流式细胞仪在猪性别控制中的应用初探

在国内首次报道使用流式细胞仪对猪进行性别控制研究。猪精液通过流式细胞仪进行X、Y精子分离,分离精子经过数小时保存后,用于陆川母猪输卵管授精,最后产出健康仔猪。试验结果表明:①第1次(2005-10-14)精子分离的纯度为:X88%、Y83%;活力为X35%、Y21%。第2次(2006-01-13)纯度为:X70%,Y68%;活力为:X52%,Y36%。②该试验中母猪受孕率为25%,未受孕者仍然可在生产条件下继续繁殖。③受孕母猪(1头)产出健康仔猪5头,出生成活率100%,大小均匀,活力良好,哺乳期(28 d断奶)平均日增重与生产群极为接近(151.43 g1、48.93 g),但产活仔数低(生产群为8.4头/窝)。④仔猪雌性率为60%。试验结果初步表明:在该实验室现有条件下,使用流式细胞仪分离的X、Y精子,母猪通过输卵管受精可以产出健康仔猪;精子分离对仔猪哺乳期生长无明显影响;对性别比例有一定调控作用。     

动物性别决定的分子机理及性别鉴定与控制新技术

哺乳动物中位于Ｙ染色体短臂临界区域的ＳＲＹ基因启动雄性性状的发育。该基因在生殖腺脊中的表达可激发下游基因ＭＬＳ的转录,引起缪氏体抑制物和睾酮分泌,促使睾丸组织器官的发育。针对该基因制备特异探针或产该区域片段得到引物对就可用ＦＩＳＨ或ＰＣＲ准确,快速鉴定出植入母体前的胚胎性别,以及对精子筛选分离的结果作出准确评价。与ＰＣＲ相比,ＦＩＳＨ技术更具检测优势。     

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40–60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40–60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells.     

Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

高寒地区奶牛“性控精液”应用推广试验

试验对奶牛＂性控精液＂在高寒地区的推广应用进行了研究。结果表明：成年牛的受胎率（41.64%）极显著低于青年牛（65.31%）（P〈0.01）;性控精液与常规精液相比其受胎率,青年牛组分别为65.31%和67.80%,两者之间差异不显著（P﹥0.05）,而成母牛组分别为41.64%和54.03%,两者之间差异极显著（P﹤0.01）;饲养状态（集中饲养、散养）对性控精液的受胎率有显著影响（P〈0.05）,集中饲养明显优于散养;季节对性控精液的受胎率无显著影响（P〉0.05）。     

奶牛性别控制的研究现状与展望

家畜的性别控制是提高畜牧业经济效益的重要途径。本文介绍了奶牛性别控制的途径、方法、研究现状及其应用前景。     

精子性别化对乳牛性别控制的试验

试验于1991年在华南农业大学实验乳牛场进行,初步成功地获得乳牛雌性控制率达到81.8％的国内外先进水平,计有十头乳牛产犊:二雄、九雌,其中一头产异性双椟,研究试验结果包括三个方面:一是研制成具有一定的纯度和工作效价的H-Y抗血清;二是这种H-Y抗血清IgG确实存在有抑制牛Y染色体精子的基因抗体;二是掌握H-Y抗血精IgG与牛精液发生“感作”和导致正常受胎的技术方法。试验应用免疫遗传学原理,进一步证明H-Y抗血清IgG有抑制牛Y染色体精于的受精能力而获得较高的雌性控制率,具有很大的生物科学与经济价值     

A simple PCR method for sexing Japanese quail Coturnix japonica at hatching

1. The quail is a potentially important avian model for molecular studies; a major drawback is the inability to sex visually before 3 weeks of age. Molecular sexing is therefore an absolute requirement when animals are sampled before that age.

2. A low-cost method using common laboratory equipment based on Allele-Specific Multiplex-Polymerase Chain Reaction was developed to undertake reliable molecular identification of the sex of Coturnix japonica directly at hatching.

3. This simple method works with down feathers collected from behind the neck of the newly hatched quail and includes internal controls during the PCR to limit risks of error. Males and females can be discriminated on the basis of the presence of one or two amplicons, respectively.     

Gender determination in the Paiche or Pirarucu (<Emphasis Type="Italic">Arapaima gigas</Emphasis>) using plasma vitellogenin, 17β-estradiol,and 11-ketotestosterone levels

Arapaima gigas is an air-breathing giant fish of Amazonian rivers. Given its great economic and cultural importance, the aquaculture development of this species represents an evident solution to face the decline of wild populations. In captivity, reproduction occurs generally in large earthen ponds where stocks of a few tens of brooders are maintained together at the beginning of the rainy season (December–March in the Peruvian Amazon). Fry production relies on the spontaneous formation of male and female pairs, which build a nest, delimit a territory and guard the offspring for at least 20 days from other congeners and predators. However, as sex determination of A. gigas is not possible by morphological criteria, it is very difficult to optimize reproduction conditions and fry production in each pond, which seriously hampers the culture of this species. This situation prompted us to develop sexing methodologies based on (1) the detection of female specific plasma Vitellogenin (Vtg) using an enzyme immuno assay (EIA), and (2) the determination of plasma 17β-estradiol and 11-ketotestosterone levels for immature specimens. The Vtg purification was performed by electro-elution after polyacrilamide gel electrophoresis (PAGE) from plasma of 17β-estradiol treated A. gigas juveniles. Two different Vtg molecules were isolated, (Vtg₁ and Vtg₂) with 184 and 112 kDa apparent molecular masses, respectively, and two antibodies were raised in rabbits for each Vtg molecule. Adult fish were 100% accurately sexed by Vtg EIA, while 100% of immature fish and 95% of adults were accurately sexed by 17β-Estradiol and 11-Ketestosterone ratios. We also observed different color pattern development in male and female adult fish (6-year-olds) around the reproductive period.     

苏新棕壳蛋鸡自别雌雄配套系的研究——雏鸡羽色自别雌雄准确率的测定

对苏新A、B(均带有金色基因)、C、D(均带有银色基因)4个纯系按A■×B■A■×C■、A■×D■、B■×D■、AB■×CD■等5个组合进行测定。结果表明雏鸡羽色自别雌雄准确率均在98.2％以上,证明纯系的羽色基因已高度纯合。同时观察确定了商品代雏鸡羽色自别标准型。公雏羽色:全白色或白头身有棕色斑点;白头、淡黄身;白头、背部有浅灰色到深灰色条纹。母雏羽色:全棕色;棕头、白身;棕头、棕条背、白身。