哺乳动物精子性别鉴定

性别鉴定技术与人们的日常生活及畜牧业的发展有着密切的关系。精子性别鉴定技术的迅速发展为这一技术的应用提供了广阔的前景。目前最常用的精子性别鉴定技术是流式细胞分选法，此法依据X－精子及Y－精子DNA含量的不同将两种精子分开，还可以用作分类后精子物检验。因流式细胞分选法存在一些不适于大规模应用的缺陷，人们希望能依据分类后的精子之间的对比研究找出特异的蛋白制成抗体，设计出高效免疫的精子性别鉴定技术，关于这方面的研究已经取得了一定的进展。     

利用流式细胞仪建立牦牛X、Y精子分选体系的研究

旨在探索与优化牦牛精子分选条件,建立高效的牦牛X、Y精子分选技术体系。本研究制备了牦牛精液细胞悬液,采用不同量的DNA染料Hoechst33342和诱惑红共孵育精子细胞。利用流式细胞仪分选牦牛X、Y精子,通过比较分选效率、分选后精子活力及发育潜能,优化分选条件。运用RT-PCR检测分选精子的纯度,利用精子分析系统检测分选后精子的活力。将分选后的精子与体外成熟的卵母细胞进行体外受精,统计分选后精子的发育潜力,并采用SRY片段的PCR法检测早期胚胎的性别。结果显示,10 μL Hoechst33342染色40 min后的分选效果最佳,其分选产物的准确度和分选效率显著优于其他组,分选后的X、Y精子活力与20 μL Hoechest33342染色20 min组相当,显著高于其他组（P<0.05）。添加5 μmol·L^-1的诱惑红对分选的纯度无显著影响（P>0.05）,但能显著提高分选后精子的活力（P<0.05）。分选后X、Y精子分别与卵母细胞进行体外受精,受精率和囊胚形成率与未分选组差异不显著（P>0.05）,且胚胎性别比例与分选后精子纯度吻合。综上,本研究建立并优化了流式细胞仪分选牦牛X、Y精子的方法,添加诱惑红有助于改善分选后牦牛精子的活力,为后期牦牛性控精液的制备及生产奠定了基础。     

比利时蓝白花牛XY精子分选及冷冻精液制备

本研究利用流式细胞仪分选比利时蓝白花牛X、Y精子并制备冷冻精液。选择平均年龄4岁的健康比利时蓝白花种公牛12头，假阴道法采集精液送至实验室分选，经流式细胞仪分离、冻存、解冻后精液重分析和品质鉴定。X、Y冻精（93.4%和91.1%）性别比例显著高于常规冻精（50%）；分选后精液冻后存活率、活力和顶体完整率与常规冻精差异不显著。本研究结果显示，分选前控制公牛的精液品质（活力≥70%，畸形率≤18%）可以明显改善分选效果；分选后精子纯度和冻后活力满足低剂量人工授精要求（纯度>90%，活力>30%），精子分选对比利时蓝白花牛产业的发展具有重要意义。     

养猪生产中后代性别的预选——发展现状和应用前景

动物精子的性别可通过流式精子分选仪和DNA标识进行鉴定,而利用鉴定性别的精子(即性控精子)并借助人工授精技术或其它授精技术产生的后代,在过去5年中估计已多达30000个(其中大多数是母牛)。有关文献资料证明,能有效地区分X精子和Y精子的唯一标记物是精子染色体中的DNA。众所周知,目前世界各地采用的方法是Beltsville精子分选技术,该技术根据X精子群和Y精子群中DNA相对含量上的差异,用荧光染液(Hoechst33342染液)标记精子,随后利用流式细胞仪分选经荧光标记的精子,从而达到分离精子的目的。目前,X精子或Y精子正常的生产速度是每小时1500万个,该项技术已在家畜、实验动物和动物园动物中应用,如果将该技术应用在人上,在预选后代性别比例上可达90%～95%的成功率。因动物品种不同,性控精子在动物体内的授精部位也不相同。常规的人工授精技术、宫内授精技术、输卵管内授精技术、用于胚胎移植的体外受精(in-vitro fertilization,IVF)技术或子宫角深部授精技术均能有效地使动物怀孕,至于利用哪种技术进行性控精子的精则取决于动物品种。尽管所有动物都能获得高纯度的分选精子,但是在实际生产中利用低剂量精子还难以让母猪怀孕。子宫角深部授精技术每次授精0.5～1.00亿个性控精子已能产生可喜的效果:利用特制的输精管,输入常规人工授精所需精子量的五十分之一的性控精子,足以使动物怀孕。性控精子通过常规的授精技术能够被猪成功利用前,还需重新设计输精管,同时输精的次数和每次授精时精子数量也需作进一步的研究。分选精子的低温保存技术已被牛人工授精普遍采用。尽管已能利用冷冻的性别分选精子生产小猪,但是性别分选精子经冷冻和解冻处理后在常规生产中的应用还未达到最理想的效果。本文将讨论猪精子性别分选的最新研究成果及其发展趋势,并重点探讨将性别分选技术应用于养猪生产中必须对其进行必要的技术开发。     

对奶牛性别控制方法的评价与应用建议

通过人为干预使动物按人们所需性别繁衍后代一直是人们期望达到的目标。目前哺乳动物的性别控制方法主要是早期胚胎性别鉴定和性别精子分选两种途径。本文结合国内应用现状,着重介绍奶牛性别控制的常用方法、特点、影响因素及发展建议。     

利用流式细胞仪分离奶牛精液的性别控制效果观察

目前,常见的牛性别控制方法有早期胚胎性别鉴定法和利用流式细胞仪进行X、Y精子分离法两种。流式细胞仪精子分离法是根据X、Y精子在DNA含量上的差异来分离精子。分离后的X、Y精子用于人工授精或显微授精可在授精之前就控制其性别,因而被认为是更具有实用价值的方法。此次研究通过对分离后精子的人工授精效果和性别控制效果进行试验观察,以期为该方法的进一步应用提供依据。1材料与方法1.1公牛及采精公牛为大庆田丰生物有限公司饲养的荷斯坦牛,共12头。饲以全价营养日粮,每天喂给鸡蛋1枚。采用常规的假阴道法采集精液。1.2精液的处理将采…     

动物精子分离技术及分离纯度检测方法研究进展

性别控制在畜禽生产中具有重要的应用价值。X、Y精子分离技术是目前实现动物性别控制的最有效手段。精子分离技术有多种方法,目前,最有效、最可靠的方法是应用改进后的流式细胞仪,它根据X、Y精子DNA含量的差异进行分离,其分离纯度可以达到90%以上。随着动物精子分离技术的发展,精子分离纯度检测方法也不断涌现,如重分析法、FISH法、PCR法等,但是这些检测方法的成本和准确性仍有待改进。本文主要对动物精子分离技术及精子分离纯度检测方法的研究现状和进展作以综述,供大家参考。     

性控冻精在奶牛生产中应用与展望

XY精子流式细胞分选技术是目前最有效的性别控制繁殖手段之一。本文使用冷冻后X精子人工授精,平均情期受胎率达63%,青年母牛的情期受胎率为77.45%;用性控冻精通过超排技术生产体内性控胚胎平均每头次达5.2枚。用PCR技术对生产的体内胚胎进行性别鉴定后,雌性胚胎的比率为92.98%,实际出生牛犊的母犊率在93%左右。由此表明,使用XY种畜(天津)有限公司生产的性控冻精在生产中应用效果良好,在受胎率不受影响的情况下其性控率可达90%以上。     

流式细胞分离法在哺乳动物精子分离中的应用及前景

动物性别控制技术是指通过人为干预使成年雌性动物产出人们期望的性别后代的一种生物技术,该技术在畜牧业生产中具有重大意义,已广泛应用于牛、鹿等大型动物的繁殖中。哺乳动物子代性别主要由父代的X/Y染色体决定,X/Y精子分离是控制动物性别最直接有效的一种方法。近年来,关于哺乳动物(如牛、羊等)精子分离的研究有着较大的进展,目前应用最多且最准确的方法就是流式细胞法。笔者就流式细胞法分离哺乳动物精子及其应用前景进行简单综述。     

猪的胚胎移植技术—现状与未来：与猪胚胎移植相关的生物技术

胚胎工程学是在胚胎移植技术的基础上组成的,猪的胚胎工程学是今后的研究课题.最近,一些国家正在积极进行基础研究,无疑在不久的将来这一技术可望达到完善.一、性别控制1.X、Y 精子的分离法与对其它家畜或动物一样,人们对猪也进行了利用精子以控制性别的尝试.最近试用了电泳法、流式细胞分类法(细胞流速分离器)、Percoll 密度梯度法分离 X、Y 精子.尤其在确立 F 体检测法以后,容易判断分离出的精子,弄清了各种分离法的     

The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI

The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.     

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.     

Sex ratio after insemination of bovine spermatozoa isolated using a bovine serum albumin gradient

This experiment was undertaken to determine if a method reported to successfully enrich the proportion of Y-chromosome-bearing spermatozoa in human semen could be adapted for separation of bovine spermatozoa. Semen was collected from four Angus bulls and aliquots were either separated on discontinuous gradients of bovine serum albumin (BSA) or untreated before processing for cryopreservation. Two hundred seventy-one cows or heifers were assigned randomly to be artificially inseminated (20 X 10(6) sperm/insemination) with separated or unseparated spermatozoa. The proportions of male offspring were 45 and 54% after inseminations with separated or unseparated spermatozoa, respectively. In a second phase of the experiment, pooled semen from three Holstein bulls was either extended and frozen without separation or frozen after separation using the discontinuous BSA gradient. Separated and unseparated spermatozoa were analyzed by flow cytometry to determine the ratio of X- and Y-chromosome-bearing spermatozoa based on differences in DNA content. The ratios of X- and Y-bearing spermatozoa in separated or unseparated samples were indistinguishable. We concluded that the separation method did not enrich the proportion of Y-bearing bovine spermatozoa.     

Sexing sperm of domestic animals

The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.     

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.     

Flow Cytometry Identification of X- and Y-Chromosome-Bearing Goat Spermatozoa

Flow cytometric sorting technology was used to measure the difference in DNA content between X- and Y-chromosome-bearing spermatozoa in bucks. Spermatozoa were analysed by flow cytometry to characterize X- and Y-chromosome-bearing sperm populations and to quantify the DNA difference between them. Two symmetrical, overlapping and clearly separated peaks, corresponding to X- and Y-bearing spermatozoa, were detected. The difference in fluorescence intensity between the peaks was 4.4 +/- 0.03% without any significant inter- or intra-animal variations. Therefore, the identification and selection of high-purity samples of sperm populations for sex sorting is easier in bucks compared with other domestic species.     

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.     

奶牛性别控制技术的研究和应用进展

奶牛性别的有效控制对显著提高优质奶牛的繁殖效率,提高经济效益具有重要意义。本文就奶牛性别控制有效方法、实际应用加以综述,主要介绍精子的流式细胞分离技术,性控精子应用准确率达92．3％以上;奶牛早期胚胎性别PCR鉴定,准确率达1009／5,通过环境控制对奶牛性别的影响并不显著,目前性控精子的应用是最理想的性别控制手段。     

使用流式细胞仪分离精子进行仔猪性别控制的研究

本研究旨在探索流式细胞仪分离精子在猪性别控制中的应用。使用流式细胞仪分离猪XY精子,而后通过母猪输卵管授精生产"预知"性别的仔猪,并使用吖啶橙染色法检测粗分离对精子核酸含量的影响。结果,成功利用分离获得的猪X和Y精子对母猪进行输卵管授精,母猪怀孕率、产仔率均为100%;输Y精子母猪产仔雄性率100%(♂6/6),对照母猪产仔雄性率57.14%(♂8/14);3头输X精子母猪产母仔率91.67%(♀11/12),对照母猪产雌性仔猪40%(♀2/5);使用性别分离精子不影响母猪的怀孕率、产仔率,但窝产仔数较低;吖啶橙染色法检测结果表明,流式细胞仪粗分离对猪精子核酸含量没有显著影响(P>0.05)。本研究结果提示,使用流式细胞仪分离精子授精可以有效改变仔猪的性别比例。本研究结果为猪分离精子性别控制技术的推广应用奠定了基础。     

Validating bovine sexed semen samples using quantitative PCR

In cattle, separation of X‐ and Y‐bearing sperm cells by flow‐sorting technology makes it possible to predetermine the sex of calves. Due to high costs and decrease in fertilization, the extensive use of sexed semen in livestock depends heavily on sorting purity of sperm cells. Validating the accuracy of sperm sexing requires reliable procedures, therefore a real‐time polymerase chain reaction (PCR) assay was established to calculate the male cell proportion in the sexed semen based on the relationship between the amplification of a SRY fragment and an autosomal gene (MSHR) fragment. Our results showed stable amplification of SRY for 100–1 ng of genomic DNA, which allows detection of 1% of male cells if 100 ng of target DNA is used. To account for the discrepancy in the efficiency of the MSHR and the SRY amplification correction of the difference of the mean values was performed. The ratio of male to female sperm cells in unsexed semen cells was very accurately determined. The fractions of the sexed samples, however, were different from the expected range appearing lower than estimated. Thus, the study reveals that real‐time PCR provides a good basis for the examination of sexed sperm cells, but needs to be optimized for the samples.