哺乳动物精子分离方法的研究进展

分离X、Y精子,用于人工授精或体外受精,是目前实现哺乳动物性别控制的最有效手段。本文对哺乳动物精子分离及分离精子纯度评估方法的研究历史及现状作一回顾和总结。     

流式细胞仪分离精子法的研究进展

以X精子和Y精子的DNA含量差别为基础的流式细胞仪分离精子技术是有效的哺乳动物性别控制方法。目前,流式细胞仪分离精子的速度与上世纪90年代初期相比已提高了30多倍,以3000个/秒的常规速度分离X精子或Y精子的准确率可达到85%～90%。迄今,用分离精子人工授精已产下了数万头的动物后代,而分离人的精子也开始应用到了避免X染色体连锁遗传疾病的生殖医学工程中。     

微流控芯片技术用于精子与上皮细胞分离的研究

为研究建立使用微流控芯片技术分离精子与阴道上皮细胞的方法,选用制作工艺简单的玻璃-PDMS芯片,对混合样本进行分离。加样前分别在进、出口池中加入7此和lO此缓冲液,然后在进样口加入2此混合样本。至少静置8nlin后,从出口池中取出3此形成重力驱动的微流体后开始分离,每隔5min在进样口补加1此缓冲液。达到理想分离效果时,用移液器从出口池取出分离出的精子,核酸酶去除游离DNA,经过提取、扩增和电泳分离脸测等步骤得到精子的分型结果。结果显示,使用基于重力驱动微流体原理的微流控芯片可在30min内分离出精子,不会有上皮细胞进入分离通道;通过核酸酶对分离出的精子液的去游DNA处理,可得到单一、完整的精子分型。与传统的差异裂解法相比,这种方法在很大程度上节省了检验时间,在性侵案件中具有一定的法医物证分析价值。     

流式细胞仪原理及其在奶牛性别控制上的应用

流式细胞仪分离得到的定性奶牛精子,借助人工授精技术或其他相关技术已经生产出许多预知性别的后代奶牛。流式细胞仪分离精子的原理是基于携带有X或Y性染色体精子的DNA含量不同。高性能的流式细胞仪可以达到每小时分离1.5～2×108个精子,并且准确率可达到90%。该项技术的应用使得奶牛的人工授精和低温保存技术更为便利,并且该项技术也可以用于其他养殖动物X、Y精子的分离。     

应用生殖免疫方法制作性别化冷冻精液对奶牛性别控制的研究

本实验在种公牛站精液生产过程中应用生殖免疫技术方法 ,制作性别化冷冻精液 ,结合人工授精技术进行奶牛性别控制的研究实验。根据精子DNA含量存在的差异 ,利用荧光染料与精子DNA结合 ,通过蔗糖溶液密度梯度离心法 ,分离出和鉴别出牛精液中的X与Y精子作为抗原。经与小鼠免疫后 ,制成H Y抗血清IgG ,再经酶联免疫吸附法 (ELISA)析测表明 ,获得了具有一定纯度和工作效价的阳性抗Y精子的H Y抗血清IgG ,H Y抗血清对性别化精液冷冻前 ,解冻后活力的影响与对照组无显著差异。配种受胎试验结果表明 ,性别化冷冻精液在获得与正常冷冻精液相似的情期受胎率的同时 ,对后代性别比率有显著影响 ,奶牛产母犊率可达 60 7% ,比自然产母犊性比率理论值提高 1 0 7个百分点 (P <0 0 5 )。     

家兔早期胚胎的性别鉴定

动物的性别问题,特别是哺乳动物的性别控制,一直是生物学界探究的难题之一。长期以来,国内外研究者曾进行了大量的研究和探讨,企图达到人工控制性别的目的。过去,国外主要用分离X、Y两类精子的方法来进行性控,     

牛分离精子对其IVF胚胎染色体影响的研究

本研究采用传统的细胞遗传学方法,研究了由流式细胞仪分离的、染色未分离的及作为对照用的未染色未分离的分别来自于3头公牛的精子IVF（in vitro fertilization, IVF)后产生的6~8 d囊胚的染色体异常情况,以确定流式细胞仪分离精子的过程及染色对胚胎染色体异常的影响。结果显示,分离精子、染色未分离精子和未染色未分离精子的胚胎中,染色体组成为异常,即嵌合体的胚胎分别占40.7%（59/145）、35.8%（38/106）和37.0%（37/100）,三者染色体异常的比例无显著差异。胚胎染色体异常的频率在不同公牛之间存在差异（33.0% 比 44.6%）（p<0.05）。本研究的结果证明,染料和分离过程没有影响精子的DNA进而影响胚胎的染色体组成;胚胎染色体异常的频率在不同公牛之间存在差异。     

植物基因分离的图位克隆技术

DNA是遗传的物质基础。遗传的基本单元是以基因的形式包含在 DNA序列内。要想从分子基础上来理解遗传的本质 ,基因的分离是最基本的前提。现在分离和克隆植物基因的方法很多 ,如传统的功能克隆及近年来发展十分迅速的表型克隆。但大多数情况下 ,我们并不知道基因的表达产物 ,在未知基因的功能信息又无适宜的相对表型用于表型克隆时 ,最常用的基因克隆技术有转座子示踪法、随机实变体筛选法和图位克隆法。其中 ,转座子示踪法中的转座子受其种类、活性和数量的制约 ,随机突变体筛选法随机性较大且不能控制其失活基因的种类和数量 ,也限制了…     

我国动物学家取得试管水牛性别控制成果

2月13日上午11点20分,在广西水牛研究所的一头杂交母水牛,通过分离XY精子性别控制产下了雌性水牛双犊。据悉,这在世界尚属首例。据介绍,这两头性控试管水牛犊体重均为29kg。“姊妹花”出生经历了复杂、严密的科学设计:首先,用流式细胞仪分离出良种摩拉水牛精液的X精子和Y精子;然后, 将X精子与本地母牛的卵母细胞进行体外受精生产性别控制的胚胎;最后将3枚雌性胚胎移植给一头受体母牛,经10月怀胎后生产出来。该项由国家自然科学基金、广西壮族自治区人民政府主席基金、自治区科技厅科技攻关项目资金联合资助的水牛性别控制研究项目由卢克焕教授主持,广西水牛研究所和广西畜禽品种改良站共同参与完成。该项成果的关键技术是分离XY 精子技术。这意味着水牛生公生母完全可能人为控制。     

X,Y精子分离技术的进展

自本世纪伊始,就有许多有关分离X精子(具有X染色体的精子)和Y精子(具有Y染色体的精子)的研究报告.近年来,美国、日本等国相继报道分离人和牛的X、Y精子获得成功,然而其实验效果的重复性极低.本文仅就最近有关X、Y精子的分离研究进展情况作以扼要的介绍.     

Sex selection in mammals: a review

New methods for sperm separation and embryo sexing provide the opportunity to select the gender of the offspring of domestic mammals. We review here procedures currently available for pre- and post-fertilization sex determination, including flow cytometric selection of X- and Y-chromosome bearing spermatozoa and the application of the polymerase chain reaction to biopsied embryos. Modern techniques are considered in the context of the development of the field and parallel innovations in human medicine are briefly considered.     

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.     

哺乳类性别控制的实践与进展

动物的性别控制是指通过对精子或胚胎的性别鉴定以达到调控子代性别的目的。基于在人类健康及畜牧业生产上的应用价值,动物的性别控制一直是人类期望达到的目标之一。介绍了性别控制的研究历史及最新进展,并着重就不同性别控制手段的理论基础,特点,实践应用价值及未来发展进行了评述。     

Livestock embryo sexing: A review of current methods, with emphasis on Y-specific DNA probes

Control of the sex ratio of domestic species is potentially of great commercial importance to agriculture. While sexing of spermatozoa would be the most advantageous approach, studies to date suggest that this technology is unlikely to be available in the near future. As an alternative, four methods of sexing embryos have been developed. The use of X-linked enzymes and a serological assay involving H-Y antigen are noninvasive methods which have the advantage of allowing all embryos to be sexed, but these methods are not always accurate. Cytogenetic analysis and the use of Y-specific DNA probes are invasive methods which are limited by the accessibility of embryonic material for biopsy, but they are highly accurate. Each method is reviewed, with an emphasis on the use of Y-linked probes, and each is seen to have both advantages and limitations; the difficulty is in achieving a method that provides both an accurate sexing procedure and an acceptable pregnancy rate after embryo transfer. While no single method currently available fulfills all the criteria for a commercial method of embryo sexing, the potential for the development of an ideal method does exist.     

Extreme gender-based post-fledging brood division in the toc-toc

The possibility that parents of one sex may preferentially investin offspring of a certain sex raises profound evolutionary questionsabout the relative worth of sons and daughters to their mothersand fathers. Post-fledging brood division—in which eachparent feeds a different subset of offspring—has beenwell documented in birds. However, a lack of empirical evidencethat this may be based on offspring sex, combined with the theoreticaldifficulty of explaining such an interaction, has led researchersto consider a gender bias in post-fledging brood division highlyunlikely. Here we show that in the toc-toc, Foudia sechellarum,post-fledging brood division is extreme and determined by sex;where brood composition allows, male parents exclusively provisionmale fledglings, whereas female parents provision female fledglings.This is the first study to provide unambiguous evidence, basedon molecular sexing, that sex-biased post-fledging brood divisioncan occur in birds. Male and female parents provisioned at thesame rate and neither offspring nor parent survival appearedto be affected by the sex of the parent or offspring, respectively.The current hypotheses predicting advantages for brood divisionand preferential care for one specific type of offspring arediscussed in the light of our results.     

A rapid improved method for sexing embryo of water buffalo

The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Morphology versus molecules: sexing Red-winged Blackbird nestlings

ABSTRACT.   Past studies of offspring sex ratios in birds have often relied on sexually size dimorphic species where nestling sex could be determined based on weight at a given age. DNA-based sexing techniques allow us to assess the accuracy of those techniques and to refine them for use when costs or convenience make DNA methods impractical. Using nestling Red-winged Blackbirds ( Agelaius phoeniceus ) whose sex was determined using DNA, we compared sex ratios obtained using different morphological criteria. Conservative criteria from previous studies were completely accurate, but allowed sexing of few nestlings younger than 8 d old, and were more successful for sexing males than females. A new method was developed that allowed accurate sexing of nestlings beginning at day 6 posthatching and was less biased relative to known sex ratios. Using 11 years of data, the conservative method left an average of 55% of nestlings and 36% of fledglings unsexed, compared to 31% and 9% using the new method. Furthermore, the male bias in sex ratio estimates using the conservative method was greater, both absolutely and relative to estimates based on the new method, when the proportion of unsexed nestlings (because they were not weighed when older) was higher. Thus, estimates of population sex ratios will be more accurate as the number of nestlings measured on day 8 or older increases. However, if some nestlings that were not weighed past day 7 fledge, the new method allows more of those individuals to be sexed than the conservative method, and the population sex ratio estimate should be more reliable. Although our approach should apply to other sexually dimorphic species, the criteria used must be developed based on such species-specific attributes as growth patterns and degree of hatching asynchrony.     

Nest size and hatchling sex ratio in chinstrap penguins

Variation in the sex ratio at hatching in the chinstrap penguin Pygoscelis antarctica was investigated, using molecular sexing to test predictions of sex allocation theory. The sex ratio was slightly male-biased (0.54) but did not differ significantly from parity. The proportion of males increased with nest size, an estimator of parental quality in chinstrap penguins. High-quality parents were able to produce and rear a higher proportion of male offspring, the more costly sex in this slightly sexually dimorphic species. Our results may be in agreement with Trivers and Willards (1973) argument on biases in the offspring sex ratio being contingent on parental condition or quality.