性控精液人工授精在奶牛生产中的应用

通过流式细胞仪分离XY精子进行人工授精,是目前最准确、有效、简易的控制奶牛性别的方法。本文介绍了性控精液技术的发展简史,XY精子分离的理论基础和方法,奶牛性控精液人工授精的应用效果,提高性控精液应用效率的措施以及推广性控精液人工授精的意义、技术障碍和发展前景。     

性控精液人工授精在奶牛生产中的应用

通过流式细胞仪分离XY精子进行人工授精,是目前最准确、有效、简易的控制奶牛性别的方法。本文介绍了性控精液技术的发展简史,XY精子分离的理论基础和方法,奶牛性控精液人工授精的应用效果,提高性控精液应用效率的措施以及推广性控精液人工授精的意义、技术障碍和发展前景。     

性控精液人工授精在奶牛生产中的应用

通过流式细胞仪分离XY精子进行人工授精，是目前最准确、有效、简易的控制奶牛性别的方法。本文介绍了性控精液技术的发展简史，XY精子分离的理论基础和方法，奶牛性控精液人工授精的应用效果，提高性控精液应用效率的措施以及推广性控精液人工授精的意义、技术障碍和发展前景。     

奶牛性控冻精技术在黑龙江省推广效果

奶牛性别控制技术是对奶牛正常生殖过程进行人为干预,使之按人类的愿望繁衍所需性别后代的生物技术。目前主要采用的是流式细胞分离法分离XY精子,再生产性控精液的技术。利用性控精液通过人工授精方式,使奶牛提高母犊率,从而达提高繁殖效率的目的。笔者通过对几家牧场的使用性控冻精的情况进行统计分析,以期为该技术在奶牛生产中的应用提供参考。     

两种奶牛性别控制胚胎生产方法的比较

试验分别应用PCR法和XY精子分离法生产性别控制奶牛胚胎,并通过移植观察准确率。结果显示,PCR法准确率为89%,XY精子分离法准确率为100%。试验表明,应用XY精子分离法进行性控胚胎生产,技术难度较小,成本较低,效果良好,生产中具有较大推广应用价值。     

奶牛XY精子分离的性控繁育及推广对策

1性控繁育的概念即用性别控制的方法进行繁殖育种,是性别控制和繁殖育种的简称。目前,关于奶牛性控繁育的热门话题就是XY精子分离技术。该技术是通过流式细胞精子分离仪将公牛的X精子(雌性精子)与Y精子(雄性精子)分离,是真正从生公生母的内因———基因、性染色体的角度出发,来实现性别控制的。目前X精子分离准确率已达到93%以上。据美国XY公司报道,奶牛性控冻精配种产母率在90%以上,10787头青年母牛人工授精的受胎率为53%。国内报道,产母率达到92%,受胎率达到49%~68%,平均50%以上。目前XY种畜(天津)有限公司已经引进XY公司的技术和设…     

奶牛XY精子分离后输精对超排卵子受精及人工授精受胎率的影响

就奶牛生产而言,如能将产母犊率提高10％。15％,则对迅速发展我国乳牛业能起很大作用。流式细胞仪精子分离法是根据X、Y两类精子在DNA含量上的差异来分离精子。分离X、Y精子并用于人工授精或显微授精是控制家畜性别最简单、可行的方法之一,它可在授精之前就控制其性别,避免了胚胎的浪费。现在,超数排卵利用性控精液生产性控胚胎是性控胚胎移植的主要胚胎来源。对优良种母牛进行超排生产性控胚胎,是充分挖掘良种母牛的遗传潜力和向社会提供大量优质性控胚胎的重要手段。本研究旨在检测和验证流式细胞分离仪分离精子的过程或染料对奶牛超数排卵后卵母细胞受精及胚胎质量的影响,以及通过精子分离后奶牛精液对受胎率及性别比率的控制。     

奶牛性控冻精人工授精技术研究进展及应用前景

奶牛的性别是由性染色体决定的。在生产中人们希望母牛多产母犊以加快奶牛群的扩繁速度和提高牛群的产奶量,因此对奶牛性别控制的研究就成为了奶牛繁殖科研工作者所关心的课题。奶牛性控冻精人工授精技术是将奶牛种公牛的精子通过精子分离仪使含X、含Y染色体精子得到有效的分离,将分离后得到的X精子进行分装冷冻制成冻精细管,进行奶牛的人工授精,从而使母牛怀孕产母犊的技术。     

膜联蛋白Ⅴ去除奶牛死精子技术及其在性控冷冻精液生产中的应用

本研究旨在通过膜联蛋白Ⅴ(Annexin Ⅴ)与奶牛死精子的特异结合,并尝试与免疫磁珠技术共用去除奶牛精液中死精子,从而提高性控精液生产效率。试验结果显示,通过膜联蛋白Ⅴ与免疫磁珠筛选后的精子活力、膜完整性、线粒体活性均有不同幅度改善,特别是上机分离时X/Y死精率明显下降(P0.05),说明该技术流程设计可以有效去除部分死精子,提高性控精液生产效率。同时,试验证明膜联蛋白Ⅴ与免疫磁珠筛选操作对于分离后的X或Y精子制备的性控冷冻精液各项产品技术指标均没有明显影响(P0.05),说明该技术流程并不会对精子造成额外的损伤。综上所述,本研究开发的Annexin Ⅴ蛋白的特异性结合免疫磁珠方法与技术工艺,可以有效去除奶牛精液中的死精子,显著提升奶牛性控冷冻精液生产效率、降低生产成本,为家畜性别控制冷冻精液推广应用提供新的技术支撑。     

流式细胞仪用于牛精液分选的技术要点

流式细胞仪应用于性控冻精生产是目前奶牛生产繁育性别控制技术的有效方法。本文阐述了流式细胞仪分离精子的工作原理及应用效果,并对流式细胞仪分离精子存在的问题进行了分析,提出了奶牛X精子和Y精子分离技术的展望。     

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.     

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。     

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.     

分离Y精子在茸鹿性别控制中的应用研究

为研究一种新的茸鹿繁殖方法,2007～2008年对469头马鹿,79头梅花鹿进行性别控制技术研究。结果表明：性别控制冻精情期受胎率马鹿为87．21％,梅花鹿为60．76％,两者之间差异极显著（P〈0．01）,性比率（指产公仔率）马鹿为90．95％,梅花鹿为93．75％,两者之间无显著差异（P〉0．05）;性别控制冻精与普通冻精输精比较,马鹿情期受胎率之间、性比率之间和梅花鹿性比率之间都存在极显著差异（P〈0．01）,梅花鹿情期受胎率之间则无显著差异（P〉0．05）。普通冻精受胎率马鹿为94．89％、梅花鹿为61．63％,性比率马鹿为50．22％、梅花鹿为49．06％,性别控制冻精受胎率整体接近普通冻精,性比率则提高40％以上,比较分析认为对茸鹿X、Y精子分离进行性别控制试验是成功的。     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

奶牛性控冻精人工授精影响因素研究

用分离X和Y精子的性控精液进行人工授精是控制家畜性别之最简单可行的方法.然而,低密度性控精液输精效果还不如常规人工授精,许多技术环节都有待改进.以常规冻精和稀释常规冻精为对照,研究解冻方法、输精时间和部位、不同精液来源和输精员以及育成和经产牛等因素对性控冻精人工授精妊娠率的影响.结果显示,精液解冻水浴温度和持续时间对人工授精效果有显著影响,性控精液对解冻水浴温度更敏感;性控冻精和稀释常规冻精比常规冻精对输精时间要求更严格;3种精液输精到排卵卵泡同侧子宫角基部受胎率都显著高于输精于子宫体和同侧子宫角前端;3种精液育成牛受胎率(80%)都显著高于经产牛(50%);于输精同时注射促排卵素3号明显提高性控冻精受胎率;经严格挑选、能够从事胚胎移植操作的技术熟练输精员之间性控冻精受胎率差异不显著;在所设计的不同条件下,性控冻精与稀释同样倍数的常规冻精行为相似,说明精子分离过程没有对精子造成特殊损伤.研究结果说明,精确控制人工授精各个技术环节可以实现消除性控与非性控、低密度与高密度精演之间的差别,获得高妊娠率.     

哺乳动物性控DNA疫苗研究现状和构想

介绍了DNA疫苗的定义、组成、机理、应用策略,综述了X、Y精子间蛋白和mRNA的研究现状,指出X、Y精子间基因表达差异的存在,利用mRNA差异显示技术可对X、Y精子构建性别差异表达的消减cDNA文库,大规模筛选性别差异表达基因,在此基础上应用DNA疫苗技术,既避开蛋白质水平寻找X、Y精子弱差异蛋白这个困难,又能够特异性地抑制X或Y精子,更经济地实现对性别的控制.性控DNA疫苗是新的研究方向.     

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.     

性别控制与体外受精技术快速繁育高产奶牛群

使用流式细胞分离仪可以将X精子从Y精子中选离出来,分离精度高达90%或更高。这种性别控制(性控)技术可以用来生产性别预选的家畜以及优化育种的方案。然而,精子分离的速度以及其性控冷冻精液的不同受精力仍然限制着该技术的广泛应用,尤其是在传统的人工授精育种方案中。体外受精技术(Invitro fertilization,IVF)提供了一个可行而有效的手段来提高奶牛性控精子的受精效率。本文介绍了有关利用IVF技术来控制奶牛性控胚胎发育率和提高雌性胚胎质量的新方法,从而提高胚胎生产效率,达到快速繁育高产奶牛群的目的。这些IVF程序包括以优化的方法遴选出用每头公牛X精子进行IVF所需的最佳肝素浓度;减少IVF受精液的体积以确定每枚卵子获得受精所需的最少精子数目;在体外培养液中使用果糖来替代葡萄糖,以及使用能超速降温的玻璃化冷冻技术。     

哺乳动物精子分离技术研究进展

精子分离是控制动物性别简单而有效的方法之一,本文综述了X、Y精子分离的生物学基础,分离方法及其应用效果的研究进展,并阐述了精子分离技术存在的问题及其发展前景。