牛活体采卵技术研究进展

应用活体采卵技术可以从活体母牛反复采集大量系谱明确的卵母细胞,用于胚胎的体外化生产。本文将对牛活体采卵技术的研究进展和操作方法进行综述,分析影响活体采卵效果的因素和存在问题,并论述活体采卵在家畜育种和胚胎生物技术等方面的应用前景。     

影响水牛活体采卵效果的因素分析

水牛活体采卵是继水牛体外受精(IVF)、胚胎移植(ET)等技术之后推出的一项良种胚胎产业化生产的关键技术。本文叙述了水牛活体采卵技术在水牛繁殖中的应用概况,并分析了影响水牛活体采卵回收效果和胚胎生产的各项因素。这套技术可以充分挖掘优良母水牛的繁殖潜力,缩短世代间隔,加速育种进程,同时也能为水牛的胚胎生物工程、基因工程等研究提供大量的试验材料,从而促进水牛科学研究和相关产业的持续发展。     

采卵间隔时间对山羊活体采卵效果的影响

目前从离体卵巢采集卵母细胞是体外受精研究所需卵母细胞的主要来源之一.一般是从屠宰场采集母畜的卵巢,抽取卵巢上的卵母细胞.但我国缺乏良种羊的卵巢资源,故所采卵母细胞种质一般较差,而且其系谱不明.而活体采卵技术(Ovum Pick-up OPU)正是解决当前我国体外受精研究中存在的卵母细胞资源缺乏、系谱不明等问题的有效手段.羊的活体采卵主要有三种方法:常规手术法、B型超声波法和腹腔镜法.腹腔镜法活体采卵(Laparoscopic Ovum Pick-up LOPU)操作相对简便,对羊的伤害较小,可重复采卵;而常规手术法对羊的伤害较大,较难重复采卵; B型超声波法活体采卵技术需要大量的练习才能掌握,可见卵泡数和获卵数较少.因此羊的活体采卵在国外应用最为广泛的方法当属LOPU.Snyder和Dukelow在1974年首先将腹腔镜用于绵羊的活体采卵,获得了6枚卵母细胞,然而当时并没有得到充分的发展,随着胚胎体外生产技术的发展,直到近些年在国外才逐渐得到应用.而在国内,这方面的研究甚少,因此本实验开展了山羊LOPU的探索研究.     

奶牛活体采卵-体外受精效率的影响因素研究

为建立稳定高效的活体采卵-体外受精技术体系,提高体外胚胎生产效率,本研究先利用屠宰场采集的新鲜卵巢卵母细胞进行体外受精,通过胚胎发育潜力来筛选最佳的体外胚胎培养液;再进一步研究不同种公牛精液和供卵母牛对活体采卵-体外受精效率的影响。结果显示,CR1aa培养液和mCR1aa培养液卵裂率差异不显著（P>0.05）,但mCR1aa组的囊胚发育率显著提高（28.1% vs 20.6%,P<0.05）;选取的3头荷斯坦种公牛精液的活体采卵-体外受精胚胎的卵裂率差异不显著（P>0.05）,但1号种公牛精液体外受精后囊胚率（38.7%）显著高于2号和3号（23.8%&22.9%）（P<0.05）;随机选择的3头活体采卵供体母牛（H1、H2、H3）获得的头均可用卵母细胞数无显著差异,但H1和H2供体母牛体外受精胚胎的卵裂率和囊胚率均显著高于H3供体牛（P<0.05）,且H1供体牛体外受精囊胚率显著高于H2供体牛（P<0.05）。结果表明,mCR1aa培养液能显著提高体外受精囊胚发育率,适用于体外胚胎生产;种公牛精液和供体母牛个体差异会直接影响活体采卵-体外受精胚胎的生产效率,为奶牛活体采卵-体外受精生产技术体系的优化提供参考。     

牛活体采集卵母细胞技术研究进展

活体采卵是良种胚胎产业化生产的关键技术,具有生产胚胎成本低、效率高,对供体母畜生产和繁殖性能无不良影响等特点,本文综述了牛活体采卵方法、影响采卵效果因素及存在问题等方面的研究进展,对当前肉牛、奶牛发展具有重要指导意义。     

奶牛非手术受精卵移植试验研究

用 FSH 诱发供体母牛超数排卵,授入冷冻精液后6至8天用两路采卵管进行非手术子宫采卵,用非手术法即将牛用玻璃输精器通过子宫颈将受精卵移植到与供体母牛同期发情的受体母牛子宫内。1978年对26头超排母牛采卵,有17头共采到38个卵,其中受精卵33个。1979年对21头超排母牛采卵,有18头共采到86个卵,其中受精卵83个。1978年将20个受精卵移植给18头受体母牛,4头妊娠,除1头流产外,其余3头于1979年8至9月产下4头健康犊牛,其中一对双胎。胚胎存活率20％。     

牛活体采集卵母细胞技术研究进展

活体采卵是良种胚胎产业化生产的关键技术，具有生产胚胎成本低、效率高，对供体母畜生产和繁殖性能无不良影响的特点。本文综述了牛活体采卵方法、影响采卵效果因素及存在问题等方面内容，并对其前景做了展望。     

影响牛活体采卵效果因素的研究进展

利用活体采卵技术可以连续采卵数周,每周两次,而不影响供体的正常生产,而且几乎在任何体况下都可以采卵,因此,活体采卵是目前唯一一种可以替代超数排卵的技术。目前活体采卵主要用于牛,在羊、马、水牛等体型较大的家畜也有应用,但应用相对较少,在这以牛为例总结一下影响活体采卵的各种因素,主要包括以下四个方面。1供体牛的体况1.1年龄青年母牛(13～15月龄)甚至性成熟前的犊牛(6～9月龄)都可作为OPU的卵母细胞供体,且不会影响这些牛卵巢的功能和以后的妊娠率(Brown等,1996)。年龄对可采卵泡数的影响报道不完全一致。Kruip等(1994)、Bunga…     

水牛活体采卵及体外受精研究

应用B型超声波采卵法进行水牛活体采卵,42头3～17岁良种水牛间隔3 d采卵1次,连续采卵2～3次,共进行96次,穿刺卵泡762个,回收卵母细胞513枚,其中可用卵母细胞305枚,回收率67.3%,可用卵占回收卵总数59.5%,平均每头次获卵母细胞5.34±4.25枚,其中可用卵母细胞3.18±2.89枚.采集的可用卵母细胞以MC和SOF 2种系统培养,受精分裂率和囊胚率分别为45.2%、30.6%和70.8%、18.8%.用8枚胚胎移植受体母牛8头,妊娠2头,产下了世界第1例由沼泽型水牛孕育生产的河流型水牛.     

加拿大太平洋遗传育种中心首批胚胎牛在本溪试验基地诞生

【本刊讯】(记者聂迎利)为了缓解我国良种奶牛缺乏,购买进口奶牛成本高等问题,加拿大太平洋遗传育种中心于2009年在辽宁省本溪市山区建立了一个奶牛良种繁育试验基地。该基地占地40余亩,实验和养殖设施齐全;新建了四栋牛舍;可以饲养育成牛、成母牛和犊牛400余头。试验基地于2010年起成功地引进了胚胎性别鉴定、性控胚胎生产、胚胎移植、活体采卵和体外受精等技术,开     

羔羊卵泡诱导发育、活体采卵和体外发育潜能研究

本研究旨在探讨聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)作为缓释剂在羔羊卵泡诱导发育中的应用效果,以及品种、年龄、激素剂量和超排次数对活体采集卵母细胞数量和体外发育能力的影响。选用4~12周龄无角道塞特、萨福克母羔120只,分为15%PVP一次肌注组、30%PVP一次肌注组和常规递减注射组,每组40只,注射剂量均为120 IU,对30%PVP一次肌注组按品种、周龄、激素剂量和重复次数分为3~4小组,分别进行卵泡诱导发育和活体采卵,并与屠宰厂采集的卵母细胞对比进行体外受精和胚胎移植。结果表明,采用30%PVP缓释FSH进行羔羊超排,可以起到与常规超排一致的效果,二者差异不显著(P>0.05),但采用15%PVP缓释FSH进行羔羊超排没有取得理想的效果,在各项指标上均显著低于30%PVP组和常规递减注射组(P<0.05);采用30%PVP缓释FSH分别超排道塞特和萨福克羔羊,只均获卵数和可用卵数差异不显著(P>0.05),4、6和8周龄羔羊只均获卵数和可用卵数显著高于12周龄羔羊组(P<0.05),40 IU组只均获卵数显著低于80、120和240 IU剂量组(P<0.05),240 I...     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

Clinical Application of in Vitro Embryo Production in the Horse

The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor mares (historically <25%; now >50%) and in the successful culture of zygotes to the blastocyst stage in vitro (historically <10%; now >20%). It has also been established that immature oocytes can be “held” at room temperature for at least 24 hours, allowing overnight transport to a laboratory with expertise in IVEP. Moreover, because in vitro–produced embryos can be cryopreserved with no appreciable reduction in viability, they can be shipped and stored until a suitable recipient mare is available for transfer. Most importantly, in an established equine ovum pick-up intracytoplasmic sperm injection (OPU-ICSI) program, blastocyst production rates now exceed 1 per procedure, and posttransfer foaling rates exceed 50%, such that overall efficiency betters that of either embryo flushing or oocyte transfer. Moreover, OPU-ICSI can be performed year round and allows embryo production from mares with severe acquired subfertility and extremely efficient use of scarce or expensive frozen semen. Cumulatively, these factors have stimulated rapid growth in demand for IVEP among sport horse breeders.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.     

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.     

牛胚胎的体外生产技术

应用体外受精技术，可以有效地利用淘汰的高产奶牛和屠宰的青年母牛卵巢中的未成熟卵，大量，廉价地实验室生产胚胎，这对加速我国高产奶牛群和良种肉牛群的繁殖，是一项行之有效的胚胎工程技术，借鉴国外实验室生产牛胚胎的经验和根据我们实验室的实践，本文比较详细地叙述了体外受精牛胚胎（试管牛）的生产过程，包括卵母细胞的获得，培养，精子获能，体外受精以及受精后的胚胎培养，超低温冷冻保存与解冻后的胚胎移植等。     

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.     

谷氨酰胺和IGF-I对晋岚绒山羊卵母细胞体外成熟的影响

为了探讨在成熟培养液中添加谷氨酰胺和IGF-I对晋岚绒山羊母羔卵母细胞体外成熟的影响。随机选择12只6-8周龄晋岚绒山羊母羔为试验动物,用FSH进行超数排卵,活体采集卵母细胞,并对未成熟的卵母细胞进行体外成熟培养。在培养液中添加0、50、100和150μg/mL的谷氨酰胺及0、25、50和100 ng/mL的IGF-I。结果表明：培养液中谷氨酰胺添加量为100μg/mL时,能显著提高卵母细胞成熟率（P〈0.05）;在培养液中添加100μg/mL谷氨酰胺的基础上,再添加50 ng/mL的IGF-I可以进一步显著提高卵母细胞的成熟率（P〈0.01）。