从江香猪生精上皮周期及睾丸发育的形态学分析

试验旨在探究初情期前后生精上皮周期差异及睾丸发育过程中的形态学变化。通过测定15、30、60和90 d睾丸相关指数,结合睾丸组织形态学特征,判断香猪初情期,划分从江香猪生精上皮周期。结果显示,30 d的睾丸指数较15 d极显著升高(P0.01),睾丸重、长轴及短轴的增长率分别为298.05%、66.42%和65.45%,60和90 d两个阶段睾丸重增长率相对稳定。形态学观察表明,从江香猪30 d时生精小管出现游离精子,完成第一次生精并进入初情期;与15 d相比,30 d生精小管面积和生精上皮厚度极显著增加(P0.01),增长率分别为136.12%和40.19%,在60和90 d均处于稳定增长状态。睾丸细胞数统计显示,日龄增加不影响支持细胞(setoli cells,SC)数量(P0.05),而30 d生殖细胞数(germ cells,GC)较15 d极显著增加(P0.01)。相关分析结果发现,生殖细胞数量增加与生精小管面积增大、生精上皮厚度变化之间呈明显正相关(r=0.994;0.96)。根据生殖细胞组合形式差异,将初情期前后生精上皮分为3和8个阶段。初情期前生殖细胞以第一次减数分裂前期为主,A、B型精原细胞、SC、初级精母细胞(primary spermatocyte,Ps)、前细线期(preleptotene,PI)、细线期(leptotene,L)等生殖细胞在初情期前后生精上皮中均存在,而圆形精子(round spermatids,R)、延伸精子(elongating spermatid,E)、精子细胞(spermatozoa,S)仅存在于初情期后的生精上皮。本研究结果表明,从江香猪30 d初情,睾丸发育以生殖细胞和生精小管面积的迅速增加为主,该结果对从江香猪早熟性状挖掘、种猪选育及开发利用等有重要的指导意义。     

香猪睾丸发育的形态学变化

为了解香猪睾丸发育过程中的形态学变化,外科手术取30、40、50、60、90、110日龄（每个年龄组n=3～4）香猪右侧睾丸经中性多聚甲醛固定,石蜡包埋,组织切片采用HE染色,40倍物镜下观察细胞数量变化,经显微照相后用Scion image软件测量生精小管、输出小管和附睾管的管壁面积。结果发现30～50日龄时睾丸生精...     

从江香猪附睾发育中凋亡相关因子Caspase-3的表达模式

细胞凋亡可及时清除机体内多余和受损伤的细胞,以维持组织器官的稳定性。为探讨凋亡过程在公猪附睾发育过程中发挥的潜在作用,本实验以从江香猪为研究对象,利用实时荧光定量PCR(qRTPCR)、免疫组织化学（IHC）和WesternBlot技术分析了附睾15d（初情前期）、30d（初情期）、60d（初情后期）和180 d（性成熟期）4个时期凋亡相关因子Caspase-3、Bax和Bcl-2的差异表达模式。qRTPCR结果显示：Casp3和Bax基因mRNA在从江香猪60d表达量最高,Casp3在15d表达量最低,15d和180 d差异不显著;Bax在180 d表达量最低,且15 d、60 d和180 d差异不显著;Bcl-2在60 d表达量最低,在15 d表达量最高,4个日龄段差异显著。免疫组化结果显示：Caspase-3蛋白在30 d主要表达在附睾管腔的微绒毛,而在180 d主要表达在管腔中的精子上。Western Blot结果显示：Caspase-3蛋白表达丰度依次为60 d>30 d>15 d>180 d,且各日龄段差异显著。综上,本实验发现不同日龄从江香猪附睾凋亡相关...     

贵州香猪睾丸发育中支持细胞和生精细胞数量变化观察

为了解香猪睾丸发育过程中生殖细胞和支持细胞数变化规律,用手术取出30,40,50,70,90和110日龄(每个年龄组n=3～4)香猪右侧睾丸,经中性多聚甲醛固定,石蜡包埋,组织切片采用免疫组化SP法,用单克隆抗体GATA-4检测睾丸支持细胞的特异生长转录因子-4,经DAB显色、苏木素复染。光镜下核呈棕色者为支持细胞,核呈蓝色者则为生殖细胞;经显微照相并用Scion image软件测量生精小管及管壁面积。结果:30～110日龄睾丸支持细胞数维持在稳定水平(P>0.05),而生殖细胞数随日龄增加而增多,70日龄生殖细胞数快速增多(P<0.05),持续到110日龄。同样,从70日龄开始睾丸生精小管和管壁面积显著性增大(P<0.05)。香猪睾丸支持细胞快速增殖发生在30日龄前,而生殖细胞数随着日龄的增长而增多。     

从江香猪气喘病血清学调查

为了解从江香猪气喘病(支原体)感染情况,2014年9月至2015年8月分别从江县和榕江县采集香猪血清共640份.用ELISA的方法对全部样品进行检测,结果显示,抗体阳性总数为526份,阳性率为82.19%.表明在从江县和榕江县香猪普遍存在猪支原体感染,因此,采取相应的防控措施,保证从江香猪的健康发展.     

促进从江香猪生产发展的技术措施及对策

香猪是我县的特色地方品种,它以其“体型矮小,肉质细嫩,基因纯合,纯净无污染”等特点而闻名全国,自1998年以来,为了发挥香猪品种资源优势,县委、县政府结合县情实际,把发展香猪生产作为农村脱贫致富的一项支柱产业来抓,在国家、省、地县的关心和重视下,先后投入600多万元,建成面积达600m2,年屠宰50万头猪的现代化加工生产线和贮存200t的冷库一座,组建香猪开发公司,研制出腊香猪、烤香猪、香猪香肠、香猪火腿,香猪腊肉、美味心肝等系列风味食品,在香猪     

从江香猪细小病毒血清学调查

为了解从江香猪细小病毒病感染情况,2014年9月至2015年8月分别从江县和榕江县采集香猪血清共640份.用ELISA的方法对全部样品进行检测,结果显示,抗体阳性总数为244份,阳性率为38.13%.表明在从江县和榕江县香猪存在不同程度的细小病毒病感染,因此,采取必要的防控措施,对香猪资源的保护与发展具有重要意义.     

高产从江香猪资源调查及其FSH基因型分析

对贵州省从江县养殖的从江香猪资源进行了全面的调查,发现当地存在特殊的高产从江香猪群体。对同龄的高、低产香猪的体尺进行对比,同时采用PCR分析2个群体的促卵泡激素β亚基(follicle stimulating hormone,FSHβ)的基因型,结果表明,高产从江香猪群体的产仔数明显高于低产群体,同时,从江香猪高产群体的体长、体重、胸围、腰围4项指标明显较高(P<0.05),纯合的AA基因型频率也较高。结果表明:高产从江香猪不仅产仔数多,同时生长速度较快,基因更为纯合。从江香猪高产群体的深入研究将为香猪资源的多方位开发利用奠定基础。     

从江香猪圆环病毒Ⅱ型血清学调查

为了解从江香猪圆环病毒病Ⅱ型感染情况,2014年9月至2015年8月分别采集从江县和榕江县非免疫香猪血清318份,用ELISA方法对全部样品进行检测。结果:抗体阳性率为66.04%(210/318)。表明在从江县和榕江县香猪主产区存在不同程度的圆环病毒Ⅱ型感染,且规模场的香猪感染率明显低于散养户。     

从江香猪SIM1基因SNPs筛查及生物信息学分析

本研究以从江香猪为研究对象,二元杂交猪(从江香猪×野猪)、外三元杂交猪(杜×长×大)为对照,采用DNA池和直接测序技术对3个群体的SIM1基因7个外显子、部分内含子以及3'非翻译区序列进行多态性检测;利用生物信息学软件预测多态位点对SIM1基因mRNA二级结构和蛋白质一级、二级结构的影响。结果表明,在3个群体的SIM1基因中筛查到12个SNPs,C77T位于第5外显子,T29186C、A29195C位于第9内含子,C63T、C225T位于第10外显子,C107T、A426G、T583C、A586C、A605C、A615C位于第11外显子,G267T位于3'非翻译区。其中C77T、T29186C、A29195C、C63T、C225T、C107T、G267T为同义突变,A426G、T583C、A586C、A605C、A615C为错义突变;5个错义突变分别导致异亮氨酸(Ile)变为缬氨酸(Val)、亮氨酸(Leu)变为脯氨酸(Pro)、谷氨酸(Glu)变为丙氨酸(Ala)、谷氨酸(Glu)变为天冬氨酸(Asp)、天冬酰胺(Asn)变为组氨酸(His);根据在线软件预测,突变前后的SIM1基因mRNA二级结构和蛋白质一级、二级结构均会发生改变。     

野乔菜替代部分基础日粮对从江香猪肌肉脂肪酸及氨基酸的影响

试验旨在探究野乔菜替代部分基础日粮对从江香猪肌肉脂肪酸和氨基酸的影响。选取体重约20 kg的从江香猪40头,随机分为4个组,每组10头。对照组饲喂基础日粮,试验前期试验Ⅰ、Ⅱ、Ⅲ组分别以野乔菜替代10%、20%和30%的基础日粮,试验后期分别用野乔菜替代20%、30%和40%的基础日粮。育肥期结束后,每组随机抽取3头屠宰,取背最长肌用于脂肪酸和氨基酸测定。结果显示:①野乔菜替代不同比例基础日粮对从江香猪肌肉脂肪酸含量有一定影响。与对照组相比,试验Ⅰ组亚油酸、试验Ⅰ、Ⅱ组亚麻酸、试验Ⅱ组花生二烯酸和试验Ⅰ组花生四烯酸分别提高了54.69%、43.57%、62.50%、25.00%和127.10%;各试验组中,饱和脂肪酸(SFA)、多不饱和脂肪酸(PUFA)及必需脂肪酸(EFA)总含量均高于对照组(P>0.05)。②野乔菜替代部分基础日粮对氨基酸含量有一定影响。对照组天门冬氨酸显著低于试验Ⅰ(P<0.05)、极显著低于Ⅲ组(P<0.01);试验Ⅲ组甘氨酸显著高于试验Ⅰ、Ⅱ组(P<0.05),且高于对照组(P>0.05),甲硫氨酸、赖氨酸含量分别显著高于对照组(P<0.05)和试验Ⅰ组(P<0.05);试验Ⅰ组EAA/TAA显著低于其他3组(P<0.05)。综上所述,野乔菜替代部分基础日粮能在一定程度上提高从江香猪肌肉PUFA和EFA含量,显著提高了部分氨基酸含量。     

Effects of Partial Substitution of Basal Diet with Wild Buckwheat on Fatty Acids and Amino Acids in Congjiang Xiang Pigs

This study investigated the effects of replacing partial basal diet with wild buckwheat on fatty acids and amino acids in longissimus dorsi muscles of Congjiang Xiang pigs.Forty Congjiang Xiang pigs with 20 kg initial body weight,were randomly assigned to 4 groups,10 heads per group.The pigs in control group were fed the basal diet.In the other groups (groupⅠ,Ⅱ and Ⅲ),diets were substituted with fresh wild buckwheat at 10%,20% and 30% on the early fattening stage and 20%,30% and 40% on the late fattening stage,respectively.After feeding trial,three pigs from each dietary treatment were slaughtered to determine the fatty acids and amino acids in longissimus dorsi muscle.The results showed that:①The partial substitution of basal diet with wild buckwheat had some effects on fatty acids in porcine muscle.Compared with the control group,the content of linoleic acid in the group Ⅰ,the content of linolenic acid in group Ⅰ and Ⅱ,the content of peanut diene acid of group Ⅱ and arachidonic acid of group Ⅲ were increased by 54.69%,43.57%,62.50%,25.00% and 127.10%,respectively.The saturated fatty acid (SFA),polyunsaturated fatty acid (PUFA)and essential fatty acid (EFA)of all testing groups were higher than that of control group (P>0.05).②The amino acids were affected in some degree by the substitution rate of wild buckwheat.The content of asparaginic acid in control group was significant lower than that in group Ⅰ(P<0.05) and extremely significant lower than that of group Ⅲ (P<0.01).The content of glycine in group Ⅲ was significantly higher than that of groupⅠ and Ⅱ (P<0.05),higher than that of control group (P>0.05).The content of methionine and lysine in group Ⅲ were significantly higher than that in control group and group Ⅰ (P<0.05),respectively.The EAA/TAA in group Ⅰ was significantly lower than the other groups(P<0.05).In conclusion,partial substitution of basal diet with wild buckwheat could increase the contents of PUFA and EFA,and improve the amino acid profile of Congjiang Xiang pigs.     

从江香猪γ干扰素基因克隆与序列分析

试验旨在研究从江香猪γ干扰素（interferon gamma,IFN-γ）基因克隆与序列分析。试验提取贵州从江香猪肝脏组织总RNA,反转录生成cDNA,采用2对特异性引物进行巢式PCR扩增从江香猪IFN-γ（CJ-poIFN-γ）基因编码区,将其克隆至pUCm-T载体上,获得重组质粒pUCm-CJpoIFN-γ,并测序鉴定;利用NCBI、SOPMA、SignalP-4.1等在线服务器软件、DNAStar软件对CJ-poIFN-γ进行序列分析。结果表明,CJ-poIFN-γ基因编码区长501 bp,编码166个氨基酸;核苷酸序列比对结果显示,CJ-poIFN-γ与梅山猪、剑白猪、藏猪、成华猪、荣昌猪、印度猪、长白猪、内江猪同源性为99.4%~100.0%;进化树分析结果显示,CJ-poIFN-γ与梅山猪、剑白猪、藏猪、成华猪亲缘关系较近;CJ-poIFN-γ基因编码蛋白不存在跨膜结构,为分泌蛋白,前23个氨基酸为信号肽序列;CJ-poIFN-γ基因编码蛋白二级结构主要以α-螺旋（50.60%）和无规则卷曲（33.14%）为主,B细胞表位主要位于62-65、84-87、113-115、144-156和162-166位氨基酸。试验结果为进一步研究IFN-γ的生物活性、加快从江香猪品种资源的有效利用奠定基础。     

SNPs Screening and Bioinformatics Analysis of SIM1 Gene in Congjiang Pigs

In this study, Congjiang pig was served as a research object, crossbred pigs (Congjiang pig×boar), outside crossbred pigs (Duroc×Landrace×Yorkshire) as controls.Using DNA pools and direct sequencing detected polymorphism of 7 exons, part of introns and 3'untranslated region (3'-UTR) sequences of SIM1 gene for three groups.Bioinformatics software predicted what impact polymorphic loci had to mRNA secondary structure and protein primary, secondary structure of SIM1 gene.The results showed that 12 SNPs were screened in SIM1 gene of three groups, C77T located in exon 5, T29186C and A29195C located in intron 9, C63T and C225T located in exon 10, and C107T, A426G, T583C, A586C, A605C and A615C located in exon 11, G267T located in 3'-UTR. C77T, T29186C, A29195C, C63T, C225T, C107T and G267T were silent mutations, A426G, T583C, A586C, A605C and A615C were missense mutations.The five missense mutations respectively led to isoleucine (Ile) into valine (Val), leucine (Leu) into proline (Pro), glutamate (Glu) into alanine (Ala), glutamic (Glu) into aspartic (Asp), asparagine (Asn) histidine (His), and according to online software forecast, mRNA secondary structure and protein primary, secondary structure of SIM1 gene changed in mutations before and after.     

从江香猪IFN-β基因的序列分析及原核表达

试验旨在探明从江香猪β-干扰素（interferon-beta,IFN-β）基因编码区分子序列及原核表达产物特征。以从江香猪为研究对象,提取肝脏总RNA并反转录为cDNA,设计特异性引物扩增IFN-β基因编码区,将目的基因片段克隆至原核表达质粒pET-28a上,获得重组质粒pET28a-CJpoIFN-β,并利用生物学软件对江香猪IFN-β基因编码区进行序列分析;将鉴定正确的重组质粒pET28a-CJpoIFN-β转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导表达、SDS-PAGE与Western blotting分析原核表达蛋白。结果表明,从江香猪IFN-β基因编码区长为561 bp,编码186个氨基酸;该蛋白为分泌性蛋白,前21个氨基酸为信号肽序列;二级结构主要以α-螺旋（77.42%）和无规则卷曲（17.74%）为主。从江香猪与其他猪源IFN-β基因核苷酸序列同源性为99.5%~100.0%,与禽的同源性最低（35.2%）;从江香猪与巴马猪、梅山猪IFN-β氨基酸同源性均为100.0%,但与贵州白香猪IFN-β同源性为99.5%,存在E43Q、K73R和C161R 3处氨基酸的差异。Western blotting结果显示,带His标签的重组表达蛋白能被His单抗识别,条带大小约为24 ku。本试验结果为进一步研究IFN-β基因生物学活性及加快从江香猪这一品种资源的有效利用提供参考依据。     

从江香猪胴体及肉品质性状研究

为探究从江香猪胴体性状和肌内脂肪含量变化规律,通过测定117头从江香猪皮厚、10～11肋间背膘厚、眼肌深度、眼肌面积、背最长肌游离水分和肌内脂肪含量指标,分析性别和屠宰体重对各指标的影响及各性状间的相关关系。结果表明,性别对从江香猪10～11肋间背膘厚、眼肌深度和眼肌面积有显著影响(P<0.05),对游离水分和肌肉脂肪含量(IMF)均无显著影响(P>0.05)。随着屠宰体重的增加,除肌肉游离水分外,从江香猪各项测定性状均呈现出增加趋势,其中屠宰体重对10～11肋间背膘厚和眼肌面积有显著影响(P<0.05),对IMF含量无显著影响(P>0.05)。综合各项测定指标,从江香猪屠宰体重处于65～75kg阶段屠宰较优。IMF与游离水分呈现出强负相关关系(相关系数为-0.624),二个指标间回归曲线模型Y=594.93589-15.58725X+0.10251215X2,决定系数R2为0.8783,模型准确度较高。     

从江香猪基因组甲基化修饰与体重之间的相关性研究

为了探索从江香猪生长慢的表观遗传学机理,本试验应用甲基化敏感扩增多态性(methylation-sensitive amplifying polymorphism,MSAP)技术,将60头20 d从江香猪分为高体重组和低体重组,研究血液和肝脏基因组DNA中胞嘧啶的甲基化程度,并测定差异的甲基化片段核苷酸序列.结果发现,MSAP分析得到5 977条带;与低体重组相比,高体重组的血液和肝脏总甲基化水平较低,两组的血液半甲基化水平均较肝脏中的高,全/总甲基化水平较低.提示香猪血液和肝脏基因组的总甲基化程度随体重的增加而下降.经克隆测序得到5条有差异的甲基化条带A1-A5,其中A1和A2源自血液,A3-A5来自肝脏;A1和A4片段为高体重组特有,其余3条片段为低体重组特有;A3片段未找到相关基因,其他4个片段处于代谢或免疫相关的基因内部或5'侧翼区.提示从江香猪基因组的甲基化水平存在个体差异,并与20 d体重相关;得到的4条甲基化片段,将有助于香猪甲基化调控机制的研究.     

贵州从江香猪PDK4、FGF10基因的克隆及组织表达分析

试验旨在克隆获得PDK4、FGF10基因,并研究大白猪与从江香猪不同组织中PDK4、FGF10基因mRNA的表达差异。采用RT-PCR分别克隆从江香猪PDK4、FGF10基因并进行生物信息学分析,利用实时荧光定量PCR技术检测PDK4、FGF10基因在大白猪和从江香猪不同组织中mRNA的相对表达量。结果显示,从江香猪PDK4基因的编码区全长1 224 bp,编码407个氨基酸;FGF10基因的编码区全长636 bp,编码211个氨基酸。经BLAST软件进行同源性比对,发现从江香猪PDK4基因与羊、马、人的核苷酸序列同源性分别为93%、92%和91%;FGF10基因与羊、牛、人、鼠的核苷酸序列同源性分别为94%、93%、93%和90%。由PDK4基因系统进化树可知,从江香猪与牛、绵羊亲缘关系较近;由FGF10基因系统进化树可知,从江香猪与绵羊、牛、人、猕猴亲缘关系较近,与小鼠和鸡亲缘关系较远。实时荧光定量PCR结果显示,在从江香猪不同组织中,PDK4基因在肾脏中的表达量最高,在胃和脂肪中表达量较高,FGF10基因在胃中表达量最高,在肾脏和脂肪中表达量较高,两个基因在背最长肌中的表达量均最低;在大白猪的不同组织中,PDK4、FGF10基因在脂肪中的表达量均最高,PDK4基因在背最长肌中的表达量最低,而FGF10基因在心脏中表达量最低。本试验成功克隆了从江香猪PDK4、FGF10基因,并检测了其在大白猪与从江香猪不同组织中的表达,为进一步研究PDK4、FGF10基因在脂质代谢及脂肪沉积等方面的调控作用提供科学依据。     

Cloning and Tissue Expression Analysis of PDK4 and FGF10 Genes in Guizhou Congjiang Xiang Pig

The objective of this study was to clone PDK4 and FGF10 genes, and investigate the expression level of PDK4 and FGF10 genes mRNA in different tissues of Large White pig and Congjiang Xiang pig. The PDK4 and FGF10 genes were cloned by RT-PCR and analyzed by bioinformatics, the relative expression of PDK4 and FGF10 genes were detected by Real-time PCR. The results showed that the coding region of PDK4 gene was 1 224 bp, encoding 407 amino acids; The coding region of FGF10 gene was 636 bp and encoded 211 amino acids. The homologies of nucleotide sequences of PDK4 gene with sheep, horse and human were 93%, 92% and 91%,respectively. The homologies of nucleotide sequences of FGF10 gene with sheep, cattle, human and mouse were 94%,93%, 93% and 90%, respectively. The phylogenetic tree of PDK4 gene showed that the genetic relationship of Congjiang Xiang pig, cattle and sheep were very close, the phylogenetic tree of FGF10 gene indicated that the genetic relationship of Congjiang Xiang pig, cattle, sheep, human and macaque were very close, but the genetic relationship of Congjiang Xiang pig, rat and chicken were far away. Real-time PCR results showed that, in different tissues of Congjiang Xiang pig,PDK4 gene expression in kidney tissue was higher than other tissues, with a higher expression in stomach and adipose as well,FGF10 gene expression in stomach tissue was higher than other tissues, with a higher expression in kidney and adipose as well, but both of PDK4 and FGF10 genes expression were the lowest in longissimus dorsi. In different tissues of Large White pig, both of PDK4 and FGF10 genes were expressed the highest in adipose than other tissues, PDK4 gene expression in longissimus dorsi was the lowest, while the FGF10 gene expression the lowest in heart. This study successfully cloned the PDK4 and FGF10 genes of Large White pig and Congjiang Xiang pig,and detected the relative expression of PDK4 and FGF10 genes in different tissues of Large White pig and Congjiang Xiang pig, and also provided scientific basis for further study on regulation of PDK4 and FGF10 genes on lipid metabolism and deposition.