The W‐ and Z‐linked EE0.6 sequences used for molecular sexing of captive Japanese crested ibis on Sado Island

The Japanese crested ibis Nipponia nippon is a critically threatened bird. Accurate sexing is necessary to perform effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis on Sado Island. A PCR‐based sexing method targeting a 0.6 kb EcoRI fragment (EE0.6) sequence on W chromosome with AWS03 and USP3 primers has been developed for the Japanese crested ibis. However, the primers were selected from the EE0.6 sequences from bird species other than the Japanese crested ibis. In this study, we determined the W‐ and Z‐linked EE0.6 sequences in the Japanese crested ibis, and clarified Japanese crested ibis sequence mismatch in the binding sites of the primers. Further, we found no polymorphism in the primer binding sites among five founder birds for the Sado captive Japanese crested ibis population. These findings validated the PCR‐based sexing method with the AWS03 and USP3 as accurate molecular sexing methods of captive Japanese crested ibis on the Sado Island. Additionally, we designed a primer set for a novel PCR‐based sexing, based on the EE0.6 sequences obtained in this study. This novel sexing method may be useful for future ecological research following the release of Japanese crested ibis on Sado Island. This is the first report to show the EE0.6 sequences in Japanese crested ibis.     

家鸡CHD1基因PCR快速性别鉴定方法的建立及其在早期鸡胚中的应用

本试验以优化PCR条件,建立快速性别鉴定方法为目的,从家鸡雏鸡血样、鸡胚羊水和尿囊液中获得DNA进行性别鉴定.通过对家鸡的两个性别鉴定候选基因CHD1和xho Ⅰ重复片段进行PCR扩增引物筛选和反应体系的优化.结果显示CHD1基因PCR法可稳定、准确地用于鉴定家鸡性别,该体系经改进和优化后,缩短了鉴定时间,提高了鉴定的稳定性和准确性.在此基础上利用12.5日龄鸡胚羊水和尿囊液建立了早期胚胎的性别鉴定体系.该方法为家鸡早期胚胎性别鉴定批量化的快速操作提供了可行的依据,同时也为鸟类性别决定机制的研究奠定了基础.     

哺乳动物性反转分子机制的研究进展

哺乳动物性反转的分子机制的研究是目前遗传学、医学、生物学等多学科的研究热点。随着分子生物学等学科的快速发展和分子生物学方法的运用,以及对一些自然性反转动物及其人工方法诱导的性反转小鼠的研究,研究者们陆续发现并证实了一些性反转相关基因,如SRY、SOX9、DAX-1、SF-1、DMRT、WNT-4、Wt-1、AMH(MIS)等,并在一定程度上阐述了性反转的分子机制。文章从分子水平综述了哺乳动物的性反转的研究进展。     

Immunolocalization of sex steroid receptors in the epididymis and ductus deferens of immature and mature Japanese Quail, Coturnix Japonica

The goal of this study was to determine whether in the Japanese quail the male genital tract contains receptors for progesterone, androgen and estrogen (PR, AR and ER, respectively), which have significant roles in reproductive functions, and whether their localization changes during sexual maturation. The epididymis and ductus deferens (middle and ampulla regions) of immature (approximately 30-day-old) and mature male Japanese quail were collected and frozen sections of them were immunostained for PR, AR and ER. The immunoreaction products for AR and PR were found in the nuclei of epithelial cells in the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens of mature and immature birds. In the mature birds, the epithelial cells of the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens were positive for ER, although some of the cells in the ductus deferens were negative. The epithelial cells of the ductules in the epididymis stained positive for ER, but the immunoreactions were negligible in the ductus deferens of immature birds. These results suggest that the epididymis and ductus deferens in quail possesses PR, AR and ER receptors. Each receptor is expressed before sexual maturation, although enhancement of ER expression may occur during maturation.     

Allele frequencies of the extension locus encoding the melanocortin-1 receptor in Japanese and Korean cattle

In order to estimate the influence of the Extension (E) locus in cattle coat color, the melanocortin‐1 receptor (MC1R) gene in Japanese Black, Japanese Brown and Korean (Hanwoo) cattle were sequenced. The sequences of the coding region revealed three alleles (E^D, E⁺ and e), which were previously reported. Polymerase chain reaction‐restriction fragment length polymorphism was performed to investigate the gene frequencies of the three breeds. Japanese Black was almost composed of E^D and E⁺ individuals, E^D = 0.481 and E⁺ = 0.514, and no homozygous e/e, therefore that is consistent with the hypothesis that E^D and E⁺ induce black pigment synthesis. Allele frequencies between Japanese Brown and Hanwoo were obviously different; however, recessive red e allele frequency was 0.038 for Japanese Brown and 0.948 for Hanwoo, even though both breeds have quite similar coat colors (ranging from yellowish brown to dark brown including a red coat color). This result suggested that other genes are also associated with a coat color of red and brown in cattle.     

江淮流域9个家鸭品种的分子生物地理演化过程分析

通过对江淮流域9个家鸭品种106个样本的线粒体D-loop区的序列分析，探讨了各品种体现在该特征的遗传多样性的现代分布格局及其地理演化过程。对检测出的31个单倍型的分析表明：长江中游地区的家鸭品种遗传多样性高于东部地区的家鸭品种。品种之间基因流同地理距离之间呈弱负相关。歧点分布分析和Tajima＇s D值中性检验符合中性突变，提示江淮流域鸭在过去没有出现群体扩张，群体大小稳定。各单倍型在最大简约树中的分布和实际地理分布无显著关系，各地理单元的单倍型都相互散布在不同的分布群中，呈现一种混杂的分布格局。     

Pool‐based genome‐wide association study identified novel candidate regions on BTA9 and 14 for oleic acid percentage in Japanese Black cattle

Fatty acid composition is an important indicator of beef quality. The objective of this study was to search the potential candidate region for fatty acid composition. We performed pool‐based genome‐wide association studies (GWAS) for oleic acid percentage (C18:1) in a Japanese Black cattle population from the Hyogo prefecture. GWAS analysis revealed two novel candidate regions on BTA9 and BTA14. The most significant single nucleotide polymorphisms (SNPs) in each region were genotyped in a population (n = 899) to verify their effect on C18:1. Statistical analysis revealed that both SNPs were significantly associated with C18:1 (p = .0080 and .0003), validating the quantitative trait loci (QTLs) detected in GWAS. We subsequently selected VNN1 and LYPLA1 genes as candidate genes from each region on BTA9 and BTA14, respectively. We sequenced full‐length coding sequence (CDS) of these genes in eight individuals and identified a nonsynonymous SNP T66M on VNN1 gene as a putative candidate polymorphism. The polymorphism was also significantly associated with C18:1, but the p value (p = .0162) was higher than the most significant SNP on BTA9, suggesting that it would not be responsible for the QTL. Although further investigation will be needed to determine the responsible gene and polymorphism, our findings would contribute to development of selective markers for fatty acid composition in the Japanese Black cattle of Hyogo.     

Analysis of 46-kDa protein in the perivitelline membrane of Japanese quail (Coturnix japonica)

The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. The PL is constructed with two major glycoproteins, ZPC and ZP1, which are synthesized in the ovarian granulosa cells and the liver, respectively. Although the properties of the major components in the PL have been examined, knowledge about the nature of its minor constituents is lacking. In this study we focused on PL protein, which migrates at 46‐kDa in the gel of SDS‐PAGE. N‐terminal sequence analysis demonstrated that the 46‐kDa protein is the C‐terminal fragment of ZP1. Analysis of lysylendopeptidase digests or cyanogens bromide‐degraded fragments of ZP1 confirmed this postulate. Western blot analysis using antiserum against 46‐kDa protein indicated the absence of 46‐kDa protein in the serum. Moreover, small immunoreactive bands, thought to be cleaved fragments of ZP1, were detected in the PL lysate by western blot analysis using antiserum against the N‐terminal peptide of ZP1. These results indicated that the N‐terminal proteolytic processing of ZP1 might take place after the arrival of ZP1 at the ovary, and the resulting product, 46‐kDa protein, is incorporated into the PL.     

我国部分地区禽戊型肝炎病毒分子流行病学分析

为了解我国鸡群中禽戊型肝炎病毒(avain hepatitis E virus,aHEV)的分子变异情况,对2018年从山东、河南、河北、辽宁、吉林、黑龙江、陕西、山西、江苏等地疑似aHEV感染鸡群中采集的679份肝脏、脾脏病料样品进行RT-PCR检测,从中选取PCR阳性产物,对其ORF1基因进行序列测定与遗传进化分析...     

Effect of Anserine and Carnosine on Sperm Motility in the Japanese Quail

Sperm motility is considered as one of the most important traits for successful fertilization, but the motility of an ejaculated sperm decreases with time when stored as liquid. It is reported that seminal plasma serves as a nutrient rich medium for sperm and plays an important role in sperm motility and its fertilization ability. Several studies have reported that imidazole dipeptides such as anserine and carnosine affect sperm motility and its fertilization ability in mammals. In this study, we report the presence of anserine and carnosine in the male reproductive tract of the Japanese quail. Abundant levels of anserine (44.46 µM) and carnosine (41.75 µM) were detected in the testicular fluid and seminal plasma respectively using the amino acid analyzer; however, seminal plasma solely contained carnosine. When the ejaculates were incubated with anserine or carnosine, we found that both the dipeptides improve sperm motility parameters such as straight line velocity, curvilinear velocity, average path velocity and amplitude of lateral head displacement after in vitro sperm storage at 15°C. These results indicate that imidazole dipeptides are present in the male reproductive tract and may improve sperm quality during in vitro sperm storage in the liquid states.     

Effect of supplemental vitamin E on the peripheral blood leukocyte population in Japanese Black calves

We investigated the effect of supplemental vitamin E on the peripheral blood leukocyte population in Japanese Black calves. Twenty-six calves kept at the same farm were studied. They were divided into two groups; thirteen calves received 300 IU/day of vitamin E orally from 1 to 3 months of age (VE group), and the other thirteen calves did not receive the vitamin E supplement (control group). The VE group showed a higher serum vitamin E concentration at 2 and 3 months of age compared with the control group (P<0.01). The numbers of CD3⁺ cells and CD4⁺ cells were higher in the VE group than in the control group, and the difference was statistically significant at 3 months of age (P<0.05). The numbers of CD21⁺ cells were higher in the VE group than in the control group, and the difference was statistically significant at 2 months of age (P<0.05). The numbers of CD335⁺ cells tended to be higher in the VE group than in the control group. The numbers of CD8⁺ cells and CD14⁺ cells tended to be higher in the VE group than in the control group at 3 and 4 months of age. This study demonstrated that the supplementation of suckling Japanese Black calves with vitamin E might affect the numbers of some immune cell types in the peripheral blood.     

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.     

自噬调节剂对感染日本脑炎病毒小鼠脑部细胞凋亡的影响

旨在研究自噬调控药物对感染日本脑炎病毒(Japanese encephalitis virus,JEV)小鼠脑部细胞凋亡的影响,本试验建立自噬调控药物处理的感染日本脑炎病毒小鼠的动物模型,其中,雷帕霉素为自噬诱导剂,渥漫青霉素及氯喹为自噬抑制剂.实验动物分成8组:DMEM对照组(Control);JEV感染组(JEV)...     

分子生物学技术在乳酸菌鉴定中的应用

介绍了几种分子生物学技术在乳酸菌鉴定中的应用,并对这些方法的优缺点进行了简单的阐述,并对该技术今后的发展方向做了明确的讨论。     

猪乙型脑炎病毒LS株的理化特性及其在BHK-21细胞上增殖特性的研究

对猪乙型脑炎病毒(Japanese encephalitis virus,JEV)LS株的理化特性及其在BHK-21细胞上的增殖特性进行研究,结果表明,该病毒对温度(56 ℃)、酸(pH 5.0以下)、乙醚、胰蛋白酶敏感,反复冻融(-65～20 ℃)3次几乎不影响病毒效价;该毒株在BHK-21细胞上连续传20代,仍能维持较高的病毒滴度(TCID₅₀=10^7.75/mL)和血凝效价(2⁸),且根据其在BHK-21细胞上的增殖规律,得到最佳收毒时间为接毒后28～40 h。本试验为进一步研究JEV LS株的生物学特性奠定基础。     

免疫学与分子生物学技术在弓形虫病诊断中的研究进展

弓形虫病是一种呈世界性分布、危害严重的人兽共患寄生原虫病。弓形虫病给养殖业、公共卫生及经济发展带来严重危害。目前在国内还尚未有弓形虫相关疫苗。弓形虫感染的常用诊断方法包括临床诊断、病原学检查和血清学诊断等方法。但由于传统检测方法在特异性、敏感性和早期诊断方面存在一定的缺陷。因此,建立简便、快捷、敏感、特异的弓形虫病的检测方法对于检测弓形虫的感染显得尤为重要。论文主要从免疫学和分子生物学方面对该病的诊断和应用做一简要介绍。     

Effects of probiotic Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail (Coturnix japonica)

Effects of a commercial probiotic, Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail fed either a commercial control diet (crude protein (CP), 23.5%; metabolizable energy (ME), 11.7MJ/kg) or a high‐energy diet (CP, 23.0%; ME, 13.8MJ/kg) were investigated. Four‐week‐old male birds (n = 108) were divided into four diet groups: control diet, control with probiotic, high‐energy diet and high‐energy diet with probiotic. At 8 weeks and 12 weeks of age (4 and 8 weeks of probiotic supplementation period, respectively), abdominal fat, muscles and the liver were weighed. The weight of total fat and protein in the muscles and liver were also determined. A rectal temperature was recorded weekly. Bodyweight and feed intake in both diets were not affected by probiotic supplementation. At 8 weeks of age, birds fed the control diet with probiotic had significantly less abdominal fat than those fed without the probiotic, and a similar tendency was seen for both diet groups at 12 weeks of age. The decrease in abdominal fat coincided with an increase in rectal temperature. These results suggest that greater metabolizable energy was consumed through elevation of heat production by supplementation of B. cereus toyoi, with hardly any energy stored as body fat. Probiotic supplementation appears to induce greater muscle weight, higher protein and lower lipid content in muscles by 8 weeks of age, and lower lipid content in muscles and greater liver weight by 12 weeks of age, in the Japanese quail.     

lncRNA在乙脑病毒感染PK15细胞过程中的作用研究

本研究旨在初步探索乙脑病毒（JEV）感染PK15细胞后的增殖情况,筛选与病毒感染相关的lncRNA,并对其进行亚细胞定位及靶基因预测。通过免疫荧光试验来检测病毒结构蛋白E的表达情况,采用TCID₅₀法检测PK15细胞中病毒的增殖情况,利用实时荧光定量PCR检测病毒感染后lncRNA的表达水平,在NONCODE数据库对lncRNA进行亚细胞定位,通过starBase、NONCODE、KEGG等数据库对其进行靶基因预测和信号通路分析。结果显示,JEV感染PK15细胞后,24~36 h为病毒滴度指数增长期,感染后36 h病毒滴度已达10^-5.75 TCID₅₀/mL。PK15细胞在感染JEV 12 h后,lncRNA A、B、C表达水平均无显著变化（P>0.05）,lncRNA D表达水平极显著下降（P<0.01）;感染JEV 24、36和48 h后lncRNA A、B、C表达水平极显著上升（P<0.01）,lncRNA D表达水平极显著下降（P<0.01）。lncRNA A主要定位在胞质溶胶,lncRNA B在细胞核和细胞质中均有分布,lncRNA C主要定位在细胞质中,在细胞核中也有可能分布,lncRNA D可能在细胞内呈现广泛性分布。通过靶基因预测和信号通路分析,lncRNA A、B、C的靶基因主要为OAS1、OAS2、OASL、COX1等,lncRNA D的靶基因主要为DST、ND1、ND2、ND4等。信号通路分析发现lncRNA可能通过肿瘤坏死因子（TNF）、NF-κB和Toll样受体（TLR）等信号通路参与病毒感染后的增殖过程。本研究为进一步探索宿主细胞lncRNA对病毒增殖的影响奠定一定的基础。     

不同生理发育期日本大耳白兔红细胞免疫学研究

为探讨不同生理发育时期日本大耳白兔红细胞免疫功能的变化规律,应用红细胞C3b受体花环（E—C3bRR）和红细胞免疫复合物花环（E—ICRR）试验以及红细胞免疫粘附调节因子活性测定试验．对不同生理发育期日本大耳白兔红细胞免疫学进行了研究。结果显示：随着年龄的增长,日本大耳白兔的红细胞C3b受体花环（E—C3bRR）和红细胞免疫粘附促进因子（RFER）显著降低（P〈0．01）,而红细胞免疫复合物花环（E—ICRR）和红细胞免疫粘附抑制因子（RFIR）显著升高（P〈0．01）。日本大耳白兔红细胞E—C3bRR和RFER与年龄呈显著负相关,而E—ICRR和RFIR与年龄呈显著正相关。