性别控制（综述）

一、X、Y 精子的分离(一)两类精子的差异人们从生物学、生物化学、形态学、生物物理学、免疫学等方面对 X、Y 精子的不同点做了大量研究,归纳起来主要有以下几方面。(1)DNA 量:已测出猪、牛、羊的 X 精子中 DNA 量比 Y 精子高2.5～4.5%。(2)头部大小:据报道,哺乳动物细胞的分裂中期赤道板上 X 染色体比 Y 染色体大得多.例如,牛的 X 染色体面积为7.85μ~2,Y 染色体为     

Toll样受体7/8在公猪生殖系统中的表达及其配体对猪X/Y精子分选效果评价

旨在分析Toll样受体7/8(TLR7/8)在公猪精子及生殖器官中的表达情况，并探讨是否能通过其配体处理的方式分离猪X精子和Y精子。本研究采用qRT-PCR分析3头健康成年公猪睾丸、附睾头、附睾体、附睾尾以及精子中TLR7和TLR8基因的mRNA表达水平，利用免疫组化检测公猪睾丸和附睾中TLR7和TLR8蛋白的表达，并通过细胞免疫荧光分析其在不同物种(健康成年小鼠、公牛和公猪)精子中的表达情况，将其配体R848与猪精子共孵育，研究其对精子活力以及X/Y精子分离的影响。结果表明，TLR7和TLR8 mRNA在公猪睾丸、附睾头、附睾体、附睾尾组织以及精子中均表达；免疫组化结果显示，TLR7/8蛋白在睾丸中主要表达于生殖细胞，在附睾中主要表达于柱状细胞微绒毛中；细胞免疫荧光结果表明，TLR7和TLR8蛋白只表达于小鼠和牛X精子尾部，Y精子中不表达，但TLR7和TLR8蛋白在公猪X和Y精子中都表达且表达模式无显著差异，TLR7蛋白主要表达于猪精子头部顶体区域，TLR8蛋白主要表达于猪精子尾部；与对照组相比，用TLR7和TLR8配体R848孵育猪精子后，精子活力降低，但上层精子的性别比例无显著差...     

核酸疫苗研究进展

核酸疫苗是利用基因重组技术生产的疫苗,又称为基因疫苗,包括DNA疫苗和RNA疫苗。目前研究最多的是DNA疫苗,因它不需要任何化学载体,所以又称为裸DNA疫苗。先将编码抗原蛋白的基因连接到真核质粒表达载体上,然后导入宿主细胞内,抗原基因就可以在宿主细胞内被表达,从而诱导宿主对     

琉式细胞技术在哺乳动物性别控制中的研究进展

在动物性别控制的各种技术中,应用流式细胞仪分离的X、Y精子授精可较准确地控制动物后代的性别.文章简单阐述了流式细胞仪分离X、Y精子的原理及其在动物性别控制中的应用,并结合最新研究进展详细地探讨了这项技术在实际生产与应用中的注意事项,旨在为这项技术的研究与应用提供必要的参考与建议.     

流式细胞技术在哺乳动物性别控制中的研究进展

在动物性别控制的各种技术中,应用流式细胞仪分离的X、Y精子授精可较准确地控制动物后代的性别.文章简单阐述了流式细胞仪分离X、Y精子的原理及其在动物性别控制中的应用,并结合最新研究进展详细地探讨了这项技术在实际生产与应用中的注意事项,旨在为这项技术的研究与应用提供必要的参考与建议.     

X、Y精子差异膜蛋白及其分离方法的研究进展

X、Y精子差异膜蛋白的分离是利用抗原抗体反应进行性别控制的关键环节,H-Y抗原是最早被发现并应用于性别控制的Y精子特异抗原,但由于抗原特异性较差,未能在生产中得到广泛应用。雄性性别传递偏移、性别偏移及其他相关研究表明,尽管在精子形成过程中,细胞间桥使两类精子部分基因的表达产物被共享了,但X、Y精子膜蛋白差异仍然存在。同时,差异膜蛋白分离技术的进步及这些技术的优化集成,使这种差异膜蛋白的分离成为可能。     

奶牛性别控制技术研究及其利用

奶牛的性别控制主要有三条途径，一是在人工授精前通过对X精子与Y精子分离以控制性别；二是在胚胎移植前对胚胎的性别进行鉴定以控制性别；三是通过控制外环境来控制性别。１人工授精前的性别控制奶牛染色体的数目为６０条，其中５８条为常染色体，另外两条为性染色体即X、Y染色体，一般认为雄性动物胚胎的性染色体为XY型，雌性动物胚胎为XX型。由于Y染色体只在公牛才含有，因此，精卵结合时精子的类别就决定了奶牛的性别。X精子与Y精子之间存在着微弱的生物学差异，根据精子的形态、DNA含量、比重、膜电荷、抗原性、体积等差异，实施…     

双顺反子表达质粒在DNA疫苗研究中的应用

简述了DNA疫苗的研究概况，介绍了真核双表达质粒的结构特点及其在基因佐剂和二价DNA疫苗中的应用情况，总结分析了双表达质粒的优点和存在的问题，并对其今后在DNA疫苗中的研究方向和前景进行了展望。     

哺乳动物XY精子分离技术的研究

近几十年来人们对动物的性别控制进行了大量的研究,其中X精子和Y精子的分离技术被广泛应用于哺乳动物性别的控制。论述了哺乳动物X精子和Y精子分离的方法,并简述了X精子和Y精子分离技术存在的问题及其发展前景。     

水牛精液中X和Y精子比率的鉴定

利用流式细胞仪分析水牛分离和未分离精液中精子的DNA含量,所得的直方图用高斯曲线拟合,分析计算出样本中X和Y精子的比率。结果表明:未分离的水牛精液中X精子的比率为50%,与正常水牛后代性别比率没有显著差异;而水牛的分离X精液样本中X精子占93%,分离Y精液样本中Y精子占89%。实验结果显示了流式细胞仪DNA分析法鉴定水牛精液中X和Y精子比率的可靠性,而流式细胞仪分离精子程序和方法在水牛上的应用是可靠而有效的。     

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。     

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.     

低剂量梅花鹿XY精子马花杂交人工授精试验

采用流式细胞分离仪分离的梅花鹿X和Y型冷冻精液与常规冻精对62头3组同期化处理的马鹿进行直肠把握人工授精。结果表明,0.25 mL/支含106个有效精子的X和Y型冻精产仔率分别为43%和37%,而0.25 mL/支含107个有效精子的常规冻精产仔率为55%,X、Y型冻精与常规冻精组间差异显著(P<0.05),所产后代性别比率分别为0∶10,9∶0和5∶6,X、Y型冻精与常规冻精组间差异显著(P<0.05),X、Y型冻精与常规冻精所产后代出生及60 d时的体重差异不显著(P>0.05)。     

Sex Preselection in Swine: Altered Sex Ratios in Offspring following Surgical Insemination of Flow Sorted X- and Y-Bearing Sperm

Flow cytometric sorting of X and Y chromosome-bearing sperm into separate populations, followed by surgical insemination into the isthmus of the oviduct of mature gilts, produced litters with phenotypic sex ratios skewed in the direction of male or female according to the predicted sex of the sperm population inseminated. The skewing of the sex ratio of the offspring was predicted from flow cytometric DNA reanalysis of flow sorted sperm populations. Gilts inseminated with intact, viable flow-sorted X-bearing sperm produced litters of off spring that were 74% female (P < .01). Gilts inseminated with intact, viable flow-sorted Y-bearing sperm gave off spring that were 68% male (P < .04). These results validate the use of DNA as a marker for pre-selecting the sex of offspring when used in conjunction with flow cytometric sorting of sperm .     

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.     

Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.     

Deep insemination with sex‐sorted Cashmere goat sperm processed in the presence of antioxidants

Flow cytometrically sex‐sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex‐sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex‐sorting and freezing process, including ascorbic acid‐2‐glucoside AA‐2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex‐sorted fresh and frozen‐thawed sperm. Based on these experiments, we performed deep insemination with sex‐sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA‐2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA‐2G supplementation showed an evident protection against the sorting process‐induced membrane and acrosome damage. In Experiment 2, AA‐2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen‐thawed sex‐sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA‐2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA‐2G supplementation efficiently protects quality and function of both fresh and frozen‐thawed sex‐sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.