西农萨能奶山羊SERPINA1基因克隆、生物信息学及组织表达分析

本研究旨在克隆西农萨能奶山羊SERPINA1基因的CDS区,采用生物软件和在线预测工具进行生物信息学分析,通过实时荧光定量PCR技术检测SERPINA1基因在西农萨能奶山羊各组织间mRNA的表达水平。根据GenBank中山羊SERPINA1基因CDS区序列（登录号：XM_018066209.1）,利用Primer Premier 5.0软件设计特异性引物,RT-PCR扩增目的基因,构建原核表达载体测序后对序列进行生物信息学分析;采集西农萨能奶山羊心脏、肝脏、脾脏、肺脏、乳腺、肾脏、肌肉、瘤胃和小肠组织,提取组织RNA,反转录为cDNA模板,设计特异性定量引物,进行实时荧光定量PCR,检测SERPINA1基因在不同组织中的表达差异。结果显示,西农萨能奶山羊SERPINA1基因CDS区全长1 326 bp,编码441个氨基酸;同源性比对分析显示,西农萨能奶山羊与山羊、绵羊、牛和小鼠SERPINA1基因核苷酸序列同源性分别为100%、98.6%、95.7%和71.6%,与山羊亲缘关系最近,其次是绵羊。SERPINA1蛋白分子质量为48.71 ku,等电点为5.71,为跨膜亲水蛋白;SERPINA1氨基酸序列分别有62个磷酸化位点,3个跨膜区结构。组织表达分析显示,SERPINA1基因在西农萨能奶山羊肝脏组织中显著高表达（P<0.05）,其次是乳腺组织,在肺脏组织中表达量最低。研究结果为进一步探究SERPINA1基因在奶山羊乳蛋白合成代谢中的作用提供理论依据。     

干扰CREB基因对奶山羊乳腺上皮细胞脂质合成相关基因表达及甘油三酯合成的影响

旨在筛选奶山羊cAMP应答元件结合蛋白CREB（cAMP response element binding protein）基因的siRNA,揭示干扰该基因后对乳腺上皮细胞中乳脂合成相关基因表达及甘油三酯合成的影响。本研究通过qRT-PCR方法从西农萨能奶山羊乳腺组织中扩增CREB基因完整的CDS区,进行序列分析和不同泌乳时期表达水平分析,合成靶向CREB基因的siRNA,利用荧光定量PCR筛选有效siRNA,并检测脂质合成相关基因的表达,采用试剂盒检测细胞内甘油三酯含量。结果表明：1）克隆得到全长为984 bp的奶山羊CREB基因的CDS区（GenBank登录号：MK158073）,并对其进行生物信息学分析。2）该基因在奶山羊泌乳盛期乳腺组织的表达量为干奶期的1.93倍（P<0.05）。3）成功筛选到靶向CREB基因的有效siRNA,干扰效率为72%（P<0.01）;并将其在奶山羊乳腺上皮细胞进行转染,通过qRT-PCR检测脂质合成相关基因的表达,与对照组相比,干扰CREB基因后显著抑制了FASN、ACACA、SCD1、FABP3、LPL、CPT1B、GPAM和DGAT2基因表达量（P<0.05）,并显著增加了HSL基因表达量（P<0.05）;且细胞内甘油三酯含量被显著下调（P<0.05）。综上所述,CREB基因在奶山羊原代乳腺上皮细胞中很可能通过调控脂质代谢相关基因的表达及甘油三酯含量对山羊的乳脂合成过程发挥重要作用。     

西农萨能奶山羊泌乳中期和后期乳腺组织差异基因的筛选

旨在利用抑制性削减杂交(SSH)技术筛选西农萨能奶山羊泌乳中期和后期的差异表达基因,并用实时定量PCR(Q-PCR)分析差异基因的表达丰度,探讨奶山羊不同泌乳阶段乳腺组织的基因表达规律。本研究以西农萨能奶山羊泌乳中期和后期乳腺组织的mRNA互为检测子(Tester)和驱动子(Driver)构建cDNA消减文库(M-L和L-M),随机挑选克隆测序,进行序列比对分析,并检测部分差异基因在乳腺组织中的表达丰度。结果,成功构建了泌乳中期和后期的cDNA文库,随机挑选30个克隆测序,得到M-L和L-M文库中与细胞凋亡、抗氧化、脂类代谢、能量代谢等生理过程相关的差异基因,对其中的6个基因进行实时定量分析,发现5个均为阳性克隆,表达水平增加了1.3~5.5倍不等。结果表明,利用SSH技术成功构建了泌乳中期和后期乳腺组织正反向消减cDNA文库,筛选出24个差异基因,为进一步研究奶山羊乳腺组织基因调控机理奠定了基础。     

Effect of Bone Marrow Mesenchymal Stem Cells Transplantation on the Mammary Tissue Development and Serum Hormone Level of Saanen Dairy Goat

The study was aimed to analyze the effect of transplanted bone marrow mesenchymal stem cells (BMSCs) on the mammary gland development and serum hormone level of Saanen dairy goat.16 healthy goats with 2 months old and similar body weight were randomly divided into control group and experimental group. The BMSCs which was amplified to the P6 generation was injected to the goat via jugular vein,and the control group was injected with the same amount of normal saline. Feeding and management of the two groups were the same. Blood samples were collected monthly,and mammary gland tissue samples were collected at 3,5 and 7 months old. Frozen sections of mammary gland tissue was prepared, the ducts and acinus numbers were observed and recorded. The results showed that after transplantation of BMSCs,at 5 and 7 months old,the ducts and acinus number of goat in experimental group were significantly higher than that of control group (P<0.05).At 5,7,8 and 9 months old,the serum E₂ level of goat in experimental group was significantly higher than that of control group (P<0.05);At 5 to 9 months old,the serum P level in experimental group was significantly higher than that of control group (P<0.05).At 7 to 9 months old,the serum PRL and INS levels in experimental group were significantly higher than that of the control group (P<0.05);At 8 and 9 months old,the serum levels of GnRH level in experimental group was significantly higher than that of the control group (P<0.05).The serum LEP,CRH and ACTH levels were higher than that of the control group at the whole test process,but the difference was not significant (P>0.05).In conclusion, injection of BMSCs could increase the duct and acinus number of dairy goat mammary,improve the hormone level in serum,and promote mammary gland development.     

移植骨髓间充质干细胞对萨能奶山羊乳腺组织发育及血清激素水平的影响

试验旨在研究移植骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）对萨能奶山羊乳腺发育的影响。选取体重接近的2月龄健康萨能奶山羊16只,随机分为对照组和试验组。将扩增至P6代的BMSCs经颈静脉注射至奶山羊体内,对照组注射等量生理盐水,两组间饲养管理均相同。每月进行空腹采血,并分别在3、5和7月龄采集乳腺组织样品,制备乳腺组织冰冻切片,观察记录各组乳腺组织导管数和腺泡数。结果显示,移植BMSCs后,5和7月龄时,试验组奶山羊的导管数、腺泡数均显著高于对照组（P<0.05）。在5、7、8、9月龄时,试验组奶山羊血清中雌激素（E₂）水平显著高于对照组（P<0.05）;在5~9月龄时,试验组血清中孕酮（P）水平显著高于对照组（P<0.05）;在7~9月龄时,试验组血清中催乳素（PRL）和胰岛素（INS）水平均显著高于对照组（P<0.05）;8、9月龄时,试验组血清中促性腺激素释放激素（GnRH）水平显著高于对照组（P<0.05）;整个试验过程中试验组血清中瘦素（LEP）、促肾上腺皮质激素释放激素（CRH）和促肾上腺皮质激素（ACTH）水平均高于对照组,但差异不显著（P>0.05）。综上所述,注射BMSCs能增加奶山羊乳腺导管数和腺泡数,提高血清中激素水平,从而促进乳房发育。     

B‐Mode and Doppler Sonography of the Mammary Glands in Dairy Goats for Mastitis Diagnosis

This study aimed to evaluate the sonographic characteristics of the udder and teats and to determine the Doppler indexes of mammary artery in healthy and undergoing subclinical and clinical mastitis goats. Thirty animals among Saanen and Alpine Brown goats were arranged in three groups, healthy goats (HG), goats with subclinical mastitis (SMG) and goats with clinical mastitis (CMG). Using the B‐mode, the sonographic characteristics (echotexture and echogenicity) and biometry (diameter and area of the udder cistern, diameter and area of the teat cistern and thickness of the teat wall) were evaluated. Using Doppler ultrasonography, the vascular indexes of the mammary artery were obtained. It was observed hyperechogenicity with solid component in the gland cistern when comparing animals with clinical mastitis and healthy mammary tissue. Regarding the echotexture of the breast tissue, there was heterogeneity in the mammary parenchyma on the three groups, for the milk, it was observed homogeneity for animals on HG and SMG and heterogeneity for animals on CMG. Grey‐scale quantitative assessment revealed increase in echogenicity (mean value) for all the structures when comparing the three groups. Biometry did not reveal statistical difference between groups, for none of the evaluated structures. Doppler examination of the mammary artery showed the decrease of end diastolic velocity and raise of pulsatility index between groups. The association of B‐mode and Doppler ultrasonography is useful for the evaluation of the udder of dairy goats with mastitis. It is a sensitive and specific method for the study of this disease. Doppler mode was unable to establish reliable criteria for diagnosis of subclinical mastitis. Moreover, the quantification of echogenicity is a useful technique for the evaluation of the milk in animals with mastitis; therefore, it is suggested that it can be used as complementary technique for the diagnosis of mastitis in goats.     

Coexistence of Tuberculosis and Mammary Carcinoma in a Goat

Synchronic occurrence of tuberculosis mastitis and mammary cancer is rare in humans and, to the best of our knowledge, not reported in domestic animals. Here, we present a case of a female adult goat of Serrana breed with simultaneous occurrence of a granulomatous mastitis, due to Mycobacterium caprae, and a mammary carcinoma. Both pathological conditions are rare in goats and should be included in differential diagnosis of mammary lesions.     

西农萨能羊两个泌乳阶段差异表达基因SAA3的克隆和序列分析

为了研究不同泌乳期乳腺组织中差异表达基因，利用抑制性削减杂交技术构建西农萨能羊泌乳60d乳腺组织及泌乳28d乳腺组织中差异表达基因文库，用Real-Time PCR技术验证阳性克隆血清淀粉样蛋白A3（SAA3）基因，通过RT-PCR方法克隆西农萨能羊乳腺组织SAA3，并进行序列比对和功能预测。结果成功构建了西农萨能羊不同泌乳期乳腺组织中差异表达基因文库，筛选克隆了乳腺组织SAA3基因，GenBank登录号为：DQ839400，编码区长度为396bp，含有131个氨基酸。西农萨能羊乳腺组织中SAA3与牛（GenBank：NM_181016）、兔（GenBank：M64696．1）、人（GenBank：BC020795）、鼠（GenBank：NM_011315）核苷酸同源性分别为95％、84．3％、81.3％和81.9％，氨基酸同源性为93％、76％、72％、72％；在编码区261-287位较人、兔、鼠SAA编码区多27个碱基，连续大于5个氨基酸的保守区域有6个，较人SAA多3个潜在功能基序。     

奶山羊ACSL6基因克隆及表达调控分析

研究旨在获得西农萨能奶山羊长链脂酰CoA合成酶6（long-chain acyl-CoA synthetase 6，ACSL6）基因CDS序列，初步探究其对奶山羊乳腺上皮细胞脂质代谢的影响。以西农萨能奶山羊原代乳腺上皮细胞为试验材料，对ACSL6基因进行扩增和克隆，并利用实时荧光定量PCR方法对ACSL6基因进行组织表达分析，针对ACSL6基因的碱基序列利用在线软件进行生物信息学分析，采用RNA干扰技术改变奶山羊乳腺上皮细胞中ACSL6基因mRNA的表达水平。结果显示，ACSL6基因CDS区全长为2 169 bp，编码722个氨基酸；生物信息学分析表明ACSL6蛋白为碱性不稳定蛋白。组织表达分析显示，ACSL6基因在奶山羊乳腺组织中高度表达，其次为脾脏。将设计合成的siRNA转染乳腺上皮细胞，发现干扰ACSL6基因的表达使脂质代谢相关基因乙酰辅酶A羧化酶（acetyl CoA carboxylase，ACC）、硬脂酰辅酶A去饱和酶1（stearyl-CoA dehydrogenase 1，SCD1）、脂肪酸转运蛋白36（cluster of differentiation 36，CD36）、固醇调节元件结合蛋白1（sterol regulatory element binding proteins 1，SREBP1）的mRNA表达水平极显著下调（P<0.01）。本研究结果为从蛋白及个体水平上研究ACSL6基因对奶山羊乳腺脂质代谢的影响提供理论依据。     

Occurrence and diversity of coccidia in indigenous, Saanen and crossbred goats in South Africa

Occurrence and diversity of Eimeria species in two groups of indigenous South African goats kept under traditional management systems, as well as in a mixed herd of Saanen, indigenous and crossbred goats kept under an intensive management system were examined. Infection rates ranged from 88.7 to 100% in the various groups. Mean OPG of immature goats (< 1 year old) exceeded that of adult goats at all three sites. There was no consistent difference between adult OPG counts at the three sites. Under the intensive system, adult crossbred goats had significantly higher OPG counts than adult Saanen or indigenous goats. Overall, OPG counts of immature goats were significantly higher during the dry season (winter) than during the wet season (summer). Ten Eimeria species were identified, Eimeria arloingi being the most prevalent species at all three sites, followed by Eimeria hirci. Up to seven Eimeria species were recovered from individual specimens.     

Failure of young goats to acquire resistance to Haemonchus contortus

Two experiments were carried out to investigate the acquisition by goats of resistance to Haemonchus contortus. In Experiment 1, five Saanen wethers reared worm-free and averaging 5 1/2 months of age at the start of the experiment, were dosed with 200 H. contortus infective larvae three times per week for 10 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) and then given anthelmintic treatment. Each goat and an equal number of worm-free controls were then challenged with 10,000 infective larvae. Post mortem worm counts were carried out 30 days later. In Experiment 2, eight worm-free Saanen wethers, 14 months old at the start of the experiment, were dosed with 825 infective larvae per week for 14 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) except for one week when only 300 larvae were given and one week when no larvae were given. After anthelmintic treatment, each received, together with seven worm-free control animals, a challenge dose of 15,000 infective larvae. Post-mortem worm counts were carried out 28 days later. There were no significant differences in post-mortem worm counts between previously infected and uninfected groups in either experiment. In both experiments, serum pepsinogen values rose significantly as a result of infection but there was no significant (p>0.5) correlation between worm counts and pepsinogen values on the day of slaughter.     

Failure of young goats to acquire resistance to Haemonchus contortus

Two experiments were carried out to investigate the acquisition by goats of resistance to Haemonchus contortus. In Experiment 1, five Saanen wethers reared worm-free and averaging 51/2 months of age at the start of the experiment, were dosed with 200 H. contortus infective larvae three times per week for 10 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) and then given anthelmintic treatment. Each goat and an equal number of worm-free controls were then challenged with 10,000 infective larvae. Post mortem worm counts were carried out 30 days later. In Experiment 2, eight worm-free Saanen wethers, 14 months old at the start of the experiment, were dosed with 825 infective larvae per week for 14 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) except for one week when only 300 larvae were given and one week when no larvae were given. After anthelmintic treatment, each received, together with seven worm-free control animals, a challenge dose of 15,000 infective larvae. Post-mortem worm counts were carried out 28 days later. There were no significant dii- ferences in post-mortem worm counts between previously infected and uninfected groups in either experiment. In both experiments, serum pepsinogen values rose significantly as a result of infection but there was no significant (p>0.5) correlation between worm counts and pepsinogen values on the day of slaughter.     

Experimental infection of pregnant and lactating goats with Leptospira interrogans serovars hardjo and szwajizak

Pathogenesis of 2 Leptospira serovars, hardjo and szwajizak, was studied in pregnant and lactating goats. Although clinical signs of leptospiral infection were minimal, cultural isolations were made from the mammary gland of 2 goats and the kidney of 1 goat inoculated with serovar hardjo (C846). The isolations were made only on solid bovine albumin polysorbate-80 medium supplemented either with rabbit serum or sodium pyruvate. Cultural isolations of serovar szwajizak were made from kidney, liver, brain, urine, and mammary gland samples of 1 goat and the liver and kidney samples of its kids. These isolations were made in only the solid bovine albumin polysorbate-80 medium which had been supplemented with normal goat serum.     

Serum pepsinogen concentrations in strongyle-free French dairy goats

Serum samples from 533 strongyle-free goats were analysed to assess the normal values of pepsinogen concentration. Results obtained from adult goats ( > 12 months) indicated higher values of pepsinogen compared with sheep and cattle, with more than 25% of the animals exceeding 1000 milliunits of released tyrosine (mU TYR). Weaned goats less than 6 months old had lower pepsinogen concentrations than the adult goats: 490 ± 175 mU TYR vs. 825 ± 414 mU TYR for the French Alpine breed and 397 ± 135 mU TYR vs. 709 ± 274 mU TYR for the Saanen breed. Saanen goats showed lower pepsinogen values than French Alpine goats. Additional sources of variation included farm and duration of lactation. Pepsinogen determinations were highly repeatable for 4.5 months, suggesting a pronounced stability of pepsinogen levels for the same non-infected animals.     

The aetiology of a keratoconjunctivitis occurring in goats in the Sudan

An infectious keratoconjunctivitis occurring in goats in the Sudan is described. Experimental transmission using infective ocular discharges resulted in a conjunctivitis in young kids and a severe keratoconjunctivitis in an adult goat. A pleomorphic organism which resembled Rickettsia spp was found in conjunctival smears from naturally and experimentally infected animals. Neither Mycoplasma nor Chlamydia spp were isolated in microbiological cultures.     

Pathogenicity of different species of staphylococci in caprine udder

Aseptic foremilk samples were collected from Finnish landrace goats. Ten different species of staphylococci, causing subclinical infections were detected. Twelve goats with persistent subclinical staphylococcal infection were followed on a monthly basis and compared with foremilk samples of nine goats suffering from clinical mastitis. Parameters of inflammation based on the activity of the California Mastitis Test (CMT). N-acetyl-beta-D-glucosaminidase (NAGase) and antitrypsin were determined from the milk. Staphylococci were further classified using the API STAPH system. On the basis of elevations of activities of CMT, NAGase and antitrypsin, Staphylococcus aureus was the most pathogenic in clinical and subclinical mastitis, S. hyicus showed only marginal pathogenicity. Subclinical infections were persistent and the infective organism was not always detected from milk by culture. The biochemical reactions of subclinical staphylococci seemed to vary within the same gland by time. Antitrypsin was most effective in differentiating between subclinical and clinical infection. A teat cistern puncture technique was found to be suitable for the goat.     

转基因山羊研究进展

近年来,动物转基因技术发展日淅完善,在牛、羊、猪等家畜上的应用日益增多.为全面了解山羊转基因技术的研究进展,论文简要回顾了国内外转基因山羊生产的里程碑事件,总结了生产转基因山羊的方法和原理,并对转基因山羊研究状况、应用前景及存在问题进行了较为系统的概括和分析,从而为借助现代生物技术丰富我国山羊育种手段,加快山羊新良种培...     

Identification of sex-specific polymorphic sequences in the goat amelogenin gene for embryo sexing

Amelogenin (AMEL) is a conserved gene located on the sex chromosomes of mammals. It is involved in the formation of enamel, which is the hard, white material that forms the protective outer layer of each tooth. In this study, we first cloned and determined the intron sequences of the goat AMELX and AMELY genes from female and male ear tissues. The polymorphic AMEL alleles were further analyzed by PCR-based RFLP and Southern blot hybridization analyses. Results showed that intron 5 nucleotide sequences of the goat AMELY gene contains multiple deletions/insertions and shares only 48.5% identity to intron 5 of the goat AMELX gene. Based on the polymorphic AMEL intron sequences, a set of sex-specific triplex primers was designed to PCR amplify a single fragment of 264 bp from the X chromosome of female goats and 2 fragments of 264 and 206 bp from the X and Y chromosomes, respectively, of male goats. An increased sensitivity for sex determination was reached with a single blastomere at the blastula stage isolated from goat embryos. A total of 43 goat embryos were used to estimate a 100% accuracy rate of this method confirmed by chromosomal karyotyping and live births. The embryo sexing technique has been successfully applied in different strains of goats including Alpine, Saanen, Nubian, and Taiwan goats.     

Caprine beta-mannosidosis: phenotypic features

The clinical features of caprine beta-mannosidosis were evaluated in 10 newborn goats, one stillborn goat and one goat fetus. The phenotypic abnormalities observed in all 10 live affected animals included an inability to rise from a recumbent position, moderate to marked intention tremor, eye movements resembling pendular nystagmus, clinical deafness, bilateral Horner's syndrome, carpal contractures, pastern joint hyperextension, thickened skin and to a varying degree, a dome-shaped skull. Subjective evaluation suggested that most animals had a decreased muscle mass. Together, these characteristics represent a common phenotype which is expressed at birth in caprine beta-mannosidosis.