间的核苷酸序列同源性为95.8%~99.5%,与国内株的核苷酸序列同源性为94.6%~99.6%,与NZ2参考株的核苷酸序列同源性为94.6%~96.4%。基于基因核苷酸序列的遗传进化分析表明,10株ORFV F1L基因与福建省分离株、山西株和新疆株亲缘关系较近;10株ORFV B2L基因与新疆、山西、德国毒株亲缘关系较近;10株ORFV VIR基因与台湾、新疆株亲缘关系较近。结果提示,当前福建省流行的ORFV F1L、B2L和VIR基因尚未出现明显变异,但是其F1L、B2L和VIR基因核苷酸序列之间普遍存在异质性。