水牛性控精液微生物分析

研究针对目前分离水牛X和Y精子性别控制过程中,容易导致微生物快速繁殖进而污染性控精液的问题,对水牛新鲜及冷冻性控精液中的微生物进行分析研究,以期改进水牛分离精子保存方法。结果表明:新鲜采集的水牛精液、常规冷冻精液及分离后的冷冻精液中,平均菌落密度分别为10 619个/mL、293个/mL和18935个/mL,三者差异显著(P0.05)。新鲜精液检测到的菌落中,革兰氏阳性球菌占48%,阴性杆菌占41%,阳性杆菌占9%,霉菌占2%;性控冷冻精液中的菌落100%为革兰氏阴性杆菌。对性控冻精菌落药敏检测发现,菌落对妥布霉素和庆大霉素均呈现耐药性,而对丁胺卡那霉素以及药物环丙氟哌酸药物敏感性较强。本研究结果,为改进水牛分离精子稀释液以及冷冻保存液中的抗生素使用提供了有价值的参考。     

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。     

浅谈优化分离精液的冷冻保藏方法

正动物的性别控制技术是通过对动物的正常生殖过程进行人为干预,使成年雌性动物产出人们期望性别后代的一门生物技术。现在一般多采用流式细胞仪进行X、Y精子分离法,该技术是利用动物含有X染色体和含有Y染色体的精液中DNA含量的差异,借助特异性染色剂染色,然后根据发出的荧光强度,通过计算机预测精子类别,再通过给精液液滴附加电荷,根据静电原理,将含有X染色体的精液和Y染色体的精液分离,得到分离精液,如果得到的分离精液在短     

流式细胞仪分离哺乳动物精子的研究进展

目前,分离精子性别准确率在90%以上,受精率是未分离精子的70%～80%。尽管动物分离精子还存在许多亟待解决的问题,但随着精子分离仪的改进以及与各项辅助生殖技术的结合,动物分离精子必将在实践中得到广泛的应用,并产生巨大的社会效益和经济效益。主要概述了近年来用流式细胞仪分离哺乳动物X、Y精子的研究进展、存在的问题和应用前景。     

世界首例通过分离XY精子进行性别控制的试管水牛在广西诞生

本刊辑:2006年2月13日,广西水牛研究所的一头杂交母水牛,通过分离XY精子,进行性别控制产下了雌性水牛双犊。这在世界尚属首例。这两头性控试管水牛犊体重均为26kg,它们的出生经历了复杂、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,将三枚雌性胚胎移植给一头受体母牛,经十月怀胎后产出。该项成果的关键技术是分离XY精子技术。目前,广西大学动物繁殖研究所已掌握了这一技术,分离水牛XY精子的准确率可达90%以上,这意味着水牛生公生母…     

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…     

流式细胞仪分离处理对种公牛精子活力、顶体完整率及线粒体活性的影响

Johnson等最早报道用流式细胞仪分离兔X和Y精子,并使用分离精子受精后产下后代.目前,牛精子分离和性别控制技术已经开始商业化应用,这对于奶牛和肉牛业的发展都有着巨大的应用价值.但是,大多数研究证明,分离精子的受精能力要低于未分离精子.     

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)。     

不同染色与冷冻方法对关中奶山羊X、Y精子的分离及应用效果研究

为探究不同Hoechst 33342的染色浓度与冷冻方法对关中奶山羊X、Y精子进行分离及应用效果的影响，本实验采集6只关中奶山羊精液，使用浓度分别为10、11、12、13μL/m L的Hoechst 33342(5 mg/m L)染色液对精液样本进行染色，以流式细胞仪分离X精子和Y精子，应用3种不同冷冻程序冷冻精液，应用体外受精与胚胎发育技术评估关中奶山羊X、Y精液的分离准确性。结果表明：12μL/m L Hoechst 33342染色条件下，奶山羊X、Y精子分离效率达到91.02%，高于10μL/m L和13μL/m L(P<0.01)，最高分选速度为4 134个/s(P<0.05)；公羊个体的精液品质越好，其精子的分离速度越快，但对分离准确率的差异不显著；使用冷冻程序2进行关中奶山羊的X、Y精子冷冻的效果较好（P<0.05）；体外受精48 h后，使用Y精子进行受精的卵细胞的卵裂率高于使用X精子（P<0.05），在胚胎培养第9天，使用Y精子进行受精的卵细胞的囊胚发育率高于使用X精子（P<0.05）。总之，当Hoechst 33342浓度为12μL/m L...     

马鹿XY精子对梅花鹿腹腔内窥镜输精试验

41头梅花鹿被分为3组,CIDR+PMSG法同期发情,用流式细胞仪分离的马鹿X和Y型冷冻精液与常规精液对梅花鹿进行腹腔内窥镜子宫角人工授精.结果表明,0.25 mL/支含有106个有效精子的X和Y型冻精产仔率分别为100 %(2 / 2)和92 %(22 / 24),而含有107个有效精子0.25 mL/支的常规冻精产仔率为 93%(14/ 15),X和Y型冻精与常规冻精组间的受胎率差异不显著(P＞0.05).X和Y精子受精后产出后代全部为预定性别,所产后代雌雄比率分别为:X组为0∶2,Y组为22∶0,与对照6∶8相比性别比显著偏离.X和Y型冻精与常规冻精所产后代出生重及60 d时的体重差异不显著(P＞0.05).     

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.     

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...     

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.     

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.     

Detection of Water Buffalo Sex Chromosomes in Spermatozoa by Fluorescence in situ Hybridization

In order to identify X‐ and Y‐bearing spermatozoa in water buffalo by fluorescence in situ hybridization (FISH), some available probes of closely related species were examined. An X‐ and Y‐specific probe set, made from flow sorted yak chromosomes, labelled in somatic metaphases of water buffalo the whole X and Y, respectively, except their centromere regions. A cattle Y‐chromosome repeat sequence (BC1.2) showed strong signal on the telomere region of the buffalo Y‐chromosome, demonstrating the evolutionary conservation of this locus in water buffalo. In hybridization experiments with spermatozoa from five buffaloes, the yak X‐Y paint set demonstrated clear signals in more than 92% (46.8% X and 45.8% Y) of the cells. Using the cattle Y‐chromosome specific BC1.2 probe, clear hybridization signal was detected in more than 48% of the cells. Statistical analysis showed that there was no significant difference between bulls or from the expected 50 : 50 ratio of X‐ and Y‐bearing cells. The probes presented here are reliable to assess separation of X‐ and Y‐bearing spermatozoa.     

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.     

梅花鹿XY精子分选及冷冻精液制备

为分选梅花鹿XY精子及制备冷冻精液,2006年9月21~29日,对电刺激采集的梅花鹿精液经过长距离运输,流式细胞仪分离,冷冻,解冻后精液重分析和品质鉴定。结果显示:常规冻精和X、Y型冻精性别比分别为50%、91.21%和94.08%,常规冻精与X、Y型冻精组间差异显著(P<0.05);活率分别为0.43±0.04、0.45±0.04和0.43±0.03,常规冻精与X、Y型冻精组间差异不显著(P>0.05);存活指数分别为0.25、0.05和0.05,常规冻精与X、Y型冻精组间差异显著(P<0.05);顶体完整率分别为(77.6±1.0)%(、84.6±0.5)%和(82.2±0.6)%常规冻精与X、Y型冻精组间差异不显著(P>0.05)。用流式细胞技术可以分选获得梅花鹿XY冷冻精液,性别比和品质达到低剂量人工授精的要求。     

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 .     

Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric Sorting

The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37 degrees C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37 degrees C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 +/- 6.9% vs 30.3 +/- 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 +/- 5.2%, 36.0 +/- 12.5% and 35.1 +/- 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 +/- 6.3%, 7.8 +/- 4.7% and 7.4 +/- 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 +/- 7.5%, 23.4 +/- 5.4% and 28.8 +/- 6.3% vs 25.9 +/- 14.4%, 38.5 +/- 16.7% and 79.8 +/- 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 +/- 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa.     

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.