家蚕微孢子虫丙酮酸激酶基因的克隆与表达特征分析

丙酮酸激酶(pyruvate kinase,PK)在糖酵解最后一步起着不可逆的作用,可以催化磷酸烯醇式丙酮酸将高能磷酸基团转移给ADP生成ATP和丙酮酸。通过在NCBI和家蚕微孢子虫(Nosema bombycis)数据库中对丙酮酸激酶基因的比对分析,选择家蚕微孢子虫丙酮酸激酶M2亚型的一个基因(NbPK-2,GenBank登录号EOB11679.1)进行克隆。生物信息学分析显示,该基因序列长度为1 359 bp,包含一个完整的开放阅读框,编码452个氨基酸,预测蛋白质等电点为7.13,相对分子质量为51.7 kD,没有信号肽;NbPK-2蛋白的二级结构预测显示其含有38%的螺旋、21%的延伸片段和40%无规则卷曲。通过qRT-PCR检测感染微孢子虫后家蚕不同发育时期的NbPK-2表达水平,发现该基因在感染后24 h内处于相对较高的表达水平,而此后表达水平开始降低,至144 h达到最低值,在168 h又有所升高。研究结果可促进对家蚕微孢子虫糖酵解途径的研究,为家蚕微粒子病的防控奠定理论基础。     

鸡软骨细胞体外分离培养鉴定以及过表达miR-15a对软骨细胞的影响

为分析miR-15a在肉鸡不同组织中的表达情况,并探究过表达miR-15a对体外培养鸡软骨细胞的影响。本研究首先通过倒置显微镜观察、PCR、凝胶电泳和甲苯胺蓝染色对体外分离培养的软骨细胞进行鉴定。通过实时荧光定量PCR检测miR-15a在肢体内外翻畸形(valgus-varus deformity, VVD)组和健康组肉鸡中(各3只)各组织的表达量。之后通过CCK8和EDU方法分析软骨细胞过表达miR-15a后细胞增殖情况。软骨细胞转染miR-15a mimics后,通过qPCR检测软骨细胞的标志基因Collagen-2、Aggrecan、Collagen-10,成熟分化基因Runx2、Sox9、VEGF、MMP9,炎性因子IL-1β、IL-6、IL-8、IL-10、TNF-α、TGF-β3以及凋亡基因Fas、FasL、Bcl-2的表达量。并构建FKBP5 3'UTR的野生型载体和突变型载体,通过双荧光素酶检测报告检测miR-15a与FKBP5的靶向关系。结果表明,本研究所用的胰蛋白酶、Ⅱ型胶原酶和透明质酸酶联合消化法成功分离得到状态良好的软骨细胞。荧光定量结果显示,miR-15a在各组织中均有表达,与健康组相比,miR-15a在VVD组的肝(P < 0.01)、脾(P < 0.05)、胸腺(P < 0.01)中的表达量显著升高,在心和胸肌组织中的表达量显著降低(P < 0.01)。CCK8与EDU分析结果显示,与NC组相比,过表达miR-15a组软骨细胞增殖速度显著降低(P < 0.01),增殖细胞数量显著减少(P < 0.01)。qPCR结果显示,与mimics NC组相比,miR-15a mimics组的软骨细胞标志基因Aggrecan、成熟分化基因Sox9、Runx2表达量显著降低(P < 0.05),Fas基因表达量极显著上升(P < 0.01),FasL基因和抗凋亡基因Bcl-2极显著下降(P < 0.01)。成功构建了FKBP5 3'UTR野生型和突变型载体,双荧光素酶检测报告结果显示预测的靶基因FKBP5与miR-15a没有靶向关系。本研究成功分离并鉴定了鸡软骨细胞,过表达miR-15a抑制鸡软骨细胞增殖、成熟和分化并促进细胞凋亡。     

家蚕同源异型结构域蛋白基因Bmvis的克隆及组织表达和亚细胞定位

同源异型结构域蛋白(homeobox,HOX)基因是对生物体的生长发育从时间和空间上进行调控的一类基因。Vis(vismay)属于同源异型结构域蛋白转化生长影响因子(transforming growth interacting factor,TGIF)家族成员,在脊椎动物的研究中表明TGIF是转化生长因子-β(transforming growth factor β,TGF-β)信号转导途径中的转录抑制子,但在果蝇(Drosophilamelanogaster)的研究中发现其是一种转录激活子。基于为探究家蚕(Bombyx mori)中vis基因的功能提供基础信息的目的,在电子克隆的基础上通过RT-PCR从家蚕5龄幼虫精巢cDNA中克隆了长度为847 bp的vis基因(Bmvis)片段(GenBank登录号:JF412700)。蛋白结构分析表明Bmvis在HOX结构域的螺旋区和TALE区段内高度保守,同时在HOX结构域外的C末端大约20个氨基酸也是高度保守的。进化分析显示昆虫的Vis与Achi(achintya)聚为一类,但Vis与Achi根据物种分类关系聚在一起。对Bmvis在家蚕5龄第3天幼虫组织中表达的RT-PCR检测显示其仅在精巢中表达。将Bmvis进行原核表达后获得抗Bmvis多克隆抗体,亚细胞定位显示Bmvis在细胞核和细胞质中均有表达,但主要分布在细胞核中。     

Molecular prevalence,risk factors assessment and haemato-biochemical alterations in hepatozoonosis in dogs from Punjab,India

Hepatozoonosis caused by Hepatozoon canis is an important tick-borne disease of dogs in tropical and sub-tropical regions throughout the world. In the present study evaluation of blood samples collected from 225 dogs presented at Small Animal Clinics, GADVASU, Ludhiana, Punjab (India) was done for the presence of H. canis by PCR based assay targeting a portion of 18S rRNA gene. Of the total samples subjected to PCR, an amplicon of 666 bp was detected in 13.78% samples whereas, routine blood smear examination revealed gamonts in 5.78% samples. Furthermore, prevalence of H. canis infection was found to be significantly associated with season, being highest in summer and lowest in winter while other risk factors e.g. age, sex and breed showed non-significant association. In terms of various clinico-pathological parameters, significant drop in haemoglobin, total red blood cell count, packed cell volume and lymphocytes were recorded in positive cases whereas the total white blood cell count was non-significantly increased. The haematological alterations in the positive cases were lymphopenia, anaemia, thrombocytopenia, relative neutrophilia, neutrophilic leucocytosis, eosinophilia, monocytosis and lymphocytosis while the biochemical profile revealed hypoproteinemia and increased levels of blood urea nitrogen and creatinine (in positive cases) pointing towards renal failure.     

Cloning and Expression of CMKLR1 Gene and Its Correlation with Intramuscular Fat in Goat (Capra hirus)

In order to enrich basic date in goat (Capra hirus) CMKLR1 gene and investigate the correlation between CMKLR1 gene expression and intramuscular fat (IMF) content in muscles, the CMKLR1 gene was cloned from the goat of subcutaneous adipose tissue by RT-PCR, characterized by bioinformatics methods. The expression profiles of CMKLR1 gene of goat in various tissues were constructed liver was benchmark, the GAPDH (GenBank accession No.: AJ431207.1) as a reference gene.Then, the correlation between CMKLR1 mRNA expression and IMF content in muscles was analyzed. The results showed that the CMKLR1 gene CDS of goat (GenBank accession No.: KT165374) was 1 089 bp. Real-time PCR indicated that CMKLR1 was widely expressed in various tissues, including heart, liver, spleen, lung, kidney, adipose, longissimus dorsi, biceps femoris and triceps brachii, and was the highest in lung (P< 0.05). Similar expression variation trend of CMKLR1 was observed between the muscles from 1 to 3 and 24 months old goat, and was the highest in biceps femoris. Reversely, in 8 to 10 months old CMKLR1 was the highest in triceps brachii. The IMF of longissimus dorsi from 24 months old was the highest. Correlation analysis demonstrated that different correlations were observed between expression of CMKLR1 mRNA and IMF content in longissimus dorsi, biceps femoris, and triceps brachii in goat. The CMKLR1 gene did not participate in the deposition of IMF in goats. The research built the theoretical basis for further studies about the CMKLR1 gene.     

Effects of guanidinoacetic acid on growth performance,creatine metabolism and plasma amino acid profile in broilers

The objective of this study was to assess the effects of guanidinoacetic acid (GAA) on growth performance, creatine deposition and blood amino acid (AA) profile on broiler chickens. In Exp. 1, a total of 540 one‐day‐old Arbor Acres male broilers (average initial body weight, 45.23 ± 0.35 g) were divided randomly into five treatments with six replicates of 18 chicks each. Broilers were fed corn–soybean meal‐basal diets supplemented with 0, 600, 800, 1,000 or 1,200 mg/kg GAA for 42 days respectively. Results showed that dietary GAA inclusion increased average daily gain (ADG) and improved gain‐to‐feed ratio (G:F) from 1 to 42 days (p < 0.01). However, average daily feed intake was unaffected by dietary supplementation of GAA. As GAA inclusion increased, the contents of creatine in plasma and kidney were increased (linear, p < 0.01), while the contents of GAA and creatine in liver were decreased (linear, p < 0.01). Similarly, GAA supplementation was inversely related to concentrations of most essential AA in plasma. In Exp. 2, a total of 432 one‐day‐old Arbor Acres male broilers (average initial body weight, 39.78 ± 0.58 g) were divided randomly into four treatments with six replicates of 18 chicks each. Birds were fed a corn–soybean meal‐basal diet supplemented with 0, 200, 400 or 600 mg/kg GAA for 42 days respectively. Dietary inclusion of 600 mg/kg GAA significantly increased ADG and G:F of broilers (p < 0.05). In conclusion, dietary supplementation of 600–1,200 mg/kg GAA can effectively improve the growth performance in broiler chickens by affecting creatine metabolism and utilization efficiency of essential AA, and 600 mg/kg GAA is the minimum dose for improving performance.     

Effects of dietary supplementation of emulsifier and carbohydrase on the growth performance,serum cholesterol and breast meat fatty acids profile of broiler chickens

A total of 540 2‐day‐old male Ross 308 broilers were used in a 35‐day experiment and were randomly divided into five treatments: (i) NC (low energy); (ii) PC (high energy diet); (iii) P1 (NC + 0.1% carbohydrases); (iv) P2 (NC + 0.05% emulsifier); and (v) P3 (NC + 0.1% carbohydrases + 0.05% emulsifier). From days 0 to 21, body weight gain in PC and P3 treatments increased (P < 0.05) compared with NC treatment. The chicks fed PC, P1, P2 and P3 improved (P < 0.05) feed conversion ratio compared with the NC treatment throughout the whole experiment. Abdominal fat weight was heavier (P < 0.05) in PC, P2 and P3 treatments than in NC and P1 treatments. On day 35, serum total cholesterol and low density lipoprotein cholesterol concentration were higher (P < 0.05) and high‐density lipoprotein cholesterol was lower (P < 0.05) in NC and P2 treatments than in PC, P1 and P3 treatments. The concentration of oleic acid, linoleic acid and total unsaturated fatty acids were highest (P < 0.05) in PC than in the other treatments. In conclusion, the results indicate that low energy density diet had lower growth performance, while the inclusion of emulsifier and carbohydrases in low energy diets can partially improve growth performance.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

Variation of Hematochemical Profile and Vitamin E Status in Feral Giara Horses From Free Grazing in the Wild to Hay Feeding During Captivity

Wildlife protection and management are important priorities for landscape identity and biodiversity preservation. Feeding practices of fauna confined in facilities during temporary captivity are fundamental to support animal health and natural behavior. Appropriate provision of feedstuffs appears to be necessary to support the best practices in respect of animal species-specific natural diet. This investigation explored the variation of the metabolic profile by means of selected metabolite and respective circulating levels in a group feral Giara horses undergoing the change of the diet, moving from natural free grazing in the wild to temporary captivity. Six Giara horses (4 mares and 2 stallions; estimated age: 2.5–3 years; body weight: 163–170 kg) were captured to monitor the serological reaction to equine infectious anemia (EIA; screening at Coggins test). Animals were sheltered in a wildlife rescue center for a duration of 4 weeks, and all received the same hay-based diet (ad libitum). On 0 and 28 days of captivity, blood serum alpha-tocopherol (α-TOH) concentration was determined alongside selected metabolites (liver enzymes, total protein and fractions, cholesterol, triglycerides, and macrominerals and trace elements). Comparative feces quality and composition were also assessed. Both serum samples (0 vs. 28 days) displayed α-TOH levels below (<2 μg/mL) adequacy established for the domestic horse. Initial levels markedly (P = .020) decreased after the 4 weeks of captivity (Δ = −32.5%). Vitamin E status and ALT levels varied significantly, but serum protein fractions did not point to significant variations before and after captivity. All horses tested negative to EIA. Monitoring of vitamin E status of wild and feral herbivores may be recommendable in the context of adequate feeding practices during captivity to prevent potential deficiency or excessive depletion.     

Branched‐chain amino acid ratios in low‐protein diets regulate the free amino acid profile and the expression of hepatic fatty acid metabolism‐related genes in growing pigs

Liver metabolism is affected by nutrients. The aim of this study was to explore the effects of low‐protein diets (17% crude protein, CP) supplemented with branched‐chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile) and valine (Val), on hepatic amino acid profile and lipid metabolism in growing pigs. The ratio of Leu : Ile : Val in all groups was 1 : 0.51 : 0.63 (20% crude protein, CP), 1 : 1 : 1 (17% CP), 1 : 0.75 : 0.75 (17% CP), 1 : 0.51 : 0.63 (17% CP) and 1 : 0.25 : 0.25 (17% CP) respectively. Results revealed that compared to the positive control group (1 : 0.51 : 0.63, 20% CP), the low‐protein diets significantly augmented the concentrations of most essential amino acids and non‐essential amino acids (p < .05), with the greatest values observed in the 1 : 0.25 : 0.25 group. Moreover, relative to the control, the low‐protein diets with the Leu : Ile : Val ratio ranging from 1 : 0.75 : 0.75 to 1 : 0.25 : 0.25 markedly downregulated the mRNA abundance of acetyl‐CoA carboxylase (ACC), lipoprotein lipase (LPL) and fatty acid‐binding protein 4 (FABP‐4) (p < .05), and upregulated the mRNA expression of hormone‐sensitive lipase (HSL), peroxisome proliferator‐activated receptor‐g coactivator‐1α (PGC‐1α), uncoupling protein 3 (UCP3) and liver carnitine palmitoyltransferase 1 (L‐CPT‐1) (p < .05). Therefore, our data suggest that protein‐restricted diets supplemented with optimal BCAA ratio, that is, 1 : 0.75 : 0.75–1 : 0.25 : 0.25, induce a shift from fatty acid synthesis to fatty acid oxidation in the liver of growing pigs. These effects may be associated with increased mitochondrial biogenesis.