犬科动物和猫科动物繁殖生物技术现状

家猫和家犬主要是作为伴侣动物。在一些研究机构和私人公司都建立了犬科动物精子库( Farstad,1 996) ,在猫上则仅限于研究机构的实验室之间的动物交换 ,冷冻精液的商业交易还很少。通过辅助繁殖技术保持野生犬科动物的数量 ,还没有引起足够的重视。因为大多数的犬科动物在野生或圈养状态下都能够很好地繁殖 ,而繁殖生物技术又面临着一些问题 ,尤其是有关配子和胚胎的体外模型问题。36种野生猫科动物中的大多数被划分为受威胁的、易濒危的和濒危的 3种类型 ( Nowell等 ,1 996)。在野生猫科动物中 ,通过建立保护计划等生物技术研究正进展顺利…     

犬卵母细胞体外成熟及其体外受精研究进展

犬科动物胚胎对于改进犬科珍贵物种及保护濒危犬种十分重要,因此对犬科动物体外受精(IVF)以及辅助生殖(ART)的研究逐渐变得必要.通过体外受精和核移植已经成功获得能够发育的犬科动物胚胎,克隆的犬胚胎移植到受体母犬能成功产仔,但是这一技术的效率仍然很低.主要原因是犬科动物胚胎在体外发育能力很低.犬卵母细胞早期发育和其他动物不同,犬卵巢排出的卵母细胞处于生发泡(GV)期,在输卵管中恢复减数分裂.犬卵母细胞核难以观测并且不容易确定排卵时间,所以很难确定精子入卵的确切时间,并且多精受精的情况时常发生.回顾犬卵母细胞体外成熟(IVM)和体外受精取得的进展,对于提高其胚胎生产效率很有必要.     

猪卵母细胞体外成熟体系研究简述

随着胚胎生物技术的日臻完善并逐步应用于家畜的育种和繁殖领域,人们对动物胚胎工程的认识逐渐加深,一系列更深层次的胚胎生物技术越来越受到人们的重视。20世纪80年代以来,人们在家畜卵母细胞体外培养、体外受精、     

胚胎工程技术在畜牧业上的应用

胚胎工程或叫胚胎技术 ,是以胚胎移植为基础 ,以转基因动物为中心 ,通过人工操作胚胎 ,定向改变动物性状使之为人类造福的一系列生物技术的总称。主要包括 :胚胎移植、体外胚胎生产 (体外成熟、体外受精和胚胎培养 )、胚胎冷冻、胚胎分割、动物克隆、胚胎性别鉴定、胚胎嵌合、显微受精和转基因动物技术等。1　目前可以在畜牧业上应用的技术胚胎移植、体外胚胎生产 (体外成熟、体外受精和胚胎培养、胚胎冷冻 )、胚胎分割等胚胎工程技术在国外已经商业化 ,牛和羊的胚胎移植技术在我省的畜牧业上的应用前景最大。要开展胚胎移植技术 ,还需要开…     

动物繁殖技术的新进展

动物繁殖技术是生物技术的重要分支。传统的生物技术革新换代是从20世纪70年代初期开始的。分子生物学的某些突破使人们能够分离基因,并在体外进行重组。这些突破迎来了生物技术的时代。作为繁殖技术从20世纪50年代人工授精技术到70年代胚胎移植技术的应用,似乎才真正开始动物繁殖技术的新纪元。本文在这里集中阐述了人工授精技术(AI),发情控制,体外受精,性别控制,胚胎低温保存,无性繁殖(克隆),胚胎分割和融合技术7个方面的动物繁殖技术的进展。1家畜的人工授精(AI)人工授精是家畜繁殖应用最为广泛的一项技术。近年来,人工授精头数逐年增…     

狐狸胚胎生物工程研究进展

胚胎移植及其它生物技术研究已广泛涉及到各种哺乳动物,该技术应用于狐狸等毛皮动物上,利用胚胎生物技术可获得较高经济价值,另一方面,可将种间胚胎移植等生物技术应用到濒危动物上,为拯球和保护濒危动物提供可借鉴的模式。本文主要综述了狐狸胚胎生产过程中的超数排卵、胚胎收集和涉及体外受精技术的卵母细胞体外成熟培养、体外受精、胚胎体外发育培养以及胚胎移植和胚胎冷冻技术等,探讨狐狸胚胎生物工程的研究现状和应用前景     

犬繁殖生物技术概述

繁殖生物技术,亦称人工辅助繁殖技术(Assisted repro- ductioive technique;ART)。人工授精(Artificial insemination;AI)、胚胎移植(Embryo transfer,ET)以及所有围绕配子和胚胎进行的操作技术都属此范畴。本文对犬的繁殖生物技术进行概述,主要集中在胚胎、精液冷冻保存、人工授精(AI)、卵母细胞体外成熟(In vitro maturation;VM)、体外受精(In vitro     

繁殖生物技术进展与我国犬繁殖技术研究

繁殖生物技术,亦称人工辅助繁殖技术(ART)。人工授精、胚胎移植以及所有围绕配子和胚胎进行的操作技术都属此范畴。本文简述犬的ART的进展,主要集中在人工授精、卵母细胞体外成熟、体外受精、胚胎体外培养、胚胎冷冻保存和胚胎移植等方面,并对今后的发展,尤其是对我国犬的繁殖技术研究和应用,提出一些观点和想法。一、犬繁殖生物技术的进展和现状(一)精液冷冻和人工授精第一次有关犬的精液处理和短期保存研究出现在1960年。此后,逐渐开发和改进了犬精子冷冻保存的方法。1969年,第一批冷冻精液人工授精犬仔在美国诞生。至今,在世界范围内…     

低氧分压通过低氧诱导因子(HIF)影响卵母细胞的体外成熟和早期胚胎的发育

卵母细胞和早期胚胎的体外培养(IVC)是哺乳动物胚胎工程的一项关键技术,是生殖生物学和转基因与克隆技术等生物技术研究的基础.影响卵母细胞和早期胚胎体外培养的因素包括培养液组成成分、离子浓度和渗透压,培养环境气相组成和温度等.最近研究表明,培养系统中的氧分压可以显著影响胚胎的发育,而这种影响主要由低氧诱导因子(hypoxia-inducible factor,HIF)进行调控.作者综述了氧分压及HIF对卵母细胞和早期胚胎体外发育的影响,并对HIF的结构及调控机制进行讨论,为研究低氧培养技术在卵母细胞和早期胚胎体外培养中的应用提供依据.     

褪黑素清除氧化/硝化应激对配子和体外胚胎生产的影响

本文从活性氧(ROS)和活性氮(RNS)在体外胚胎培养过程中对胚胎氧化应激和硝化应激的影响及添加不同浓度的褪黑素与胚胎共孵育过程中具有有害或有利作用,研究褪黑素对人类和其它哺乳动物不同体外受精步骤的影响因素,从而更好地理解,特别是神经—激素系统高度协调统一的季节性繁殖动物生殖生理学原理。     

Selected Aspects of Advanced Porcine Reproductive Technology

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.     

Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus)

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild‐derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non‐reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non‐reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2‐ and/or 4‐cell stage using both fresh and frozen‐thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non‐reproductive seasons. This study is the first to develop IVF–IVC wild large Japanese field mice beyond the 2‐ and/or 4‐cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.     

利用JIVET技术快速繁育萨福克羊的研究

JIVET技术是一项利用生殖激素刺激幼畜卵巢,从而使其快速、高效生产卵母细胞的技术。对4只1月龄萨福克幼羔注射FSH和PMSG激素,48 h后采用手术法抽取幼羔卵母细胞,并在体外成熟;将成熟的卵母细胞与精子进行体外受精,将正常分裂的2~8细胞胚胎移植到6只同期发情的受体小尾寒羊子宫内。结果显示,采用激素法处理萨福克幼羔后共采集到可用卵母细胞109枚,体外成熟培养后得到成熟卵母细胞79枚,成熟率为72.48%;体外受精后得到正常卵裂胚胎34枚,卵裂率为43.04%;胚胎移植后有3只受体母羊怀孕,受胎率为50%,并分娩得到3只萨福克幼羔。综上提示,利用JIVET技术可以缩短萨福克羊的繁育周期,从而加快其繁育速度。     

Embryo Production in Dogs: from In Vitro Fertilization to Cloning

Increased availability of canine embryos would be desirable to develop research and to apply assisted reproductive technologies in the treatment of infertility and in the improvement of reproductive performances in valuable Canids, both domestic and non-domestic. Embryo production through in vitro fertilization and nuclear transfer has been technically achieved in the dog, and the transfer of cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. This is mainly because of the peculiar characteristics of the canine oocyte and the lack of its full acquisition of developmental competence in vitro. This paper discusses the latest results and aspects on which further research should be focused to provide advances in the field.     

Expression of the Orexigenic Peptide Ghrelin and the Type 1a Growth Hormone Secretagogue Receptor in Sheep Oocytes and Pre-implantation Embryos Produced In Vitro

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro . Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.     

Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.     

Developmental competence of porcine blastocysts produced in vitro

The establishment of in vitro embryo production (IVP) system in pigs enables us to generate viable embryos with a quality equal to that of in vivo derived embryos. This technology contributes not only to developments in reproductive physiology and agriculture but also to biotechnologies for producing cloned or genetically modified pigs. The birth of piglets from in vitro matured and fertilized embryos at the two- to 4-cell stage was first achieved about 10 years ago, but it was only quite recently that piglets were produced after the transfer of IVP blastocysts. This improvement to the blastocyst stage of the in vitro culture system after in vitro maturation and fertilization can be expected to play a part in the development of an advanced IVP system. Here, we discuss the developmental ability of porcine embryos produced by our improved IVP system and the utilization of this technique in the new biotechnology.