L-精氨酸对大鼠胃肠激素分泌及食欲的影响

本试验旨在研究L-精氨酸(L-Arg)对大鼠胃肠道钙敏感受体(calcium-sensing receptor,CaSR)基因表达、胃肠激素分泌及采食量的影响,试验分为两个部分。试验I,选择32只SD雄性大鼠,随机分为4组,即对照组(灌胃0 mmol·kg^-1 L-Arg·HCl),低剂量组(5 mmol·kg^-1 L-Arg·HCl),中剂量组(10 mmol·kg^-1 L-Arg·HCl)和高剂量组(20 mmol·kg^-1 L-Arg·HCl),记录大鼠灌胃后不同时间点采食量,筛选显著影响大鼠采食的L-Arg最适剂量用于试验II。试验II,选择30只雄性大鼠随机分为两组,L-Arg·HCl高剂量组(20 mmol·kg^-1)及对照组(0 mmol·kg^-1),灌胃大鼠一周(每天一次),记录大鼠体重变化及日采食量。试验结束后采集血液、胃、小肠及下丘脑组织,检测血液中胃肠激素水平,胃肠道CaSR及下丘脑食欲调节因子基因表达。结果显示:试验I,10和20 mmol·kg^-1的L-Arg·HCl分别在灌胃后的0~1 h和0~24 h显著降低大鼠采食量(P<0.05),其中20 mmol·kg^-1效果最显著;试验II,与对照组相比,20 mmol·kg^-1 L-Arg·HCl显著降低了大鼠体增重及前两天的累加采食量(P<0.05),促进了胃肠道CaSR、厌食因子POMC基因表达和胆囊收缩素(CCK)分泌(P<0.05),胃泌素(gastrin)及葡萄糖依赖性促胰岛素多肽(GIP)的分泌虽有增加但差异不显著。相关分析结果显示,血清中CCK浓度与十二指肠、空肠CaSR及下丘脑POMC基因表达量呈显著正相关(P<0.05);GIP浓度与十二指肠CaSR及下丘脑POMC基因表达量呈显著正相关(P<0.05);GLP-1浓度与空肠CaSR及下丘脑POMC基因表达量有正相关的趋势(0.05<P<0.1)。说明L-Arg可能通过上调CaSR基因表达,影响胃肠激素分泌,上调厌食因子POMC基因表达,从而下调大鼠的采食量。

Cultivation-Independent Analysis of the Development of the Lactobacillus spp.Community in the Intestinal Tract of Newborn Piglets

Molecular diversity and development of the Lactobacillus community in the intestinal tract, as influenced by age and intestinal compartment, were studied in one litter of 12 conventionally raised piglets. Piglets were euthanized at each week （3 animals per time）. Digesta and tissue samples from stomach, duodenum, jejunum, ileum, caecum, colon, and rectum were collected and analysed by using 16S ribosomal RNA-based methods. DGGE （denaturing gradient gel electrophoresis） profiles revealed that the Lactobacillus communities throughout the GI tract from duodenum to rectum showed good stability at same age. This indicates that fecal Lactobacillus communities can effectively represent the intestinal community. Two dominant bands were found in tissue samples of the small intestine, suggesting that the lactobacilli can adhere to the small intestinal wall. The Lactobacillus communities in different GI tract compartments developed over time. A successional change of Lactobacillus communities was observed from birth, through creep feeding to one week after weaning, showing a trend from simple to complex and back to simple. Furthermore, a clone library of Lactobacillus spp. 16S rRNA gene sequences were generated from jejunal and colonic chymes. Six dominant DGGE bands generated from jejunal chymes were matched with sequences that show 94-98% similarity to the bands derived from L. reuteri, L. delbrueckii, and L. crispatus. Seven dominant DGGE bands generated from colon chymes were matched with sequences that show 88-99% similarity to those derived from L. reuteri, L. delbrueckii, L. amylovorus/L. sobrius, and L. acidophilus. Amplicons related to L. reuteri were found in all DGGE fingerprints from jejunal digesta of age of weeks 1, 3, and 4. Amplicons related to L. amylovorus/L. sobrius were present in all DGGE fingerprints from colonic digesta of age of week 1, 3, and 4. Amplicons related to L. delbrueckii were found before weaning, L. crispatus after creep feeding before weaning and L. acidophilus after weaning. This indicates that L. reuteri and L. amylovorus/L. sobrius probably belong to the permanent composition, while L. delbruckii, L. acidophilus, and L. crispatus probably belong to the temporal groups of Lactobacillus communities in the GI tract of piglets.

Gut Bacterial and Lactobacilli Communities of Weaning Piglets in Response to Mannan Oligosaccharide and Sugar Beet Pulp In vitro Fermentation

Microbiota in the gastrointestinal tract （GIT） of piglets during weaning transition can experience a sharp change which could result in growth reduction and diarrhea of weaned piglets. Dietary manipulations can play an important role in attenuating such changes caused by weaning stress. Therefore, ileal and colonic contents of weaned piglets were used as inocula, mannan oligosaccharide （MOS） or sugar beet pulp （SBP） was supplied as single energy sources to investigate effects of MOS or SBP on the shifts of gastro-intestinal microflora and lactobacilli populations. The universal bacteria- and lactobacilli-specific PCR/denaturing gradient gel electrophoresis （DGGE）, cloning and sequencing techniques were used. DGGE profiles of the universal bacteria showed that great changes were found in the position, numbers and intensity of dominant bands after fermentation. The similarity of bacterial community between ileum and colon was increased to 85-97% by MOS or SBP treatment after fermentation from the similarity with 20% before fermentation. MOS treatment significantly increased the bacterial diversity and band number in both ileal and colonic fermentation （P〈0.05）. SBP treatment significantly increased the bacterial diversity and band number in colon （P〈0.05）. It implies that some species were enriched by the addition of MOS or SBP to increase the similarity and diversity of bacterial community in weaned piglets. Five specific bands appearing in MOS or SBP treatment group after fermentation were cloned and sequenced, the changes of species related to Prevotella and Ruminococcus were observed. Two bands related to uncultured bacterium with 98% similarity were detected by MOS or SBP treatment. However, there were no effects on the similarity, diversity index and lactobacilli species revealed by MOS or SBP treatment. These results imply that MOS or SBP could have beneficial effects on the weaning piglets by stablizing microbiota in the GIT microflora.

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis （DGGE） with two primer pairs （344fGC/519r and 519f/915rGC） and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE proifles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no signiifcant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs;however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A （mcrA） gene fragments ampliifed with real-time PCR showed all samples possessed a single peak at 82＆#176;C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase （including diet factor） other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

Fermentation characteristics of Megasphaera elsdenii J6 derived from pig feces on different lactate isomers

D-Lactate-utilizing bacteria play important roles in maintaining the balance of gut lactate; however, studies on gut D-lactate-uti-lizing bacteria have been limited. This study aimed to isolate and identify D-lactate-utilizing bacteria from pig gut using the Hungate rol-tube method, and to investigate their metabolic characteristicsin vitro. Six different anaerobes were isolated from pig feces, which were identiifed as related toBacteroides fragilis, Bacteroides acidifaciens,Veilonela denticariosi, Veilonela caviae,Bacteroides uniformis, andMegasphaera elsdenibased on the 16S rRNA gene sequences. Al strains had a signiifcant ability to utilize D-lactate, which was concluded afterin vitro fermentation with 25 mmol L–1 D-lactate as the primary carbon source. Of al 6 strains,M. elsdeniJ6 showed the highest efifciency of D-lactate utilization and produced a higher ratio of butyrate in total short chain fatty acids （SCFAs）. Thus, thein vitro fermentation characteristics of this strain in D-, L-, and DL-lactate mixtures （D-lactate：L-lactate=1：1 or 1：2） were further studied. The results showed that M. elsdeni J6 preferred utilizing D-lactate, and produced more SCFA when using D-lactate as the primary carbon source. The ifndings suggest that the administration of D-lactate-utilizing bacteria such asM. elsdeni J6 may have a potential advantage in the aleviation of D-lactic acidosis in the animal gut.

新冠疫情下“医学遗传学”的线上教学实践

在当前新冠病毒流行期间,各级各类学校都不能开展正常的线下教学,开展基于各种学习平台的线上教学是必然选择。借助于“超星学习通”平台开展“医学遗传学”线上教学模式的尝试,通过教学实践发现,线上教学包括课前预习准备、课上互动与讲解、课后讨论和考核等环节,具有其特有信息量大、师生互动充分、充分的多媒体使用等优势,但也存在不足之处。线上教学为以后的线上线下的混合式教学提供了经验,各高校教学部门、任课教师仍需要充分地探索、利用网络学习平台,以适应数字化新时代的教学要求。

Genomic characterization and antimicrobial susceptibility of bovine intrauterine Escherichia coli and its relationship with postpartum uterine infections

To investigate the roles of Escherichia coli in the pathogenesis of postpartum uterine diseases in dairy cows, a total of 145 E. coli isolates were recovered from 18 healthy cows （61 isolates） and 25 cows with clinical endometritis （84 isolates） at 25-35 days after parturition. Genomic characteristics including phylogenetic grouping, genetic diversity and virulence genes of E. coli isolates were screened to profile the characteristics related to uterine infections. The susceptibility of the bacteria against 23 antibiotics was also evaluated to support prevention and treatment of clinical cases. Genetic diversity of E. coli identified by random amplification of polymorphic DNA （RAPD） revealed 103 clonal types, including 3 common types to unaffected cows and endometritis cows, 39 types specific to healthy cows and 61 types in endometritis subjects. In addition, the isolates from endometritis uteri showed more genetic variability compared with that of healthy cows. Ac- cording to the findings of phylogenetic grouping, the E. coil isolates were assigned to group A （35.9%）, B1 （59.3%） and D （4.8%）. The expression of 10 of 20 virulence gens were detected positively, and only fimH gene was revealed significantly （P〈0.05） associated with endometritis. From antimicrobial susceptibility test, E. coli was found highly resistant to tetracy- cline, ampicillin, carbenicillin and amoxicillin, but sensitive to amikacin, netilmicin, tobramycin, cefepime and ceftazidime. In conclusion, E. cofiwere extensively observed in both healthy and endometritis cows, and presented a large clonal types, however, fimH was the only gene observed associated with clinical endometritis. Our results suggest that the drugs like amikacin, netilmicin, tobramycin and cefepime could be considered for preventing and treating clinical endometritis in the practical management of dairy cow.

G6PD缺乏症2种检测方法的对照研究

目的利用葡萄糖-6-磷酸脱氢酶(G6PD)/6PGD比值法与变性球蛋白小体检测法相结合,检测G6PD缺乏症。方法用改良G6PD定量比值法和变性球蛋白小体法对G6PD缺乏症个体的血液进行生化及形态学检测分析。结果利用G6PD/6PGD比值法,83例男性缺乏症个体中,16例为中度缺乏,67例为严重缺乏;而42例女性缺乏症个体中,20例为中度缺乏,22例为严重缺乏。用变性球蛋白小体检测法检测G6PD缺乏症患者,男性中度缺乏和严重缺乏分别为24例和48例,女性中度缺乏和严重缺乏分别为7例和26例。变性球蛋白小体检测法可以检测出假阳性,对检测严重缺乏症患者男女无差异。结论改良G6PD定量比值法检测结合变性球蛋白小体检测法,可以减少假阳性率,提高检测G6PD缺乏症结果的准确性。