鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

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.     

滩羊公羊特异性RAPD标记的研究

利用混合样本分析方法对滩羊进行了RAPD分析 ,筛选了 6 0种随机引物 ,结果发现OPB17 32 0这一扩增片段只出现在公羊的混合样中 ,进一步对 5 0只滩羊进行逐个检测 ,发现 2 1只公羊均出现了OPB17 32 0这一DNA片段 ,而 2 9只母羊无一出现此条片段 ,据此推断OPB17 32 0为滩羊公羊的特异性RAPD标记。这一发现为滩羊胚胎性别鉴定和Y染色体上基因的研究提供了新的方法和标记参考座位。     

A single blastomere sexing of caprine embryos by simultaneous amplification of sex chromosome-specific sequence of SRY and amelogenin genes

The primary objective of this study was to develop a simplified, rapid and authenticated protocol for sexing of caprine embryos. Polymerase chain reaction (PCR) is a powerful tool in preimplantation sex diagnosis, using embryo biopsy at the early developmental stage. Based on the amelogenin gene located on the conserved region of the sex chromosome, a primer pair was used and PCR was established to amplify a 262-bp fragment from the Xchromosome in female goat embryos and 262-bp fragments from the X chromosome and 202-bp fragments from the Y chromosome in male embryos. To validate the reliability of PCR, using the sex-determining region Y (SRY) gene located on the conserved region of Y chromosome, a primer pair was used and PCR was established to amplify a 122-bp fragment specific to the Y chromosome in male embryos. The in vitro-produced goat in vitro fertilisation (IVF)-embryos were made zona free by treating with pronase. The cell number in each embryo was counted before sexing. A single blastomere taken from these embryos was directly used as a template in PCR containing SRY and amelogenin gene-specific primers separately. Of 75 pronase-treated and 60 micromanipulated goat IVF embryos, 33 (44%) and 26 (43.33%) were confirmed as male and 42 (56%) and 34 (56.66%) as female, respectively. The sex-diagnosed embryos were kept in research vitro cleavage (RVCL) medium, and developed into 42.66% and 61.66% morulae and 13.33% and 23.33% blastocysts among pronase-treated and micromanipulated embryos, respectively. The AMELX gene-specific primer served as the internal control and did not interfere with amplification of the Y-specific sequence. In conclusion, a single blastomere sexing protocol based on the SRY and the amelogenin gene is simple, rapid, sensitive and efficient for sex determination in caprine early stage embryos.     

A novel repeated sequence located on the bovine Y chromosome: its application to rapid and precise embryo sexing by PCR

A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.     

Differential transcription of the bovine AMPD3 gene in single blastocysts cultured under various oxygen tensions

Studying gene expression during the early embryonic stages of in vitro culture is essential and of special interest, not only to gain insights in embryo development but also to find candidate genes that can be used as markers to assess embryo quality. Using differential display RT‐PCR expression of AMPD3 coding for AMP deaminase 3 was found in bovine blastocysts cultured in vitro only under 2% but not under 5 and 20% oxygen tensions. Mapping of this gene to bovine chromosome 15 confirmed its identity. The differential expression of bovine AMPD3 was confirmed by RT‐PCR with specific primers.     

Differences in genetic structure assessed using Y‐chromosome and mitochondrial DNA markers do not shape the contributions to diversity in African sires

Up to 173 African sires belonging to 11 different subpopulations representative of four cattle groups were analysed for six Y‐specific microsatellite loci and a mitochondrial DNA fragment. Differences in Y‐chromosome and mtDNA haplotype structuring were assessed. In addition, the effect of such structuring on contributions to total genetic diversity was assessed. Thirty‐five Y‐chromosome and 71 mtDNA haplotypes were identified. Most Y‐chromosomes analysed (73.4%) were of zebu origin (11 haplotypes). Twenty‐two Y‐haplotypes (44 samples) belonged to the African taurine subfamily Y2a. All mtDNA haplotypes belonged to the “African” taurine T1 haplogroup with 16 samples and nine haplotypes belonging to a recently identified subhaplogroup (T1e). Median‐joining networks showed that Y‐chromosome phylogenies were highly reticulated with clear separation between zebu and taurine clusters. Mitochondrial haplotypes showed a clear star‐like shape with small number of mutations separating haplotypes. Mitochondrial‐based F_ST‐statistics computed between cattle groups tended to be statistically non‐significant (p > .05). Most F_ST values computed among groups and subpopulations using Y‐chromosome markers were statistically significant. AMOVA confirmed that divergence between cattle groups was only significant for Y‐chromosome markers (Φ_CT = 0.209). At the mitochondrial level, African sires resembled an undifferentiated population with individuals explaining 94.3% of the total variance. Whatever the markers considered, the highest contributions to total Nei's gene diversity and allelic richness were found in West African cattle. Genetic structuring had no effect on patterns of contributions to diversity.     

Bovine foetal sex determination—Different DNA extraction and amplification approaches for efficient livestock production

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender‐specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell‐free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300–600 bp) in maternal circulation. The aim of this study was to assess this non‐invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non‐pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single‐tube extraction without silicone membranes and phenol–chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real‐time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single‐tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real‐time PCR test sensitivity in Group I was 90% and in Group II 91.6%.     

PCR扩增妊娠奶牛牙釉蛋白基因进行胚胎性别鉴定的研究

牙釉蛋白（amelogenin,简写为AML）基因是牙齿发育过程中丰富表达的多拷贝基因,AML基因的同源基因分别定位在XY染色体上。本试验利用x—Y同源的牙釉蛋白基因序列设计一对特异性引物（牛AML基因序列的扩增片段长度：雌性为只有467bp的特异性扩增片段：雄性为同时具有341bp和467bp的两条特异性扩增片段）,应用PCR技术同时扩增X和Y染色体上的特异性片段,扩增产物用PAGE电泳分离技术,经硝酸银溶液染色及扫描分析进行妊娠奶牛早期胚胎的性别鉴定。结果显示,从X染色体上扩增出467bp的片段．从Y染色体上扩增出341bp的特异性片段。由此可知,PCR扩增妊娠奶牛牙釉蛋白基因可以进行胚胎的性别鉴定。     

Sry基因和Y染色体重复序列在牛早期胚胎性别鉴定中应用效果的比较

本试验以牛Sty基因和Y染色体重复序列作为雄性特异性基因,分别设计引物,建立多重巢式PCR体系,比较二者在牛早期胚胎性别鉴定中的应用效果。试验结果表明,当扩增体系中的模板量为一个胚胎细胞的DNA量时,以Y染色体重复序列构建的扩增体系比Sry基因具有更高的灵敏度和稳定性,更适合用于牛早期胚胎性别鉴定。     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

常规PCR和巢式PCR法鉴定牛早期胚胎性别体系的建立和优化

试验利用牛Y染色体重复序列作为雄性特异性引物,_以肿蔼坏死因子(TNFα)为内标引物建立多重PCR和多重巢式PCR体系,进行牛早期胚胎性别鉴定.共设计4对引物-Y染色体重复序列外引物和内引物,其扩增片段大小分别为534 bp和480bp,肿瘤坏死因子外引物和内引物,扩增片段大小分别为357 bp和272 bp.结果表明,4对引物均有很高的特异性和稳定性;多重PCR体系灵敏度为50 pg(约8个细胞),多重巢式PCR体系灵敏度为10 pg(约2个细胞),故多重巢式PCR体系更适合于牛胚胎性别鉴定.     

狍Sry基因PCR扩增的初步研究

为研究狍性别决定机制,采用聚合酶链式反应(PCR)技术对野生狍(Capreoluscapreolus)(n♀=2,n♂=2)Sry基因(哺乳动物Y染色体DNA雄性特异区)进行扩增。根据人的SRY基因核心序列设计合成了1对引物1,2。结果在野生狍雄性个体中扩增出1条带,大小约为220bp,而在雌性个体中未见扩增带,表明了Sry基因的性别特异性,为探讨野生狍的性别决定机制提供了分子资料。     

Quantification of the DNA Difference,and Separation of X‐ and Y‐Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre‐determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome‐bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X‐ and Y‐chromosome and (iii) determine the DNA difference between X‐ and Y‐bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high‐speed cell sorter (SX‐MoFlo^®). H33342 staining concentrations of 36, 54, 72 or 90 μm did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X‐ and Y‐enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX‐MoFlo^®), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.     

随机扩增多态DNA（RAPD）技术在鹅育种上的应用

本文用4种引物（OPH5、OPH13、OPH16和OPF4）对隆昌鹅、太湖鹅和新太湖鹅进行基因组DNA、RAPD分析。结果表明：三种鹅都有特异性的条带,扩增DNA条带都表现为多态性,条带数为3-12条,大小范围在0.36-0.38kb之间,多态频率为72.14%;RAPD标记可作为一种辅助手段来比较鹅群体的遗传变异性,太湖鹅遗传变异性大于隆昌鹅;OPF-04很可能用作区分隆昌鹅与太湖鹅的标记性引物。     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis)

The aim of the present study was to assess the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8, respectively, failed to amplify in Saola. Two cattle Y‐chromosome‐specific microsatellite markers (INRA126 and BM861) were successfully amplified from both sexes in Saola. However, two additional markers (INRA124 and INRA189) on Y‐chromosome failed to amplify in the female animal. These results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population.     

应用Sry-PCR扩增鉴定狍(Capreolus capreolus)的性别

根据人的SRY基因核心序列设计合成1对引物C1、C2，应用PCR技术对野生狍(Capreolus capreolus)Sry基因(哺乳动物Y染色体DNA雄性特异区)进行扩增．结果在野生狍雄性样本扩增出1条带(221bp)．而在雌性样本未见扩增带．显示了Sry基因的性别特异性。应用这1对引物对42个未知性别狍肌肉组织样本进行了性别鉴定．结果雄性22个，雌性20个。对Sry-PCR产物克隆测序，得到185bp的Sry基因部分核苷酸序列。本试验为狍种群性别比率及其种群动态变化机制研究提供了资料。     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

H-Y单抗检测方法初探

优化间接ELISA条件 ,提高灵敏度 ,建立检测H Y抗体的检测方法。利用BALB/c公鼠免疫同系母鼠 ,制备H Y单抗。PCR验证间接免疫荧光法鉴定胚胎性别准确率。结果表明 :鉴定雄性胚胎的准确率为 83% ( 1 5 / 1 8)。鉴定雌性胚胎的准确率为 94 % ( 1 5 / 1 6)。