猪氨基酸转运载体b0,+AT-2的分子克隆及其序列分析

本研究克隆了b0,＋AT-2的全长cDNA序列。基于NCBI发布的人和鼠b0,＋AT序列,应用生物学软件进行序列比对后在同源区设计引物,获取cDNA片段;再利用RACE技术克隆了其全长cDNA序列。生物信息学分析显示：猪b0,＋AT-2的全长1 488 bp,包括90 bp的3′非翻译区（UTR）和126 bp的5′UTR;编码区（CDS）编码423个氨基酸残基,分别和人、鼠b0,＋AT以及猪b0,＋AT-1有75.1%、71.9%和86.6%的同源性。     

Markers of bone metabolism in dog breeds of different size

Serum and urinary markers of bone turnover may be of value in animals as noninvasive tools for determining the response of the skeleton to disease and injury. Although normal values for bone markers have been reported for the Beagle, concerns remain that breed to breed differences will complicate the interpretation of bone marker data in dogs. To explore this, we examined serum bone markers in two breeds of vastly different size, Pomeranians and Irish Wolfhounds. Our hypothesis was that serum concentrations of bone markers are similar in toy and giant dog breeds and fall within the same range as those reported for Beagles. Bone alkaline phosphatase (BALP) and carboxy-terminal telopeptide of type I collagen (ICTP), respectively markers of bone formation and bone resorption, were measured in age matched Pomeranians (n=14) and Irish Wolfhounds (n=14). No statistically significant differences between the mean BALP and mean ICTP serum concentrations from Pomeranians and Irish Wolfhounds were found. All BALP and ICTP concentrations were within the reference range reported for Beagles. The results of this study suggest that serum BALP and ICTP concentrations in giant and toy breeds are the same as in Beagles and that these assays may be used for dogs of all sizes.     

Analysis of a deletion in the nephronophthisis 4 gene in different dog breeds

Cone‐rod dystrophy is a progressive inherited retinal degenerative disorder that occurs in humans and dogs. The deletion in the nephronophthisis 4 (NPHP4) gene was established as a causative mutation in standard wire‐haired Dachshunds. We analyzed all varieties of Dachshunds from the Czech Republic and five other dog breeds and found that the deletion in the NPHP4 (in heterozygous state) is present not only in standard‐, but also in miniature wire‐haired Dachshunds, but not in other varieties of Dachshunds or in other breeds.     

Examination of the density of the brain parenchyma in different dog breeds using quantitative computed tomography

In the present study quantitative computed tomography was used to analyze the absorption density in different localisations of the brain parenchyma comparatively in five different dog breeds. The breeds German Shepherd Dog, Dachshund, Boxer, Labrador Retriever and Miniature Poodle were chosen as representatives of different skull shapes and sizes. The mean absorption density for the German Shepherd Dog was 35.8 HU in the cerebellum, 28.5 - 34.8 HU in several locations of the cerebrum, 39.6 HU in the brain stem and 40.8 HU in the hypophyseal region. In the Dachshund, the mean density was 33.8 HU (cerebellum), 34.3 - 44.2 HU (several locations of the cerebrum), 33.3 HU (brain stem) and 38.6 HU (hypophyseal region). The Boxer showed a mean density of 38.7 HU in the cerebellum, 30.2 - 40.8 HU in several locations of the cerebrum, 35.6 HU in the brain stem and 33.1 HU in the hypophyseal region. The mean absorption density in the Labrador Retriever was 37.2 HU in the cerebellum, 29.4 - 32.9 HU (several locations of the cerebrum), 34.7 HU in the brain stem and 47.5 HU in the hypophyseal region. In the Miniature Poodle the mean density was 33.6 HU (cerebellum), 34.9 - 45.5 HU (several locations of the cerebrum), 32.7 HU (brain stem) and 32.4 HU (hypophyseal region). The study showed that the absorption density of the cerebrum, the brain stem and of the hypophyseal region partly were influenced by the different dog breeds. In the cerebellum no difference of density could be seen in the different breeds. The standardised values for the data of absorption densities of the brain parenchyma are the base for further examinations of dogs with clinical CNS diseases.     

Analysis of the canine mdr1-1Delta mutation in the dog breed Elo

A deletion mutation in the canine multidrug resistance gene, MDR1, is associated with drug sensitivity. This was shown for several purebred dog breeds from the Collie lineage such as the Collie (rough-coated and smooth-coated), the Australian Shepherd and the Old English sheepdog. To determine whether the mdr1-1Delta mutation could be found in the newly bred German dog breed Elo which is based amongst other breeds on Old English sheepdogs, 177 blood samples representative for the Elo breed were collected. After DNA extraction, a polymerase chain reaction-based method with subsequent polyacrylamide gel electrophoresis was used for detection of the mdr1-1Delta mutation. The mdr1-1Delta allele was not observed in the Elos investigated. The probability that the mdr1-1Delta allele originated in the Old English sheepdog breed is segregating in the Elo population was estimated at 3.68 x 10(-17).     

Allele frequency distribution of the canine dopamine receptor D4 gene exon III and I in 23 breeds

Various canine breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of them are under genetic control. Although dopaminergic neurotransmission system is considered to affect animal behavior, little is known about related genes in canine. Relations between specific alleles in polymorphic regions of the dopamine receptor D4 gene (DRD4) and personality or psychiatric disorders have been reported in humans, and we first found polymorphism in exon III region of the gene in 4 canine breeds. In this study we surveyed allele frequency distribution in 23 breeds including a total of 1,535 unrelated individuals. In exon III, 8 alleles including a novel allele were identified. A group of breeds in which the alleles 447b, 498 and 549 were frequent tended toward high scores in aggression-related behavioral traits than that with frequent alleles 435 and 447a. Moreover, a polymorphism based on 24 bp insertion/deletion was found in exon I region for the first time in dogs. This information may be of use for candidate gene studies of behavioral variation in dogs.     

含赖氨酸二肽对奶牛乳蛋白合成和乳腺氨基酸转运相关基因表达的影响

试验旨在研究含赖氨酸二肽对奶牛乳腺组织中乳蛋白合成和氨基酸转运的影响。在体外培养的奶牛乳腺组织培养液中添加不同含赖氨酸二肽(赖氨酸-赖氨酸(KK)、赖氨酸—组氨酸(KH)、赖氨酸—苯丙氨酸(KF)、赖氨酸—亮氨酸(KL)和赖氨酸—苏氨酸(KT)),分别等量取代培养液中0、5%、10%、15%和20%的游离赖氨酸(赖氨酸总浓度为210μg/mL)进行培养,试验结束后收集乳腺组织用于基因表达检测。结果显示,同游离氨基酸替代量为0组相比,KH替代Lys比例为15%,KF替代比例为10%,KK、KL和KT替代比例为5%时,α_s1酪蛋白(CSN1S1)mRNA丰度最高(P<0.05);KK替代Lys比例为5%、10%、15%和20%,KH替代比例为10%和15%,KF替代比例为15%,KL替代比例为5%、20%及KT替代比例为5%、10%和15%时,均显著促进了奶牛乳腺组织中小肽转运载体2(SLC15A2)的基因表达(P<0.05);KH替代Lys比例为15%,KK、KL替代比例分别为10%,KF、KT替代比例分别为5%时,赖氨酸转运载体B⁰⁺(SLC6A14)的mRNA丰度最高(P<0.05);KH替代Lys的15%、20%,KK替代比例为5%、10%和15%,KL替代比例为5%和10%时,显著促进了雷帕霉素靶蛋白(mTOR)基因的表达(P<0.05)。结果提示,奶牛乳腺摄取赖氨酸二肽能促进氨基酸的吸收和乳蛋白的合成。     

Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany

MDR1 (ABCB1) P-glycoprotein exerts a protective function in the blood–brain barrier thereby limiting the entry of many drugs and other xenobiotics to the central nervous system. A nonsense mutation has been described for Collies and related dog breeds which abolishes this function and is associated with increased susceptibility to neurotoxic side effects of several drugs including ivermectin, moxidectin and loperamide. In order to evaluate the occurrence and frequency of this nt230 (del4) MDR1 mutation in Germany, we screened 1500 dogs. Frequency of the homozygous mutated genotype was highest for Collies (33.0%), followed by Australian Shepherd (6.9%) and Shetland Sheepdog (5.7%). Thirty-seven percent of the Wäller dogs and 12.5% of the Old English Sheepdogs were heterozygous for the mutant MDR1 (−) allele. Considering the predominant role of MDR1 P-glycoprotein in drug disposition and in particular for blood–brain barrier protection, MDR1 genotype-based breeding programs are recommended for improving the safety of drug therapy in these canine breeds.     

Effects of Lysine Dipeptides on the Expression of Genes Related to Milk Protein Synthesis and Amino Acid Transporters in Bovine Mammary Tissue

The study was carried out to investigate the effects of various lysine (Lys) dipeptides on gene expression related to milk protein and amino acid transporters in bovine mammary tissue.Mammary tissues were obtained from midlactating Holstein dairy cows and cultured under lactogenic hormones (prolactin, hydrocortisone and insulin) with different ratios of various Lys-peptide (Lys-Lys (KK), Lys-His (KH), Lys-Phe (KF), Lys-Leu (KL)and Lys-Thr (KT)).They isometrically substituted 0, 5%, 10%, 15% and 20% of free Lys (total concentration of Lys was 210 μg/mL), respectively.After 48 h treatment, mammary tissues were harvested for gene expression analysis by Real-time PCR.The results showed that:Compared with the free Lys group with no Lys substitution, when the KH replaced Lys at ratio of 15%, the KF replacement ratio was 10%, KK, KL and KT replacement ratio was 5%, the CSN1S1 mRNA abundance were the highest (P<0.05);When the KK replaced Lys at ratios of 5%, 10%, 15% and 20%, KH replacement ratios were 10% and 15%, KF replacement ratio was 15%, KL replacement ratios were 5% and 20% and KT replacement ratios were 5%, 10% and 15%, the SLC15A2 gene expression was significantly enhanced (P<0.05); when the KH replaced lysine at ratio of 15%, KK and KL replacement ratio was 10%, KF and KT replacement ratio was 5%, the abundance of SLC6A14⁺ mRNA was the highest (P<0.05);When the KH replaced Lys at ratio of 15% and 20%, KK replacement ratios were 5%, 10% and 15%, KL replacement ratio was 5% and 10%, the mRNA abundance of mTOR was significantly increased (P<0.05).The results indicated that the Lys dipeptides might be able to promote amino acid uptake and milk protein related gene expression in bovine mammary gland tissues.     

不同品种猪IGFBP-3启动子区突变位点筛查及分析

以军牧1号白猪、大白猪、东北野猪、巴马香猪和西藏小型猪为研究对象,通过PCR扩增5个品种猪IGFBP-3基因5′调控区序列(约2 000bp)并进行测序分析,来研究不同品种猪IGFBP-3基因启动子区序列的差异。同源性比对分析发现5个品种猪IGFBP-3基因5′调控区存在约50处突变;启动子预测发现在-185bp附近为核心启动子区的几率最大;突变共导致9处转录因子结合位点发生改变,其或可造成不同品种猪IGFBP-3表达量的差异,进而影响不同品种猪的体型大小。     

An evaluation of genetic diversity indices of the Red Bororo and White Fulani cattle breeds with different molecular markers and their implications for current and future improvement options

The genetic diversity of the Red Bororo and White Fulani cattle breeds of Cameroon and Nigeria was assessed with a panel of 32 markers. Estimates for the various indices of genetic diversity, total number of alleles (TNA), mean observed number of alleles (MNA), mean effective number of alleles (MNE), observed heterozygosity (H _ob) and expected heterozygosity (H _ex), were higher at microsatellite loci than at protein loci. Mean H _ex values were above 71% at microsatellite loci in all the breeds and ranged from 37% to 41.6% at milk protein loci and from 40.9% to 45.6% at blood protein loci. The highest TNA and MNA of microsatellites were recorded for the Nigerian White Fulani. MNE of milk protein loci was highest in the Cameroonian Red Bororo, while TNA of blood protein loci was highest in the Cameroonian White Fulani. The high genetic diversity levels indicate the presence of the necessary ingredients for improvement breeding and conservation. Multi-locus estimates of within-population inbreeding (f), total inbreeding (F) and population differentiation (θ) of the breeds were significantly different from zero, except for θ of blood proteins. A high level of gene flow was found between the breeds (5.829). The phylogenetic relationship existing among the four breeds is greatly influenced by location. The high gene flow between the breeds may lead to a loss of genetic diversity through genetic uniformity and a reduction in opportunities for future breed development. We propose an improvement scheme with aims to prevent loss of genetic diversity, improve productivity and reduce uncontrolled genetic exchanges between breeds.     

Myocilin protein levels in the aqueous humor of the glaucomas in selected canine breeds

Objective To compare aqueous humor myocilin protein levels in dogs with the primary glaucomas to those with the secondary glaucomas, primary cataracts, and diabetic cataracts. Materials and methods Four groups were selected, based on diagnosis by the attending veterinary ophthalmologists and included: primary glaucoma (primary open‐angle glaucoma (POAG) and primary closed angle glaucoma (PCAG); n = 155); secondary glaucoma (n = 94); primary (presumed inherited) cataract (n = 142), and diabetic cataract (n = 83). A total of 474 samples (187 males, 263 females, 24 unreported) with average ages of 117 months for the males and 101 months for the females were analyzed. Myocilin protein was measured using the Coomassie staining and Western blot methods relative to a myocilin control. Results Differences were seen between nonglaucomatous (cataractous) and glaucomatous dogs with myocilin levels in glaucomatous eyes being many times higher than those in the cataractous dogs. Primary glaucomatous dogs were found to have an aqueous humor myocilin protein level of 17.30 ± 1.03 units. Secondary glaucomas had the highest level of myocilin in the aqueous humor with 19.27 ± 1.41 units. Diabetic cataractous dogs had the lowest levels of myocilin reported with 6.60 ± 0.88 (mean ± SEM) units. Normal (cataractous) dogs had a myocilin level in the aqueous humor of 8.05 ± 0.86 units. Conclusion Aqueous humor protein levels were elevated, relative to the myocilin control, in both the primary and secondary glaucoma groups compared to the cataract and diabetic cataract groups. Like in the Beagle POAG, aqueous humor myocilin protein levels are increased. Further studies are indicated to investigate the exact role of the aqueous humor myocilin protein in the genesis in increased IOP in these primary glaucomatous breeds.     

In vitro fermentation characteristics of different carbohydrate sources in two dog breeds (German shepherd and Neapolitan mastiff)

Few studies have been published on the normal intestinal biota of canines unlike the wealth of information regarding livestock animal species. The in vitro gas production technique (IVGPT) including measurements of accumulating gas during fermentation and end‐product determinations allows obtaining a complete picture of microbial activity kinetics. The aim of this study was to study the in vitro fermentation characteristics of different carbohydrate sources using inocula from two dog breeds (German Shepherd and Neapolitan mastiff). Faeces sampled from rectum of two GS and NM adult dogs, fed the same dry food, were used as inocula. The samples, diluted and filtered, were incubated at 39 °C under anaerobic condition with nine substrates different for carbohydrate composition (rice, corn, potato, spelt, pure cellulose, beet pulp, wheat bran, inulin and fructo‐oligosaccharide). Gas production was recorded 17 times using a manual pressure transducer. After 48 h, the fermentation was stopped and fermenting liquor was analysed for pH and volatile fatty acids (VFA). Organic matter digestibility (OMD) was calculated as difference after burning the residuals. OMD, gas production and end‐products were significantly correlated with chemical composition of substrates, in particular carbohydrate fractions (total dietary fibre and starch), confirming the effectiveness of the IVGPT in evaluating dog feeds. Concerning the comparison between breeds significant differences (p < 0.01) were found for OMD, gas production, fermentation kinetic parameters and end‐products, suggesting a different pathway of fermentation and consequently, a different anaerobic population.     

三穗鸭SLC4A9基因表达量及其多态性对蛋壳品质的遗传效应分析

为探究溶质载体家族4成员9（solute carrier family 4 member 9,SLC4A9）基因的表达量及其多态性对鸭蛋壳品质的遗传效应,试验采用实时荧光定量PCR法检测SLC4A9基因在三穗鸭不同组织中的表达水平,采用PCR产物直接测序法筛查三穗鸭SLC4A9基因的SNP位点,并分析SNP位点对三穗鸭蛋壳品质的遗传效应。结果显示,SLC4A9基因mRNA在三穗鸭10个组织中均有不同程度表达,其中在心脏和胰腺中为高度特异性表达,肾脏中为中度表达,其他为低度表达。试验共检测到10个SNPs位点,其中3个位于外显子上（g.6803 C>T、g.7065 C>T和g.7089 C>G）,且均为错义突变,7个位于内含子上（g.7162 C>G、g.11044 G>A、g.11090 T>C、g.11234 C>T、g.11261 C>T、g.11349 C>T和g.11403 G>A）,其中g.6803 C>T位点突变导致丙氨酸（GCG）变为缬氨酸（GTG）;g.7065 C>T位点突变导致丙氨酸（GCC）变为缬氨酸（GTC）;g.7089 C>G位点突变导致丙氨酸（GCA）变为甘氨酸（GGA）。χ²检验结果显示,g.6803 C>T、g.11090 T>C和g.11349 C>T突变位点的基因型分布均偏离了Hardy-Weinberg平衡状态（P<0.05）,其余位点的基因型分布均符合Hardy-Weinberg平衡状态（P>0.05）。关联分析结果表明,g.11349 C>T位点的CC和CT基因型蛋壳强度显著高于TT基因型（P<0.05）。综上,SLC4A9基因的10个SNPs位点均对三穗鸭的蛋壳品质产生影响,其中g.11349 C>T位点达到了显著水平,可为蛋鸭蛋壳品质的改善及遗传标记提供参考依据。     

SNaPshot based genotyping of the RYR1 mutation in Portuguese breeds of pigs

The porcine stress syndrome or malignant hyperthermia is an inherited autosomic recessive disease, which results in neuromuscular disorders leading to death in homozygous individuals and is associated with deterioration of meat quality. The defect in susceptible animals results from modifications in the calcium release channel or Ryanodine Receptor (RYR1), with a mutation leading to a C to T transition in nucleotide 1843 of the gene. The objective of this work was to develop a method based on analysis of SNPs to detect the mutation described in the RYR1 locus in pigs, and study polymorphisms of the gene in four exotic (Large White, Landrace, Duroc and Pietrain) and three native (Bísaro, Alentejano and Malhado de Alcobaça) breeds of pigs in Portugal. The method was successful in identifying the mutation by analysis of SNPs, and results indicate a high incidence of the mutant allele in Pietrain (0.75) and, to a lesser degree, in Malhado de Alcobaça (0.34) and Landrace (0.28); frequencies in Alentejano, Bísaro and Large White ranged between 0.04 and 0.09. These results suggest the need to establish breeding programs aimed at eliminating the susceptibility allele from those populations.     

Detection and molecular epidemiology of canine parvovirus type 2 (CPV-2) circulating in Jilin Province,Northeast China

Canine parvovirus (CPV) is highly contagious and can cause haemorrhagic enteritis and myocarditis in dogs. To understand the current epidemic situation of CPV in Jilin Province, China, a total of 44 fecal or intestinal tissue samples of pet dogs suspected of being infected with CPV from February 2018 to November 2019 in Changchun and Liaoyuan City, Jilin Province were collected.All of the 44 collected samples were tested positive to CPV-2 by a PCR assay. The sequencing and analyzing of complete VP2 genes showed that CPV-2c was the most prevalent variant (n = 31;70.4 %), followed by new-CPV-2a (n = 8;18.2 %), new-CPV-2b (n = 4; 9.1 %) and CPV-2 (n = 1; 2.3 %). Phylogenetic analysis revealed that the 31 CPV-2c strains in our study are closely related to local CPV-2c isolates in cluster I. The VP2 protein of the acquired CPV 2c strains all possessed the substitutions Ala5Gly, Phe267Tyr, Tyr324Ile, and Gln370Arg only one with a novel Arg481Lys mutation. These findings demonstrate that CPV-2c was the most prominent type of CPV circulating in Jilin in 2018–2019, clustered in a separate group that is far from the vaccine strains and suggest that further and extensive epidemiological investigation among pet dogs are warranted to provide information for usage and research of current vaccines.     

Histologic morphology and involucrin, filaggrin, and keratin expression in normal canine skin from dogs of different breeds and coat types

The purpose of this study was to measure the thickness of canine epidermis at various anatomical sites according to localization of cornified envelopes (involucrin and filaggrin), keratins (keratin 10, 5), and their mRNA expression. This was done in the skin of five breeds of dogs including seven poodles, six golden retrievers, six Shih Tzus, four pugs, and four Labrador retrievers. Epidermal thickness of the stratum corneum and nucleated epidermal layer was significantly different. The greatest thickness was observed in the digital web area and the thinnest epidermis was in the axilla. Epidermal thickness was also significantly different between the breeds (p < 0.05). Immunohistochemical staining scores revealed significant decreases of involucrin, filaggrin, and keratin 10 in the ventral and weight-bearing sites, and a relative increase of keratin 5 (p < 0.05). q-PCR analysis showed that their the levels of mRNA were positively correlated with expression of the corresponding proteins in skin samples (p < 0.05). The present study is the first to report the relationship between epidermal gene expression and histologic morphology of the skin in normal dogs. Further studies will be essential to fully understand the pathogenesis of skin barrier dysfunctions in canines.