精子性别化对奶牛控制的试验

本试验获得牛犊雌性控制率达81．8％（Table1）。十头母牛产犊十一头：二雄,九雄,其中一头产异性双犊。于1．08－1．10蔗糖密度梯度之间,可以清楚地观察到Y精子能被荧光素染色（Table2);H－Y抗血清处理改变Y精子的荧光染爸,说明对Y精子有抑制作用（Table3)。制备有一定纯度和效价的H－Y抗血精IgG用以性别化处理精子;通过人工受精技术、使母牛得到受处理的精子而正常受胎。本试验结果对奶牛性别控制的前景是美好的。     

间接免疫荧光鉴别奶牛胚胎性别的研究

本实验用H—Y抗体的间接免疫荧光方法对奶牛6—7日龄胚胎进行性别鉴别。用数排卵法获得的胚胎,共选37枚在含H—Y抗血清的M2培养液中温育30min,用M2培养液清洗后,在含FITC—荧光标记的羊抗鼠IgG二抗的M2中温育30min,经洗后的胚胎在Nikon倒置荧光显微镜下鉴别有无荧光,结果21枚胚胎有特异免疫荧光,判作雄性胚胎;16枚无特异免疫荧光,判作雌性胚胎,分别占57％和43％。这与自然出生公母犊各约50％和性比率无显著差别(P>0.25),判定为雄性的5枚胚胎移植给3头受体奶牛(每头移植1或2枚)、3枚判定为雌性胚胎移植给2头受体奶牛一头移植一枚,枚、另一头移植2枚)移植后两个月时检查有3头受体怀孕,其中,一头移植有荧光胚胎的受体返植无荧光胚胎的2头受体各产一母犊(体重皆41kg)。     

精子性别化对奶牛性别控制的试验

本试验获得牛犊雌性控制率达８１．８％（Ｔａｂｌｅ１）。十头母牛产犊十一头：二雄,九雌,其中一头产异性双犊。于１．０８－１．１０蔗糖密度梯度之间,可以清楚地观察到Ｙ精子能被荧光素染色（Ｔａｂｌｅ２）;Ｈ－Ｙ抗血清处理能改变Ｙ精子的荧光染色,说明对Ｙ精子有抑制作用（Ｔａｂｌｅ３）．制备有一定纯度和效价的Ｈ－Ｙ抗血清ＩｇＧ用以性别化处理精子;通过人工受精技术、使母牛得到受处理的精子而正常受胎．本试验结果对奶牛性别控制的前景是美好的。     

牛冷冻试管胚胎性别鉴定的应用

利用双引物PCR技术对优质良种牛体外受精的冷冻胚胎进行性别鉴定。并对分割后的半胚分别进行PCR扩增和染色体分析,两种方法的判定结果相吻合,在本实验条件下,利用减少PCR体系中细胞的数量来确定最低的模板DNA的检出量,2－细胞不能检测,4－细胞检出率为62．5％,8－细胞为90％,16－细胞以上为100％。对23牧经过性别鉴定后的胚胎进行移植,获得5头纯种试管牛,产犊率为21．7％,与正常的胚胎移植 结果27．3％（3／11）无显著差异,预测性比为100％符合。     

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。     

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。     

PCR方法用于奶牛早期胚胎的性别鉴定

根据牛Y染色体上的特异DNA序列合成一对引物, 通过PCR反应对奶牛早期胚胎进行性别鉴定。预测性别与移植胚胎产犊的实际性别相符率为80%。 Abstract:We have obtained the specific DNA segment from the bovine Y-chromosome and used it to design a pair of primer.The sex of embryos at the preimplantation stage have determined by using the polymerase chain reaction.10 months after uterine transfer showed that the rate of accuracy is 80%.     

控制动物性别的原理及方法

一、性别控制的基本原理控制动物性别的原理主要是基于动物雌雄生殖细胞,特别是动物精子的差异而讲行的。高等动物的X 型精子与 Y 型精子不论在形态结构、重量密度、表面膜电荷的电量分布及电性,还是精子的抗原免疫性、运动速度和活力以及耐酸碱性等方面都存在较大的差异。(一)精子形态结构的差异哺乳动物分裂中期细胞的性染色体,一般 X 比 Y 要大。如牛的 X 染色体表面积为7.85微米~2,Y 染色体为3.47微米~2,性染色体大小的差异造成了精子形态的差异。X 精子头部大而且比较圆,体和核也较大;Y 精子头部小而且比较尖,体和核比较小。此外,精子的大小还受     

提高精子转染外源DNA效率的山羊精液冷冻方法

目的筛选一种既提高精子转染外源DNA效率,又保持解冻后精子活力的山羊精液冷冻—解冻方法。方法应用正交设计L9(34),因素分别为稀释液种类、稀释比例、降温时间和解冻液,每个因素选择3个水平,检测和比较解冻后精子转染外源DNA效率和精子活力。结果所选冷冻—解冻各因素对精液转染效率影响不显著[F(8,18)=1.032,P=0.449];平衡时间对冷冻—解冻活力影响极显著[F(2,24)=9.972,P=0.001],平衡1h极显著小于平衡2 h和4 h的精液活力(P=0.003,P=0.000),以平衡4 h最好。用筛选的冷冻—解冻方法处理精液,解冻后精子的活力极明显降低(P=0.002);生存指数降低,GOT释放量增加,菌落数减少,与鲜精相比差异显著(P=0.018;P=0.016;P=0.018);精子畸形率增加,顶体完整率降低,与鲜精相比差异不显著(P=0.494;P=0.084)。结论优化了提高精子转染外源DNA效率的山羊精液冷冻—解冻方法。     

两温度梯度多重PCR鉴别牛早期胚胎性别的技术

稳定、可靠和快速的牛胚胎性别鉴定方法在生产应用中具有重要意义.通过两温度梯度PCR方法对牛基因组、克隆胚胎、胚胎样品进行性别鉴别实验研究,建立了稳定、简便、快速的牛早期胚胎性别鉴别两温度梯度PCR方法,鉴定时间仅为57分钟.采用两温度梯度PCR方法对30枚奶牛胚胎进行了早期性别鉴别,并将鉴别的15枚胚胎(11枚为雌性,4枚为雄性)移植到同期处理的15头受体母牛体内.60天后妊娠检查,有7个受体成功受孕,5头受体怀孕晚期流产,流产犊牛全部为母犊.结果产下1公1母两头犊牛,流产个体与出生个体的性别与PCR鉴别结果完全相符.     

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley-Liss, Inc.     

Artificial insemination of Reindeer (Ragifer tarandus)

Semen collected from reindeer bulls in an artificial vagina was used for the artificial insemination of 16 reindeer cows: two with undiluted semen, five with diluted semen and nine with frozen semen. The two cows which received undiluted semen gave birth to normal calves 217 days later. The other cows returned to oestrus 23.5 days (mean) after insemination and subsequently calved, having been served by a fertile bull.     

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study

A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Low dose insemination in cattle with the Ghent device

A new artificial insemination device for semen deposition near the uterotubal junction (UTJ) in cattle (Ghent device) was developed at Ghent University (Belgium). In this study, UTJ insemination of dairy cows with the Ghent device was compared with the conventional insemination technique to evaluate the effect on pregnancy rates after insemination with different doses of semen. In each of three field trials, the cows (n=795, 659, 360) and heifers (n=253, 182, 231) were randomly assigned to receive 12 million sperm deposited in the uterine body using conventional techniques (control) or a reduced sperm dose (RSD) deposited in the same manner as the control or bilateral deposition near the uterotubal junction using the Ghent device (Ghent). Sperm dosages for RSD and Ghent inseminations were 8, 4, and 2 million sperm for field trials 1-3, respectively. In the multivariable analysis, the pregnancy rates were significantly affected by the parity of the cow (p相似文献   

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。     

In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: preliminary results of artificial inseminations

A semen extender made with low density lipoproteins (LDL) has been used instead of a standard extender that is already available on the market for the cryopreservation of bovine semen. However, in order to extend its use to artificial insemination centres, in vivo fertility studies were required. Semen was taken from three bulls and frozen-thawed in two extenders: the LDL extender and a standard Tris-egg-yolk (20%) extender used by AI centres. The quality of the semen was assessed prior to artificial insemination: motility was assessed using an image analyser (Computer Assisted Semen Analysis (Hamilton Thorne)), and the integrity of the plasma membrane was assessed using the hypo-osmotic test (HOS test). For the first time, gestations were obtained following the artificial insemination of cows in the field (n=193) with semen that had been frozen-thawed in the LDL extender. No significant difference (p>0.05) was detected between the success rates of AI between the semen that had been frozen-thawed in the LDL extender (59.2%) and the control extender, Tris-20% egg yolk (65.3%). In conclusion, the in vivo fertility of semen that has been frozen-thawed in the LDL extender is maintained since gestations are obtained following AI.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.