蓝塘仔猪IGF-1水平与组织IGF-1、GHR基因的表达

32头不同日龄(出生、3、21、35d)蓝塘仔猪,由前腔静脉采血后剖杀,取肝脏、背最长肌样品。用RIA法测血液、组织中IGF 1浓度,用放射受体法(RBA)检测肝脏、肌肉组织中GHR结合活性,用实时荧光定量PCR法检测IGF 1、GHRmRNA的表达水平。结果表明:(1)血液中IGF 1在出生日显著高于其它时期(P<0 05)。肌肉组织中IGF 1含量高于肝脏组织,肌肉组织IGF 1含量在3、21、35日龄时都显著高于出生日(P<0 05)。(2)肝脏细胞膜GHR结合活性在出生日显著高于3、21日龄(P<0 05),肝脏细胞膜GHR结合活性高于肌肉组织。(3)肝脏组织IGF 1、GHRmRNA的表达量均显著高于肌肉组织(P<0 05)。肝脏IGF 1mRNA的表达在出生日、21日龄时显著高于3、35日龄(P<0 05),GHRmRNA的表达在出生日显著高于其它日龄(P<0 05)。肌肉IGF 1、GHRmRNA的表达在出生日均显著高于其它日龄(P<0 05)。     

亚洲璃眼蜱唾液腺一新功能基因的克隆与测序

HaB1是亚洲璃眼蜱雌成蜱唾液腺差异表达基因文库中的一个片段,根据其序列设计引物HaB1-GSP1和HaB1-GSP2,以唾液腺总RNA为模板,RACE法扩增获得HaB1的未知3′-末端。测定该末端序列,进行序列拼接,设计全长引物5-′CCAGTCCGAAGGAAGGGCG-3′和5-′TCTC-CGGGCACGTGAAGTGTC-3′。以cDNA第一链为模板,扩增获得基因全长。经核查表明,该基因为一新基因(登录号AY803896)。     

山羊孤雌胚胎的体外培养

系统地研究了培养液微滴的大小、培养液体系及血清浓度对山羊孤雌胚胎早期体外发育的影响。结果表明，培养液微滴以较大体积(200～500μL)的培养效果较好；在3种培养液中，添加10％的NGS对山羊孤雌胚胎的体外发育效果较好，囊胚率分别可达62．79％(81／129)、53．52％(38／71)、13．64％(12／88)；mCRlaa组囊胚发育率和囊胚细胞数显著低于SOFaa组和CRlaa组，SOFaa组优于CRlaa组，尽管SOFaa组和CRlaa组间无显著差异。在本试验条件下，以SOFaa培养液，山羊孤雌胚胎体外培养72h时加入10％的NGs，500μL微滴培养，其发育效果较好，囊胚率可达62．79％。     

猪2型圆环病毒核酸疫苗免疫效应研究

PCR扩增PCV2 ORF2和BVP22基因。将ORF2基因克隆入真核表达载体pCDNA3．1（＋），构建了pCORF2作为核酸疫苗免疫小白鼠。同时将具有蛋白转导功能的BVP22基因分别克隆到ORF2基因的上游和下游，使二者融合表达，命名为pCORF2BVP22和pCBVP220RF2。分4组肌肉免疫BALB/c小白鼠，分别注射pCDNA3．1（＋）、pCORF2、pCORF2BVP22和pCBVP220RF2，共免疫2次，间隔2周，分别于首免后2周和二免后4周采血，ELISA法检测体液抗体。同时为在体外验证ORF2和BVP22基因的真核表达，将ORF2和BVP22基因分别克隆入pEGFP-N1载体，构建了pNORF2和pNBVP22，转染Hela细胞后在荧光显微镜下观察到了ORF2和BVP22在体外的瞬时高效表达。结果显示，通过两次免疫后，各疫苗组均产生了针对ORF2的体液抗体，同时证明BVP22可增强核酸疫苗的免疫效果，其中以BVP22在ORF2上游效果更好。本研究为防制猪2型圆环病毒感染提供了较为理想的侯选疫苗。     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.     

断奶前补饲不同直/支链淀粉比开食料对羔羊瘤胃上皮发育的影响

为了研究断奶前补饲不同直/支链淀粉比开食料对羔羊瘤胃上皮发育的影响,选取24只体况良好、胎次一致、体重相近的10日龄羔羊(湖羊),将其随机分为3组,在饲喂相同母乳的基础上,分别补饲以纯木薯(CS)、玉米(MS)和豌豆淀粉(PS)(直/支链淀粉比分别约为0.11,0.27和0.44)为唯一淀粉来源的开食料。试验期间,每天4:00-19:00将羔羊抱入补饲栏补饲不同直/支链淀粉比的开食料,并且在6:30, 10:30和15:30将羔羊抱回母羊舍哺乳1 h。羔羊自由饮水,单栏饲喂,自由采食燕麦干草。56日龄时屠宰采样,采集瘤胃组织样品制作石蜡切片进行瘤胃乳头形态测定,采集瘤胃上皮样品提取RNA测定相关基因表达。结果表明:CS组羔羊瘤胃乳头长度和表面积显著(P<0.001)高于MS和PS组,CS和MS组羔羊瘤胃乳头宽度显著(P=0.001)高于PS组。对瘤胃上皮各层厚度统计显示,CS和PS组羔羊瘤胃上皮角质层厚度显著(P=0.001)高于MS组;CS和MS组羔羊瘤胃上皮颗粒层厚度显著高于(P<0.001)PS组;CS组羔羊瘤胃上皮棘基层厚度和总厚度显著(P<0.001)高于MS和PS组。qRT-PCR结果显示:不同直/支链淀粉比开食料显著影响羔羊瘤胃上皮CDK2和CDK6的mRNA表达量(CS>MS>PS, P<0.001);CS组羔羊瘤胃上皮细胞cyclin A和CDK4的mRNA表达量显著(P<0.05)高于PS组,但与MS组无显著差异;CS组羔羊瘤胃上皮cyclin D1的mRNA表达量显著(P=0.012)低于PS组,但与MS组无显著差异。CS组羔羊瘤胃上皮类胰岛素生长因子-1(insulin like growth factor,IGF-1)的mRNA表达量显著(P<0.001)高于MS和PS组,CS和MS组羔羊瘤胃上皮IGF-1R的mRNA表达量显著(P=0.001)高于PS组。上述结果表明:较高支链淀粉比开食料与较高直链淀粉比开食料相比,在瘤胃中较易降解生成挥发性脂肪酸,促进瘤胃上皮IGF-1及细胞周期蛋白mRNA的表达,进而有利于断奶前羔羊瘤胃上皮的发育。研究结果对制订羔羊营养方案具有重要指导意义。     

Interaction between the sequence of feeding of hay and concentrate,and boiling of barley on feed intake,the activity of hydrolytic enzymes and fermentation in the hindgut of Arabian mares

The interaction between the sequence of feeding of hay and concentrate and the hydrothermal processing of barley in alleviating concentrate effects on intake, and hindgut fermentation in horses was tested. Six Arabian mares (4–10 years of age, 410 ± 35 kg body weight) were used to evaluate the effects of feeding sequence (FS) and type of barley (TB) on intake, and faecal volatile fatty acids (VFA), activities of α‐amylase (AA: EC 3.2.1.1), carboxymethyl cellulase (CMCase: EC 3.2.1.4), microcrystalline cellulase (MCCase: EC 3.2.1.91) and general filter paper degrading activity (FPD). Mares were offered a ration of air‐dried alfalfa and concentrate (70:30 as‐fed) in four subsequent periods of 14 days including 8 days of adaptation and 6 days of sampling. In each period and each meal, mares received concentrate either 30 min after (HC) or 30 min before (CH) alfalfa hay. Barley was either milled or boiled in water. Rectal samples were grabbed directly from rectum once per period. Mares subjected to CH had higher dry matter intakes than mares under HC regime. The acetate:propionate ratio (A:P ratio) in rectal content was higher with CH than HC. The AA activity was higher under CH than under HC. Mares fed boiled barley had lower rectal concentrations of VFA and propionate and a higher A:P ratio than mares fed milled barley. Furthermore, the rectal content showed a higher MCCase activity but a lower AA activity when mares were fed boiled compared with milled barley. Interactions between FS and TB were observed with respect to CMCase activity, and concentrations of propionate and valerate. In conclusion, the present results suggest that both, feeding concentrate before hay and boiling the barley, might improve the hindgut environment in Arabian mares, and that the two measures were mostly additive and sometimes even synergistic.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

Methyl donors dietary supplementation to gestating sows diet improves the growth rate of offspring and is associating with changes in expression and DNA methylation of insulin‐like growth factor‐1 gene

The study aimed to investigate the effects of maternal dietary methyl donors on the performance of sows and their offspring, and the associated hepatic insulin‐like growth factor‐1 (IGF‐1) expression of the offspring. A total of 24 multiparous sows were randomly fed the control (CON) or the CON diet supplemented with methyl donors (MD) at 3 g/kg betaine, 15 mg/kg folic acid, 400 mg/kg choline and 150 μg/kg VB₁₂, from mating until delivery. After farrowing, sows were fed a common lactation diet through a 28‐days lactation period and six litters per treatment were selected to be fed until at approximately 110 kg BW. Maternal MD supplementation resulted in greater birthweight (p < 0.05) and increased the piglet weights (p < 0.01) and litter weights (p < 0.05) at the age of day 28, compared with that in CON group. The offspring pigs in the MD group had greater ADG (p < 0.05) and tended to lower F:G ratio (p = 0.07) compared with that of CON group from day 28 to 180 of age. The offspring pigs from MD group had greater serum IGF‐1 concentrations and expressions of hepatic IGF‐1 gene and muscular IGF‐1 receptor (IGF‐1r) protein at birth (p < 0.05), and greater hepatic IGF‐1 protein (p = 0.03) and muscular IGF‐1r gene expressions (p < 0.05) at slaughter, than that from the CON group. Moreover, the methylation at the promoter of IGF‐1 gene in the liver of newborn piglets and finishing pigs was greater in the MD group than that of the CON group (p < 0.05). In conclusion, maternal MD supplementation throughout gestation could enhance the birthweight and postnatal growth rate of offspring, associated with an increased expression of the IGF‐1 gene and IGF‐1r, as well as the altered DNA methylation of IGF‐1 gene promotor.