Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

Investigation of the Role of Androstenedione-19-oic Acid in the Presence of 19-Norandrostenedione in Intact Male Horse Plasma Using Liquid Chromatography–Tandem Mass Spectrometry

A liquid chromatography–tandem mass spectrometry method was developed to confirm the presence of androstenedione-19-oic acid in intact male equine plasma and to show the source of 19-norandrostenedione in equine plasma. Androstenedione-19-oic acid was recovered from acidified plasma by liquid–liquid extraction using methyl tert-butyl ether and separated on an Ace 5 C₈ column. A triple quadrupole mass spectrometer was used to detect the analytes in negative electrospray ionization mode. Limits of detection, quantification, and confirmation of the method were 0.1, 0.5, and 1.0 ng/mL, respectively. The linear dynamic range of quantification was 0.5–50 ng/mL. The presence of androstenedione-19-oic acid was confirmed in all plasma samples obtained from intact male horses but not those from gelded and female horses; the average concentration was 3.1 ± 1.6 ng/mL, suggesting androstenedione-19-oic acid is an endogenous compound only in intact male horse plasma samples. The conversion of androstenedione-19-oic acid to 19-norandrostenedione in equine plasma was demonstrated by spiking androstenedione-19-oic acid into blank plasma and monitoring the generation of 19-norandrostenedione and its increase in concentration during storage. Results indicated that androstenedione-19-oic acid was readily converted into 19-norandrostenedione; the higher the storage temperature, the faster the conversion. The conversion was not affected by the types of plasma samples collected from gelded and female horses or by anticoagulants used in blood collection to harvest plasma. Compared with other matrices such as water, methanol, and phosphate-buffered saline, the conversion of androstenedione-19-oic to 19-norandrostenedione in equine plasma was faster, suggesting that there is an unknown factor(s) in equine plasma that enhances the conversion.     

Fatty acid patterns of dog erythrocyte membranes after feeding of a fish-oil based DHA-rich supplement with a base diet low in n-3 fatty acids versus a diet containing added n-3 fatty acids

Background

In dogs, increasing the tissue n-3 fatty acid (FA) content is associated with potential benefit in some medical conditions, e.g. atopic dermatitis, cancer or heart disease. Therefore effectively and conveniently increasing tissue n-3 FA levels in dogs is of interest. Incorporation of dietary n-3 FA into cell membranes may be studied by FA analysis of erythrocyte membranes (EM), because of the correlation of its FA composition with the FA composition of other cells. Aim of the study was to determine whether an n-3 FA additive added to a control diet is as effective in increasing EM n-3 FA content as feeding an n-3 FA enriched diet. Furthermore the time course of the incorporation of dietary n-3 FA into canine EM was investigated.

Methods

Thirty dogs were randomly divided into three dietary groups with ten dogs per group. CONT got a dry dog food diet which did not contain EPA or DHA. FO got a dry dog food diet with a high EPA and DHA content. ADD got the CONT diet combined with an n-3 FA additive rich in DHA and EPA. After a feeding period of 12 weeks the additive was discontinued in ADD and these dogs were fed CONT diet for another four weeks to observe washout effects. Erythrocyte lipids were extracted from venous blood samples and their FA composition was determined by gas chromatography. The Mann-Whitney-U-test was used to detect significant differences between the different groups and time points.

Results

After one week the proportions of n-3 FA, DHA and EPA were already significantly increased in ADD and FO, apparently reaching a plateau within eight weeks. In our study DHA and not EPA was preferably incorporated into the EM. After discontinuing the administration of the additive in ADD, the n-3 FA values declined slowly without reaching baseline levels within four weeks.

Conclusions

In dogs, an increase of dietary n-3 FA content leads to a rapid inclusion of n-3 FA into EM, regardless of whether the n-3 FA are offered as an enriched diet or as a normal diet supplemented with an n-3 FA additive.     

颈静脉灌注不同模式的氨基酸混合物对泌乳奶牛乳蛋白合成的影响

本试验旨在比较颈静脉灌注酪蛋白模式和理想模式的氨基酸混合物对泌乳中期荷斯坦奶牛乳产量、乳成分以及乳腺对氨基酸摄取利用的影响。选择8头泌乳中期[泌乳天数:(82±11)d]荷斯坦奶牛作为试验动物,试验采用随机区组设计,将试验牛随机分为2组,分别颈静脉灌注160g/d酪蛋白模式(Casein组)和理想模式的氨基酸混合物(R组)。2个试验组分别以各自灌注前作为空白对照组(C1组为Casein组的空白对照组,C2组为R组的空白对照组)。预试期14d,灌注期5d。试验采用全混合日粮(TMR)饲喂,以玉米、豆粕、棉籽粕、玉米青贮、苜蓿干草和羊草为主要原料,参照NRC(2001)奶牛饲养标准配制。结果表明:灌注酪蛋白模式的氨基酸混合物后,乳蛋白产量和含量较灌注前呈上升趋势(乳蛋白产量上升7.14%,P=0.078;乳蛋白含量上升3.27%,P=0.072);并且,奶牛动脉血浆中异亮氨酸(Ile)、亮氨酸(Leu)、赖氨酸(Lys)和组氨酸(His)的浓度较灌注前有不同程度的上升(Ile的浓度提高31.5%,P=0.097;Leu的浓度提高65.9%,P=0.041;Lys的浓度提高36.9%,P=0.088;His的浓度提高40.1%,P=0.010),而苏氨酸(Thr)、缬氨酸(Val)、蛋氨酸(Met)、苯丙氨酸(Phe)和精氨酸(Arg)的浓度在数值上虽较灌注前高但无显著差异(P0.05)。灌注酪蛋白模式的氨基酸混合物后,奶牛乳腺对天冬氨酸(Asp)和半胱氨酸(Cys)的摄取率显著升高(Asp的摄取率提高95.2%,P=0.031;Cys的摄取率提高49.6%,P=0.031),而奶牛乳腺对甘氨酸(Gly)的摄取率显著降低(降低158.3%,P=0.041)。灌注理想模式的氨基酸混合物后,乳蛋白含量比灌注前有上升趋势(提高5.78%,P=0.064),而乳脂产量显著低于灌注前(降低8.57%,P=0.015);并且,奶牛动脉血浆中Arg的浓度有上升趋势(提高18.0%,P=0.093),而酪氨酸(Tyr)的浓度呈下降趋势(降低47.8%,P=0.074)。灌注理想模式的氨基酸混合物后,奶牛乳腺对谷氨酸(Glu)、Cys和Ile的摄取率显著上升(Glu的摄取率提高118.7%,P=0.015;Cys的摄取率提高77.4%,P=0.032;Ile的摄取率提高46.0%,P=0.012),而奶牛乳腺对Ser的摄取率呈下降趋势(降低56.2%,P=0.052)。灌注氨基酸混合物后,Casein组乳脂产量增量显著高于R组(P=0.012),且Casein组的乳产量增量(P=0.095)和乳糖产量增量(P=0.091)较R组有升高的趋势,而2组间其他指标增量无显著差异(P0.05)。由此得出,在本试验条件下,颈静脉灌注酪蛋白模式和理想模式的氨基酸混合物均可提高泌乳奶牛的乳蛋白含量,而灌注酪蛋白模式氨基酸混合物同时可以促进乳蛋白产量的升高,因此,颈静脉灌注酪蛋白模式氨基酸混合物的效果优于灌注理想模式氨基酸混合物。     

荧光探针对家蚕和柞蚕核酸燐光的影响

荧光探针双乙锭、苯胺基萘-8-磺酸镁盐和溴化乙锭对家蚕和柞蚕DNA和RNA的燐光均有猝灭作用。其中溴化乙锭对燐光的猝灭作用最强,双乙锭次之,ANS(苯胺基萘-8-磺酸镁盐)的猝灭作用最小。     

生长激素对蛋白质动态代谢与氨基酸利用的调控

生长激素是调节动物生长的众多激素中最重要的一种 ,它通过在肌肉与脂肪组织之间极强的营养重分配作用 ,增加体蛋白沉积 ,减少脂肪沉积 ,从而改变这两种组织的生长状况来实现其促生长功能。动物体内蛋白沉积是蛋白合成与降解同时进行的两个过程动态平衡的结果。本文综述了近年来有关生长激素对动物蛋白质动态代谢与氨基酸利用调控方面的研究结果 ,指出生长激素使蛋白质合成与降解速率均增加 ,整体蛋白质周转代谢强度加大 ,从而导致蛋白质沉积增加。     

单宁酸对生长猪胃-小肠仿生消化中消化酶活性及饲粮粗蛋白消化率的影响

本试验旨在探讨单宁酸对生长猪胃、小肠仿生消化中消化酶活性及玉米-豆粕型饲粮干物质和粗蛋白消化率的影响，为评价单宁酸的生物学效应提供参考。试验一采用单因素完全随机设计，考察在无饲粮下2种单宁酸对猪模拟胃液、模拟小肠液消化酶活性的影响。设5个处理，单宁酸添加量分别为0 mg （胃液体积为20 mL，小肠液体积为22 mL）；单宁酸1，10 mg；单宁酸1，20 mg；单宁酸2，10 mg；单宁酸2，20 mg。测定各处理的胃蛋白酶、淀粉酶、胰蛋白酶和糜蛋白酶的活性。试验二考察玉米-豆粕型饲粮添加单宁酸对猪仿生消化中胃、小肠阶段消化酶活性及养分消化率的影响。采用单因素完全随机设计，设5个处理，单宁酸在饲粮中的含量分别为0 mg·（2 g）^-1；单宁酸1，10 mg·（2 g）^-1；单宁酸1，20 mg·（2 g）^-1；单宁酸2，10 mg·（2 g）^-1；单宁酸2，20 mg·（2 g）^-1。测定仿生消化中胃阶段0.5和4 h时胃蛋白酶活性，小肠阶段0.5、4和8 h时淀粉酶、胰蛋白酶和糜蛋白酶活性及生长猪胃-小肠仿生消化测定饲粮的干物质和粗蛋白消化率。结果表明：1）无饲粮的情况下，和空白对照组相比，2种单宁酸对模拟胃液中胃蛋白酶活性无显著影响（P>0.05），单宁酸1比单宁酸2更高地降低了模拟小肠液中淀粉酶、胰蛋白酶和糜蛋白酶的活性（P<0.05）。2）在饲粮进行仿生消化的胃消化0.5～4 h内，除4 h时10 mg·（2 g）^-1添加量外，添加单宁酸1时胃蛋白酶的活性均显著高于添加单宁酸2时的相应值（P<0.05），除单宁酸2在消化0.5 h外，2种单宁酸在添加10 mg·（2 g）^-1时胃蛋白酶活性均显著高于20 mg·（2 g）^-1添加量的相应值（P<0.05）。在小肠仿生消化0.5 h时，饲粮中添加单宁酸1、2的2个水平对消化液中淀粉酶活性无显著影响（P>0.05），但均显著降低了糜蛋白酶的活性（P<0.05）；单宁酸1的消化液中胰蛋白酶活性高于单宁酸2的相应值（P<0.05）。在小肠仿生消化4 h时，除添加水平为20 mg·（2 g）^-1时的糜蛋白酶活性外，饲粮中添加单宁酸1消化液中淀粉酶、糜蛋白酶活性高于添加单宁酸2的相应值，而胰蛋白酶活性低于添加单宁酸2的相应值（P<0.05）。单宁酸1、2的两个添加量均降低了胰蛋白酶和糜蛋白酶的活性（P<0.05）。在小肠仿生消化8 h时，饲粮中单宁酸的添加量影响了淀粉酶的活性，但单宁酸1和单宁酸2各两个添加量在淀粉酶的平均活性上无显著差异（P>0.05）。单宁酸1、2的两个添加量均降低了胰蛋白酶、糜蛋白酶的活性（P<0.05）。3）与对照组相比，两种单宁酸在两种添加水平下均显著降低了饲料粗蛋白消化率（P<0.05），且单宁酸2比单宁酸1更多地降低了饲粮粗蛋白的消化率（P<0.05）。综上所述，在有、无饲粮条件下，单宁酸对消化酶活性呈现不一致影响。单宁酸影响饲粮粗蛋白的消化率可能主要与消化液中糜蛋白酶活性降低以及单宁酸与饲粮中的化学成分形成螯合物降低了小肠消化酶的水解效率有关。     

一种治疗奶牛阴道炎症微生态制剂的临床药效学研究

为检验约氏乳杆菌SQ0048菌株所制备的微生态制剂对临床型奶牛阴道炎症的治疗作用,将其应用于患有阴道炎的病牛体内,利用临床兽医学方法观察给药前后患牛的临床症状,采用变性梯度凝胶电泳法检测给药前后患牛阴道内的微生物菌群种属,使用酶联免疫吸附技术检测给药前后患牛血清内细胞因子分泌量。结果显示,此微生态制剂不仅可以缓解临床奶牛阴道炎性症状,提高发情率及受胎率,还可以减少或杀灭阴道内的利氏卟啉单胞菌、铜绿假单胞菌、化脓隐秘杆菌及链球菌等,增加约氏乳杆菌SQ0048菌株及谷氨酸棒状杆菌的数量,改善阴道微生态环境,维持阴道的微生态平衡;同时给药后患牛血清中IL-1β、IL-6、IFN-α、TNF-α分泌量显著低于给药前,而IL-10分泌量则显著高于给药前,具有一定抑制炎症的生物学功能。     

Fibronectin modified expression of Sonic hedgehog in ATRA-mediated neuronal differentiation

In this study, the effect of fibronectin on the neurite outgrowth from embryoid bodies (EBs) in neurodifferentiated embryonal carcinoma P19 cells was examined. The neurite outgrowth on fibronectin was maintained for a longer time in comparison with those on collagen or laminin. Quantitative RT-PCR revealed that mRNA level corresponding to sonic hedgehog (Shh) in neurodifferentiated P19 cells was upregulated on fibronectin, whereas collagen or laminin did not affect. Further knockdown of integrin αv subunit in P19 cells demonstrated that expression of Shh was mediated through interaction between fibronectin and integrin. Additionally, exogenous Shh agonist accelerated neurite outgrowth from embryonic stem cell-derived EBs without large change of neuronal phenotype expression. Taken together, fibronectin could maintain neurite outgrowth via increased Shh expression.