Effects of Multi-enzyme Preparations on Growth Performance,Apparent Total Tract Digestibility and In Vitro Digestibility for Growing Pigs

The aim of this study was to investigate the effects of enzyme supplementations on performance of growing pigs, apparent total tract digestibility （ATTD） and in vitro digestibility （IVD） of nutrients. 224 growing pigs weighing 30-4-1.2 kg were allocated to fed basal diets supplemented with 0, 0.1, 0.15 or 0.2 g/kg enzyme preparations in growth trail. Those four diets were used for in vio＇o digestibility study. Pigs fed enzyme-supplemented diets improved average daily gain and decreased ratio of feed/gain （P〈0.05）. Enzyme supplementations increased ATTD of dry matter, energy, crude protein, neutral detergent fiber, acid detergent fibre and hemicellulose （P〈0.05）. Supplementation with enzymes improved reducing sugar production and the IVD of dry matter, crude protein, neutral detergent fiber, acid detergent fibre, total arabinoxylans, galacturonic acid, hemicellulose and cellulose. 0.015% enzyme supplementation in diets seemed to be sufficient to achieve maximum growth performance, nutrients digestibilities and reducing sugar production. Collectively, these results suggested that the supplementation of enzymes in diet increased the digestibilities of nutrients through the promotion of NSP degradation, thereby enhancing growth performance of growing pigs.     

Chromosome Mapping,Expression and Polymorphism Analysis of CRABP1 Gene in Pigs

Cellular retinoic acid-binding protein 1 （CRABP1） is a well-conserved member of cytosolic lipid-binding protein family. It is an important modulator of retinoic acid signaling. Long serial analysis of gene expression （LongSAGE） analysis suggested that CRABP1 gene was differentially expressed during prenatal skeletal muscle development in porcine. Here, we obtained the full-length coding region sequence and genomic sequence of the porcine CRABP1 gene and analyzed its genomic structures. Subsequently, we examined CRABP1 chromosome assignment using INRA-University of Minnesota 7 000 porcine radiation hybrid panel （IMpRH） and explored its tissue distribution in adult Tongcheng pigs and dynamical expression profiles in prenatal skeletal muscle （33, 65 and 90 days post coitus, dpc） from Landrace （lean-type） （described as L33, L65 and L90） and Tongcheng pigs （obese-type） （described as T33, T65 and T90）. The CRABPI gene was mapped to chromosome 7ql 1-q23 and closely linked to the microsatellite marker SWR1928. Quantitative real-time PCR showed that CRABP1 mRNA was highly expressed in lung and stomach, moderately expressed in placenta and uterus, and weakly expressed in other tissues. Moreover, CRABP1 gene was down-regulated during prenatal skeletal muscle development in both Landrace and Tongcheng pigs and it was expressed much higher in T33 than L33. Two single-nucleotide polymorphisms （SNPs） were detected by sequencing and mass spectrometry methods, allele frequency analysis indicated that g. 281 （G〉A） and g. 2992 （G〉A）were deviated from Hardy-Weinberg equilibrium in the Landrace and DLY （Duroc×（Landrace×Yorkshire）） pig breeds.     

Genetic Variation of EPAS1 Gene in Tibetan Pigs and Three Low-Altitude Pig Breeds in China

Endothelial PAS domain protein 1（EPAS1）, also called hypoxia-inducible factor-2, is a key regulatory factor of hypoxic responses and plays an essential role in high-altitude adaptation in mammalian species. In this study, polymorphisms of EPAS1 were detected in 217 individuals from 2 Tibetan pig populations and 3 low-altitude pig breeds by DNA pooling, PCR-SSCP, PCR-RFLP and DNA sequencing methods. A total of 14 synonymous polymorphisms were identified in the coding region. The analysis suggested that SNP1（G963A）, SNP7（C1632T）, SNP10（G1929A） and SNP11（G1947A） showed potential association with high-altitude environment because of their particular variation patterns in Tibetan pigs. Linkage disequilibrium（LD） of these SNPs was analyzed. One common LD block including 5 SNPs clustering in exon 12 was identified in all studied pig populations. Haplotype H1（AGGTC） in LD block was dominant in Tibetan pigs（76.6 and 74.2% in Linzhi（LZ） and Chayu（CY） pigs, respectively） and segregated at higher frequency than that in low-altitude pig breeds（52.3, 58.7 and 56.2% in Wuzhishan（WZS）, Min（M） and Laiwu（LW） pigs, respectively）, indicating that H1 may relate to adaptation to high altitude in Tibetan pigs. These findings raise hope that EPAS1 gene can be a candidate gene that involved in adaptation of high altitude in Tibetan pigs.     

猪线粒体DNA D-loop PCR-RFLP分析

对太湖猪（二花脸）、大约克猪、北京黑猪、五指山猪、香猪、长白猪、杜洛克猪以及太湖猪与7个品种猪的正反交杂交家系线粒体DNAD－loopPCR－RFLP分析,发现猪的线粒体DNAD－loop约为1500bp,长白猪及部分大约克猪与其他猪品种在AccⅠ酶切位点存在多态性;太湖猪、大约克猪的正反交后代的线粒体DNAD－loop在AccⅠ酶切位点存在多态性,其遗传方式遵循母系遗传特性。     

Composition of Pig Manures and Wastewaters under the Gan Qing Fen System in China

A large scale pig farm located in the North West of Beijing that applies the gan qing fen manure management system was selected and a continuous sampling of manures and wastewaters was performed from June to October 2009. Samples were collected once a week based on the pig production cycle, namely gestation, farrowing, weaning and fattening. Analysis of nutrients and heavy metals were determined by means of Inductively Coupled Plasma Atomic Emission Spectroscopy （ICP）. Total Nitrogen （TN） was determined by the Kjeldahl method. The main results showed that pig manures were characterized by high nutrients and heavy metals contents that might be due to the solid fraction separation from the liquid fraction under the gan qing fen manure management system. Further, piggery wastewaters were characterized by very low concentrations of nutrients and heavy metals as result of their dilution with flushing water mainly used for cleaning the pigsties. Manure and wastewaters samples from weaning pigs contained the highest concentrations of nutrients and heavy metals that could be due to high supplementation rates of these minerals in the weaner diets. In general, it seems that the manual daily collection of pig manures in the gan qing fen system is an efficient practice in order to maintain nutrient contents in pig manures but the use of flushing water should be reduced as it can lead to further environmental pollution.     

二花脸猪linc-NORFA核心启动子鉴定与转录调控分析

【目的】 前期研究证实linc-NORFA作为母猪繁殖性状候选基因参与调控卵泡闭锁与卵泡颗粒细胞凋亡过程,进一步鉴定二花脸猪linc-NORFA核心启动子并分析其转录调控机制,为解析linc-NORFA介导猪卵泡闭锁的分子机制奠定理论基础并提供新的研究思路。【方法】 采集二花脸猪耳组织样品并提取基因组DNA,利用PCR扩增和测序技术获得二花脸猪linc-NORFA 5′调控区序列;通过构建缺失表达荧光报告载体并利用荧光素酶活性试验鉴定二花脸猪linc-NORFA核心启动子;利用生物信息学分析二花脸猪linc-NORFA核心启动子区序列特征与潜在的转录因子结合位点;构建猪FOXO1真核生物表达载体并进一步采用Western blot、qRT-PCR以及荧光素酶活性试验分析转录因子FOXO1过表达对二花脸猪linc-NORFA转录的影响;利用染色质免疫沉淀（ChIP）技术鉴定转录因子FOXO1与二花脸猪linc-NORFA核心启动子区的结合能力。【结果】 通过克隆测序与序列拼接共获得二花脸猪linc-NORFA 5′调控区序列1 734 bp,其中包含两个潜在的CpG岛;利用荧光素酶活性试验证实linc-NORFA核心启动子位于-988 — -684 bp（转录起始位点作为+1）,生物信息学分析表明二花脸猪linc-NORFA核心启动子上包含多个转录因子的结合元件,例如ESR2、FOXO1、E2F1、BRCA1以及NFIC等;另外,ChIP试验还证实在猪卵巢颗粒细胞中FOXO1作为转录因子直接靶向结合在linc-NORFA的核心启动子区;进一步通过试验证实FOXO1过表达可显著下调linc-NORFA核心启动子区活性（P<0.01）,同时显著抑制体外培养的猪卵巢颗粒细胞中linc-NORFA的表达（P<0.01）。【结论】 鉴定了二花脸猪linc-NORFA核心启动子区,同时证实FOXO1作为转录因子能够与linc-NORFA核心启动子区特异性结合,进而抑制后者的转录活性与表达。研究结果对探究linc-NORFA在猪卵泡闭锁过程中显著下调的分子机制具有重要意义。     

猪精子黏合分子1（SPAM1）基因新SNPs的鉴别及其在12个中外猪中的遗传变异研究

精子黏合分子1 (SPAM1)是一种在受精过程中起重要作用的精子抗原,以10 个中外猪种的20个无遗传相关公猪个体的基因组DNA为模板,扩增猪SPAM1基因的部分第二外显子和第三内含子2 个DNA片段,经PCR产物直接测序并作序列比较分析后在外显子2和内含子3上分别鉴别到1个新的单核苷酸多态(SNPs)位点,其中外显子2上的T>A碱基颠换(M191T-A)可导致其编码的苯丙氨酸突变为酪氨酸.SMART软件分析显示M191T-A处在SPAM1蛋白糖基水解酶保守域的编码区.建立PCR- TasI -RFLP方法检测了4个西方商业猪种以及8 个中国地方猪种791个个体在M191T-A位点的遗传变异情况,结果在八眉猪、莱芜黑猪、米林藏猪、杭猪、剑河白香猪、大白猪、长白猪和杜洛克猪中检测到突变型等位基因A;而在二花脸猪、民猪、五指山猪和皮特兰猪中仅存在TT基因型,表现为极端的单态分布.     

民猪、长白猪及其杂种猪肢蹄性状的比较分析

通过观察民猪、长白猪、长民猪静态结构性状,分析不同品种间以及同一品种左、右肢间的差异,发现民猪前肢肢势多为X-肢势,四肢的系关节角、两个跗关节角都存在结构缺陷,民猪后蹄存在明显的左右不对称缺陷,长民猪前系关节和跗关节也存在结构缺陷。     

应用微卫星标记对中国10个品种猪遗传变异的研究

对我国7个地方猪品种(二花脸、通城、清平、阳新、皖南花、赣东黑、万安)和3个引进品种(长白、大白、杜洛克)的4个微卫星位点的多态性进行了检测,并对品种内和品种间的遗传变异进行了群体遗传学分析。结果初步表明,地方品种中二花脸、赣东黑、通城的品种内遗传变异较高,万安的品种内遗传变异最低。除万安猪外,其它地方品种的品种内变异均大于引进品种。品种间的聚类结果基本符合品种的地缘分布。     

二花脸猪Smad4基因的克隆与卵巢组织mRNA的表达水平

[目的]了解二花脸猪Smad4(common-mediator Sma4)基因的序列特征和组织表达特征,分析二花脸猪与商品猪卵巢组织中Smad4基因mRNA表达水平的差异.[方法]采用克隆测序技术获得二花脸猪Smad4基因cDNA序列,利用生物信息学方法分析二花脸猪Smad4基因的序列特征和蛋白的理化性质、高级结构,RT-PCR检测其组织表达特征,并采用real-time PCR技术检测二花脸猪与商品猪卵巢组织中Smad4基因mRNA的表达水平.[结果]二花脸猪Smad4基因编码区序列全长1 659 bp,编码一个含有5 52个氨基酸残基的蛋白质,氨基酸序列与其它哺乳动物的同源性均在98％以上;二花脸猪Smad4也包括3个典型的结构域(MH1、SAD和MH2).二花脸猪Smad4基因在所检测的组织中均有表达,且卵巢组织中mRNA的表达水平极显著高于商品猪(P＜0.01).[结论]二花脸猪Smad4基因可能拥有其它哺乳动物Smad4基因相同的功能及其可能与猪高繁殖力间有一定相关.     

猪血清蛋白质多态性与品种分类的研究

本文根据所测定的二花脸、梅山、枫泾、金华、姜曲海和长白猪的4个血清蛋白质系统——前白蛋白(Pa~A,Pa~B)、转铁蛋白(Tf~A,Tf~B,Tf~C)、铜蓝蛋白(CP~A,CP~B,CP~C)和淀粉酶(Am~A,Am~B,Am~C,Am~Y)的基因频率,估算了各品种间遗传距离和品种内平均基因多样度;并对各品种的4个血清蛋白位点的等位基因类型及分布特点进行了分析;进一步考证了我国猪种的起源与分化以及我国部分地方猪种的遗传结构和类缘关系;亦较系统地探讨了太湖猪不同类群的亲缘关系。     

猪肿瘤坏死因子α基因5'侧翼区域多态性分析

利用直接测序法研究猪肿瘤坏死因子α(Tumor necrosis factor,TNF-α)基因5'侧翼区域多态性,并作多态位点与仔猪腹泻和生长性状关联分析。发现在TNF-α基因5'侧翼区域存在6个SNPs位点,建立针对SNP:-1048T/C和SNP:-1016A/G的Hha ⅠPCR-RFLP和Taq Ⅰ PCR-RFLP分型技术。Hha ⅠPCR-RFLP研究发现,在民猪群体中存在TC和CC两种基因型,在长白猪群体中存在TT、TC和CC三种基因型。Taq Ⅰ PCR-RFLP研究发现,在民猪和长白猪群体中仅存在AG和GG两种基因型。性状关联分析结果表明,TC比CC基因型民猪具有较高的35日龄断奶重(P0.05)和日增重(P0.05);TT基因型长白猪腹泻指数高于TC基因型个体(P=0.06)和CC个体(P=0.06);TT基因型长白猪出生重显著高于TC和CC基因型长白猪(P0.05)。结果表明,猪TNF-α基因对仔猪腹泻和生长性状有一定影响,但HhaⅠ位点能否作为新遗传标记有待进一步研究。     

民猪与长白猪主要组织器官生长规律研究

选择民猪和长白猪为研究对象,利用异速生长式分析两个品种1～8月龄组织器官的早熟顺序。结果表明,民猪脂肪沉积较早,在2.5～6月龄间沉积大量的脂肪,生长发育全期腹内脂肪的沉积能力突出,而长白猪在6月龄后沉积脂肪速度加快;两个品种各脂肪组织占胴体比率在6～8月龄维持不变。民猪肉、脂、骨、皮早熟性顺序为:骨骼>肌肉>皮肤>脂肪,而长白猪为:骨骼>皮肤>肌肉>脂肪;两品种猪脂肪组织早熟性顺序均为:肠系膜脂肪>皮下脂肪>肾周脂肪>大网膜脂肪。民猪内脏器官早熟性顺序为:小肠>肾>胰>肝>脾>肺>心>胃>大肠;长白猪内脏器官早熟性顺序为:小肠>肾>肝>胰>心>脾>胃>肺>大肠。     

猪IGF-I基因SNP位点与生长性能的相关分析

【目的】分析猪胰岛素样生长因子I（IGF-I）基因SNP位点与生长性能的相关性,为后期猪的品种选育提供理论依据。【方法】采用PCR-RFLP对长白猪、环江香猪、广西巴马小型猪及长×巴F1代、F2代杂交猪的IGF-I基因SNP位点（rs322131043）进行分型,统计不同猪种该位点的基因型频率和等位基因频率,并分析长×巴F2代杂交猪的基因型频率与其生长性能的相关性。【结果】IGF-I基因SNP位点在广西巴马小型猪中以AA型为优势基因型,长白猪中以GG为优势基因型,其中A基因频率在广西巴马小型猪、环江香猪、长白猪3个品种中依次降低。不同基因型的长×巴F2代杂交猪中,基因杂合（AG）型猪的体重、体长、胸围、体高平均值在各日龄基本上都比基因纯合（AA/GG）型的低。【结论】猪IGF-I基因SNP位点与其生长性能密切相关,但该位点是否可应用于品种选育仍需进一步验证。     

Analysis of Genetic Diversity and Relationships of Seven Chinese Indigenous Pig Breeds and Three Exotic Pig Breeds Using the DNA Differential Display Technique

The genetic diversity and relationships of seven Chinese indigenous pig breeds （Meishan, Erhualian, Hezuo, Bamei, Qingping, Tongcheng, and Huainan） and three exotic pig breeds （Large White, Landrace, and Duroc） were analyzed using the DNA differential display technique by means of eight primer combinations. A total of 123 reproducible bands were used to calculate mean Nei＇s gene diversity, and mean Shannon＇s information index for each pig population. Based on these the Nei＇s standard genetic identity and distance were estimated, which was used to construct a dendrogram tree for the 10 pig breeds. The experimental results obtained and the method used in this study for evaluating the genetic diversity and relationships of pigs were also discussed.     

猪品种随机扩增多态DNA指纹分析

从 2 0个 10碱基随机引物中筛选出 12个引物对长白猪、哈白猪、大白猪、民猪、香猪、香哈杂交猪及长哈杂交猪共 4 1个个体的核 DNA遗传差异进行了 RAPD分析 ,共获得了 97个RAPD标记。品种内遗传相似度总体看来呈引入猪种→培育猪种→地方猪种递减的趋势 ,品种间遗传距离和聚类分析的结果是 :香哈杂交猪与香猪聚为一类 ,长哈杂交猪与长白、哈白、大白聚为一类 ,民猪单独聚为一类。本实验得出的结果与品种的形成历史及采样的实际情况是相吻合的     

用高通量测序技术研究松辽黑猪与长白猪背最长肌 mRNA和lncRNA的差异表达

【背景】肌肉生长和脂肪沉积是猪生产中非常重要的经济性状,同时也是复杂的数量性状,受到众多因素的影响。长白猪原产于丹麦, 是目前世界上使用最为广泛的瘦肉型猪种之一, 具有生长速度快, 瘦肉率高等特点,但肉质欠佳。松辽黑猪是以民猪、长白猪和杜洛克猪为素材经过三元杂交育成的黑色母系地方培育品种,具有繁殖率高、肉质优良、适应性强、耐粗饲等特性。二者在肉质方面存在较大差异。【目的】采用高通量测序技术对松辽黑猪和长白猪的背最长肌转录组进行分析,筛选影响猪肌肉生长、肉质和脂肪沉积的关键mRNA和长链非编码RNA（long non-coding RNA, lncRNA）,为猪肉品质研究提供新的参考信息。【方法】采集6头松辽黑猪和6头长白猪的背最长肌作为样本,提取其RNA,将每个品种内的个体取等量RNA混池,采用Illumina HiSeq 2500高通量测序技术对混池后的样本RNA进行测序,然后对所获得的Reads进行比对、注释和差异表达等分析,筛选出差异表达mRNA和lncRNA并进行相关生物学功能富集分析。而后在筛选出的差异表达mRNA和lncRNA中各挑选10个基因,包括5个上调基因和5个下调基因,采用荧光定量PCR（Real-time PCR, RT-PCR）方法鉴定所选mRNA和lncRNA的表达水平,并验证测序结果的可靠性。【结果】将得到的原始数据进行质量控制后,每个样本分别得到103 M和150 M reads,其中98%以上reads质量较高,满足进行下一步分析的要求。将reads比对到参考基因组上,共得到26 774个转录本,其中包括8 428个新的转录本。采用edgeR软件包进行差异表达分析,参考标准是错误发现率（false discovery rate, FDR）小于0.05和差异倍数大于2（fold change, FC）。在2个猪种中共得到4 239个显著差异表达的mRNA,其中在松辽黑猪肌肉中2 023个上调,2 216个下调。其中HLCS、BTD、DGKα、LPIN1、FGF1、FGF10、FGFR1和ZNF7等基因参与脂类代谢和肌肉发育相关的调控。差异表达的mRNA的生物学功能富集分析发现,其主要富集在细胞发育、生物代谢调控、肌肉发育、脂类代谢等相关的信号通路和生物代谢途径上。同时,在2个猪种中还检测到的178个差异表达显著的lncRNAs,其中在松辽黑猪肌肉组织中有84个上调,94个下调。联合差异表达lncRNAs的靶基因预测和差异表达基因结果分析,显示有4个差异表达的lncRNA与差异表达mRNA存在潜在的调控关系,分别是TCONS-00041713、TCONS-00041712、ENSSSCT00000034982、ENSSSCT00000034269。同时10个差异表达mRNA和10个差异表达lncRNA的RT-PCR结果显示：其表达水平在两组中同样差异显著,且与转录组测序中表达的趋势相似,表明高通量测序结果具有可靠性。 【结论】 获得了影响猪肌肉生长、肉质和脂肪沉积的关键mRNA和lncRNA,同时发现了几对差异表达lncRNA和mRNA之间可能存在着潜在的调控关系。这些潜在调控关系的发现,可能对于研究调控肌肉组织代谢活动的分子机制的具有一定意义,并为其提供新的参考信息。     

Genome-wide detection for runs of homozygosity analysis in three pig breeds from Chinese Taihu Basin and Landrace pigs by SLAF-seq data

Erhualian (E), Meishan (MS) and Mi (MI) pigs are excellent indigenous pig breeds in Chinese Taihu Basin, which have made great contributions to the genetic improvement of commercial pigs. Investigation of the genetic structure and inbreeding level of the 3 pig breeds is of great significance for the sustainable breeding of commercial pigs. The length and number of runs of homozygosity (ROH) as well as the frequency of genomes covered by ROH can be used as indicators to evaluate the level of inbreeding and the origin of the population. In this study, the ROH characteristics of E, MS, MI and Landrace (L) pigs were analyzed by SLAF-seq data, and the inbreeding coefficient based on ROH (F_ROH) was calculated. In addition, we have identified candidate genes in the genomic regions associated with ROH. A total of 10568 ROH were detected in 116 individuals of 4 pig breeds. The analysis showed that there were significant differences in genetic structure between 3 Taihu Basin pig breeds and L, and the genetic structure of E and MI was similar. The results of F_ROH showed that the inbreeding level of MS was the highest (0.25±0.07), while E and MI were lower than L. Compared with the other 3 pig populations, MS showed a higher frequency of long ROH (>5 Mb), indicating higher inbreeding in MS in recent generations. A large number of candidate genes related to reproductive traits are located in the genomic regions with a high frequency of ROH, and these genes are expected to be used as candidate genes in marker-assisted selection (MAS) breeding programs. Our findings can provide theoretical support for genetic conservation and genetic improvement of 3 pig breeds in Chinese Taihu Basin.