CN101797189A - Apparatus for collecting early-stage embryo or early-stage oocyte of pig and collecting method - Google Patents

Apparatus for collecting early-stage embryo or early-stage oocyte of pig and collecting method Download PDF

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CN101797189A
CN101797189A CN 201010112198 CN201010112198A CN101797189A CN 101797189 A CN101797189 A CN 101797189A CN 201010112198 CN201010112198 CN 201010112198 CN 201010112198 A CN201010112198 A CN 201010112198A CN 101797189 A CN101797189 A CN 101797189A
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egg
end
collecting
tubal
early
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CN101797189B (en )
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乔宪凤
刘西梅
华再东
华文君
李莉
熊忠良
程妮
肖红卫
魏庆信
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湖北省农业科学院畜牧兽医研究所
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Abstract

The invention relates to an apparatus for collecting early-stage embryo or early-stage oocyte of a pig and a collecting method. The apparatus comprises an ovum collecting apparatus and an ovum collecting pipe, wherein the ovum collecting apparatus is a soft colloid pipe with the length of 200-300mm, the outer diameter of 1.0-1.1mm and the pipe wall thickness of 0.08-0.1mm, one end of the soft pipe is a No.9-12 pinhead and the other end is connected with a syringe; and the ovum collecting pipe has the length of 130-140mm, the wall thickness of 0.8-1.1mm and the inner diameter of 3-4mm with an angle of 120-140 degrees bending in the middle. When collecting early-stage embryo or early-stage oocyte of a pig, firstly, one end of the ovum collecting pipe is inserted into the fallopian tube for 10-15mm from fimbriae opening of the fallopian tube, and the other end is connected with an ovum collecting container; then the syringe sucked with 15-20ml of ovum collecting solution and 5-10ml of air is connected with the ovum collecting apparatus, the pinhead of the ovum collecting apparatus is inserted into the fallopian tube for 5-8mm from the uterine horn end of the junction of the uterine and the pipe, and then ovum collection is carried out. The invention obviously improves the success ratio and the efficiency of the collection.

Description

采集猪早期胚胎或卵母细胞的器械及采集方法 Collected early embryo or pig apparatus and method of collecting oocytes

技术领域 FIELD

[0001] 本发明涉及一种用于采集猪早期胚胎或卵母细胞的器械及采集方法,属于动物胚胎工程领域。 [0001] The present invention relates to a method for the early embryo or pig collection devices and methods of oocyte collection, it is in the field of animal embryo engineering.

背景技术 Background technique

[0002] 猪早期胚胎的采集和移植最早于1951年由Kvashickii报道。 [0002] pig embryos early in the acquisition and transplantation was first reported in 1951 by the Kvashickii. 到20世纪60年代, 已经基本建立了外科手术方法采集和移植胚胎的技术。 To the 1960s, it has been basically established surgical method of collection and embryo transplant technology. 到70年代以后,北美和欧洲开始将采集和移植胚胎的技术应用于向闭锁猪群引进外血和国际间的引种。 To 70 years later, in North America and Europe began the collection and embryo transplant technology to introduce foreign blood and introduction to international lockout herd. 80年代转基因动物技术的出现,使这项技术的应用得到扩展。 80s transgenic animal technology, the application of this technology expanded. 1985年Hammer等人,首先用手术的方法采集猪的受精卵,然后将人生长激素基因应用显微注射技术注入猪的受精卵原核,再将其移入母猪的输卵管,得到转基因猪。 Hammer et al., 1985, with the first collection of fertilized pig surgical methods, and then the application of human growth hormone gene microinjected pronuclear injection technique pigs, and then move it into the oviduct of a sow, to obtain transgenic pigs. 这之后,向受精卵内注射外源基因,便成为制备转基因猪的主要方法之一。 After this, the injection of exogenous genes zygote, became one of the main methods of making a transgenic pig. 此外,用病毒感染法制备转基因猪,也需要首先采集猪的早期胚胎,然后将连接有外源基因的病毒载体注入早期胚胎,生产转基因猪。 In addition, infected with virus prepared transgenic pigs also need to collect early embryo pigs, and then connected to an exogenous gene viral vector injection early embryo, the production of transgenic pigs.

[0003] 猪卵母细胞的采集主要应用于通过体外受精技术制备“试管猪”,通过核移植技术制备“克隆猪”,通过核移植技术和转基因技术相结合制备“转基因克隆猪”和其它遗传修饰的猪。 [0003] Acquisition porcine oocyte preparation technique is mainly used in vitro fertilization "test tube pig", prepared by nuclear transfer "cloning pig", by nuclear transfer and transgenic technology combined preparation "transgenic cloned pig" by genetic and other modified pigs.

[0004] 应用体外受精技术生产出“试管猪”最早由Cheng和Polge于1983年报道,由此建立的猪胚胎体外生产技术被世界上其它研究和生产机构所应用,并得以发展。 [0004] application of in vitro fertilization technology to produce a "test-tube pigs" was first reported by the Cheng and Polge in 1983, thereby establishing a pig embryo in vitro production techniques were applied and other research institutions in the world production, and to develop. 实施猪体外受精,首先要获得猪的卵母细胞。 IVF pig embodiment, first obtain pig oocytes. 其卵母细胞的来源有二:一是体外成熟的卵母细胞,从卵巢上采集未成熟的卵母细胞,经体外培养成熟后,用于体外受精;二是体内成熟的卵母细胞。 Source of oocytes are twofold: First vitro maturation of oocytes, immature oocytes collected from ovary, mature cultured in vitro for in vitro fertilization; Second vivo matured oocytes. 本发明用于猪体内成熟卵母细胞的采集。 The present invention for collecting vivo matured porcine oocytes. 成熟的卵母细胞在体外的适宜环境下与获能的猪精子结合,即为体外受精卵;再于体外的适宜环境下培养成早期胚胎,移植给受体母猪,怀孕后产仔,即为“试管猪”。 Mature oocytes were incorporated in vitro in a suitable environment porcine sperm capacitation, namely in vitro fertilized eggs; into early embryos cultured in vitro under appropriate environment, to the transplant recipient sows after farrowing pregnancy, i.e., to "test-tube pigs."

[0005] 应用胚胎细胞作为核供体获得“克隆猪”最早由Polejaeva和Onishi于1989年报道,应用体细胞作为核供体获得“克隆猪”则最早由Prather和Onishi于2000年报道。 [0005] application of embryonic cells as nuclear donors was "cloned pig" was first reported by the Polejaeva and Onishi in 1989, as the application of somatic cell nuclear donor was "cloned pig" is first reported by the Prather and Onishi in 2000. “克隆猪”的制备,首先对成熟的卵母细胞进行去核,然后将分离的胚胎细胞或体细胞移入去核的卵母细胞,经电融合(或其它方法的融合)处理,体外培养,再移入母猪的生殖道内,生产出来的仔猪即为“克隆猪”。 Preparation of "clone pig", First matured oocytes enucleated, then the isolated embryonic or somatic cells into enucleated oocytes by electrofusion (or fused other methods) treated in vitro, then into the sow reproductive tract, piglets produced is "cloned pig." 转基因克隆猪的制备,需首先对体细胞进行基因转化,然后用转基因的细胞做为核供体,获得转基因克隆猪。 Preparation of transgenic cloned pigs, need to hold genetic transformation of somatic cells, and then used as nuclear donor cells transfected with the gene, the genetic engineering pigs. Park等人(2001,2002)用转染绿色荧光蛋白基因的成纤维细胞和耳上皮细胞做核供体,分别得到转基因克隆猪。 Park et al. (2001, 2002) were transfected with green fluorescent protein gene ear fibroblasts and epithelial cells do nuclear donor, respectively transgenic cloned pigs. 克隆技术和转基因克隆技术是猪遗传改良最具前景的手段。 Cloning and transgenic cloned pigs genetically modified technology is the most promising means. 制备“克隆猪”和“转基因克隆猪”,其卵母细胞的来源与体外受精一样,有体内成熟和体外成熟两种。 Preparation "Cloning pig" and "transgenic cloned pig", which is the source of oocytes and in vitro fertilization, as there are two kinds of maturation in vivo and in vitro maturation. 实验表明,用体内成熟的猪卵母细胞做核受体,比体外成熟的卵母细胞有较高的成功率。 Experiments show that, to do with nuclear receptors in vivo matured porcine oocytes, in vitro matured oocytes than the cells have a higher success rate.

[0006] 我国最早建立猪早期胚胎和卵母细胞采集技术的是湖北省农科院魏庆信等人, 1989年他们将采集的猪早期胚胎移入受体母猪的输卵管内,得到胚胎移植的后代;这之后,将十余种外源基因导入猪的受精卵,得到了相应整合并表达的转基因猪;赵浩斌等人1997年将早期胚胎的卵裂球移入去核卵母细胞,得到核移植后代。 [0006] China's first porcine early embryos and oocytes collection technology is the Hubei Academy of Agricultural Sciences Weiqing Xin et al., In the early 1989, they will be collected pig embryos into recipient sows fallopian tubes to obtain offspring embryo transfer; after this, the dozen foreign gene into fertilized eggs of pigs, transgenic pigs have been integrated and expressed in the appropriate; Zhaohao Bin et al. 1997 the early embryo blastomeres into enucleated oocytes, nuclear transfer to obtain progeny. 中国农科院畜牧研究所冯书堂等人,于1990年建立了猪早期胚胎的采集和移植技术,并得到胚胎移植的仔猪。 Chinese Academy of Agricultural Sciences Institute of Animal Husbandry Pingshu Tang et al., Was established in early 1990, the pig embryo collection and transfer technology, and with piglets embryo transfer. 进入21世纪以来,中国农业大学、东北农业大学、上海农科院畜牧兽医研究所等单位,应用核移植和转基因技术,获得克隆猪或转基因克隆猪。 In the 21st century, China Agricultural University, Northeast Agricultural University, Shanghai Institute of Animal Science Academy of Agricultural Sciences and other units, the application of nuclear transfer and transgenic technology, cloning to obtain transgenic cloned pigs or pig.

[0007] 国内外采集猪早期胚胎和体内成熟卵母细胞已有技术的特征是:从输卵管冲卵时,注射器与针头直接相连(中间没有软管连接),针头从宫管结合部的输卵管端插入输卵管;集卵管一般是直的、没有弯曲。 [0007] collecting domestic and porcine embryonic and mature oocytes in vivo prior art wherein: tubal from egg punch, with the syringe needle directly connected (with no hose connection), from the needle end of the fallopian tube binding portion Palace insert oviduct; tubal set generally straight, not curved. 该技术的缺陷主要有三点: The disadvantage of this technique three main points:

[0008] 1、在冲卵过程中,推动注射器时,由于注射器与针头是硬性连接,操作者稍有抖动或猪稍有騷动,针头容易划破、划穿或脱出输卵管,从而造成输卵管出血或冲卵失败。 [0008] 1, the process of the eggs in the red, the syringe is pushed, since the syringe is rigidly connected with the needle, the operator slightly shaking slightly commotion or pigs, the needle is easy to cut, scratch, or extrusion through the fallopian tube, thereby causing bleeding tubal or red eggs fail.

[0009] 2、由于针头从宫管结合部的输卵管端插入输卵管,一是容易出血;二是容易刺穿输卵管;三是如果第一次冲卵失败,不能再从相同的位置第二次插入针头,需重新选择一个部位插针,从而增加输卵管的伤口,也不容易找到合适的部位。 [0009] 2, since the end of the needle is inserted into the fallopian tube from the fallopian tube binding portion of the house, one easy bleeding; Second readily pierceable oviduct; Third, if the first punch egg fails, the same can not be inserted from the second position needles, pins need to re-select a site, thereby increasing the wound fallopian tube, not easy to find a suitable site.

[0010] 3、由于集卵管是直的,集卵皿摆放的位置受到术部狭小的局限,接卵时稍有不慎, 或猪稍有騷动,冲卵液容易流到集卵皿之外,或集卵皿内的冲卵液泼洒,造成冲卵率降低或冲卵失败。 [0010] 3, since the set of fallopian tube is straight, its placement dish eggs set by the limitations of a small portion of patients, then a slight mistake when egg, slightly commotion or pigs, readily flowing liquid egg punch set eggs outside the dish, washed eggs or egg collection liquid in the dish spill, resulting in reduced red or red eggs in egg fails.

发明内容 SUMMARY

[0011] 本发明的目的在于提供一种用于采集猪早期胚胎或卵母细胞的器械及采集方法, 能有效地克服已有技术的缺陷,大大提高采集的成功率,获卵率可达95%以上,且操作方便、安全。 [0011] The object of the present invention is to provide a method for defects early embryo or pig apparatus and method of collecting oocytes collection, can effectively overcome the prior art, greatly improve the success rate of the collected oocytes reach 95 % or more, and is easy to operate and safe.

[0012] 本发明的技术方案是:采集猪早期胚胎或卵母细胞的器械,其特征在于:包括冲卵器和集卵管;冲卵器是长200-300mm、外径1.0-1. 1mm、管壁0. 08-0. Imm的胶质软管,软管的一端为9-12号针头,软管的另一端为与普通注射器连接的接口; [0012] aspect of the present invention is: collecting oocytes or embryonic porcine instrument, characterized by: a punch and a set of tubal eggs; red egg is 200-300mm long, an outer diameter of 1.0-1 1mm. ., 0. 08-0 Imm wall gum hose, one end of the hoses 9-12 needle, the other end of the hose is connected to an interface of an ordinary syringe;

[0013] 集卵管是长130-140_、壁厚0. 8-1. 1_、内径3-4mm的管,中间弯成120° -140° 角(软管可以不用弯曲),两端烧制、研磨平滑。 [0013] is set long 130-140_ tubal wall thickness of 0. 8-1. 1_, 3-4mm internal diameter tube, an intermediate bent 120 ° -140 ° angle (the hose can not bend), both ends of the firing polishing smooth.

[0014] 如上所述的采集猪早期胚胎或卵母细胞的器械,其特征在于:集卵管是玻璃管或其它质地的管(如塑料管、胶管等),两端烧制、研磨平滑。 [0014] As described above acquisition porcine early embryos or oocytes apparatus, wherein: a set of glass or other tubal texture tube (e.g., plastic tubes, hoses, etc.), both ends of the firing, grinding smooth.

[0015] 采集猪早期胚胎或卵母细胞的方法,母猪麻醉保定后手术切口,将输卵管和卵巢引出切口之外;其特征在于:首先将集卵管的一端从输卵管伞口插入输卵管10-15mm,另一端接集卵皿;然后将吸有15_20ml冲卵液、5-10ml空气的注射器与冲卵器连接,冲卵器的针头从宫管结合部子宫角端插入输卵管5-8mm ;再实施冲卵。 Addition [0015] The method of collecting the early embryo or pig oocytes, sows after anesthesia Baoding incision, the fallopian tubes and ovaries lead incision; wherein: the first end is inserted into the fallopian tubal set tubal port from 10- 15mm, set the other end of the egg dish; then blotted 15_20ml red liquid egg, 5-10 ml of air with a syringe connected egg punch, punch needle inserted into the egg's fallopian tube from the uterine horns Palace binding end portion 5-8mm; then the implementation of red eggs.

[0016] 本发明的有益效果是:本发明有效地克服了已有采集猪早期胚胎及体内成熟卵母细胞技术的缺陷: [0016] Advantageous effects of the present invention are: the present invention effectively overcomes the deficiencies of the prior acquisition and early embryonic maturation in vivo pig oocytes art:

[0017] (1)由于冲卵器为胶质软管,向输卵管插入针头时,克服了与注射器直接连接的局限,操作自如,能将针头确实插入输卵管的内腔;在冲卵过程中,即使操作者稍有抖动或猪稍有騷动,由于软管的缓冲作用,也不会出现针头划破、划穿或脱出输卵管的现象,从而大大提高冲卵的成功率。 [0017] (1) The egg is punched glial hose, the tubal insertion needle, the syringe overcomes the limitations of direct connection, operating freely, the needle can indeed inserted into the lumen of the fallopian tube; eggs in the process of punching, even if the operator or a slight jitter slightly pig commotion, due to the cushioning effect of the hose, it will not appear needle pierced, wear or scratch fallopian tube prolapse phenomenon, thus greatly improving the success rate of red eggs.

[0018] (2)由于冲卵器的针头从宫管结合部子宫角端插入输卵管,可有效地避开血管,不引起输卵管出血;也不会刺穿输卵管;如果第一次冲卵失败,由于子宫角端面积较大,很容易重新选择插针的部位,不造成输卵管的创口。 [0018] (2) Since the needle punch egg uterine horn's end into the fallopian tube from the tube coupling part house, can effectively avoid blood vessels, fallopian tubes does not cause bleeding; not pierce the oviduct; egg fails if the first punch, due to the large area of ​​the uterine horn end, it is easy to re-select pin parts, does not cause wound oviduct.

[0019] (3)由于集卵管有弯曲,集卵皿可摆放的位置增大,局限性小,即使有时接卵不慎或猪稍有騷动,集卵皿可随之移动,不会造成冲卵液流到集卵皿之外,或集卵皿内的冲卵液泼洒的现象。 [0019] (3) Since the collector tubal bent, egg collection dish can be placed in position increases, the limitation of small, sometimes even accidentally, or pigs slightly then stir egg, egg collector dish moves can be followed, not cause flow beyond the red egg to egg dish set, collection or red eggs within egg dish liquid spill phenomenon.

附图说明 BRIEF DESCRIPTION

[0020] 图1为本发明实施例冲卵器示意图。 [0020] Fig 1 a schematic view of an embodiment of the present invention is the egg punch.

[0021] 图2为本发明实施例集卵管示意图。 [0021] Figure 2 a schematic embodiment of the present invention set oviduct.

具体实施方式 detailed description

[0022] 以下结合附图和实施例对本发明做进一步的说明。 [0022] The present invention will be further illustrated in conjunction with the accompanying drawings and embodiments.

[0023] 图1中标记的说明:1-针头,2-软管,3-接口。 Description [0023] Figure 1 labeled: 1- needles, hose 2-, 3- interfaces.

[0024] 如图1和图2所示,本发明实施例用于采集猪早期胚胎或卵母细胞的器械,包括冲卵器和集卵管;冲卵器是长200-300mm、外径1. 0-1. 1mm、管壁0. 08-0. Imm的胶质软管2,软管2的一端为9-12号针头1,软管2的另一端为与普通注射器连接的接口3。 [0024] FIGS. 1 and 2, the device for collecting cases early embryo or pig oocytes embodiment of the present invention, comprising a punch and a set of tubal egg; egg punch is 200-300mm long, an outer diameter of 1 . 0-1. 1mm, wall 0. 08-0. Imm glial hose 2, an end of the needle tube 2 is 1, the other end of the hose 2 9-12 interface for connection to an ordinary syringe 3 . 集卵管是长130-140mm、壁厚0. 8-1. 1mm、内径3_4mm的玻璃管(或其它质地的管,如塑料管,胶管等)中间弯成120° -140°角,两端烧制、研磨平滑。 Tubal set long 130-140mm, the wall thickness of 0. 8-1. 1mm, 3_4mm inner diameter glass tube (a tube or other texture, such as plastic tubes, hoses, etc.) is bent intermediate angle of 120 ° -140 °, both ends firing, grinding smooth. 冲卵器冲洗干净后自然干燥,用前在紫外灯下灭菌。 Dried natural egg is washed clean, sterilized before use under UV light. 集卵管冲洗干净后在高压灭菌锅内消毒。 Rinse set tubal sterilization in an autoclave pot.

[0025] 本发明实施例用于采集猪早期胚胎或卵母细胞时,首先将本发明实施例的集卵管的一端从输卵管伞口插入输卵管10-15mm,另一端接集卵皿;然后将吸有15_20ml冲卵液、 5-10ml空气的注射器与本发明实施例的冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管5-8mm ;再实施冲卵。 [0025] When the embodiments of the present invention for collecting porcine early embryos or oocytes, one end of the first set tubal embodiment of the present invention is inserted into the fallopian tube 10-15mm, from the other end of collection dish eggs tubal port; then, there is 15_20ml red absorption egg egg red solution, 5-10 ml of air to the embodiment of the invention the syringe is connected to a needle punch egg's house from the pipe coupling portion is inserted into the uterine horn end fallopian tube 5-8mm; washed eggs another embodiment.

[0026] 具体步骤如下: [0026] The specific steps are as follows:

[0027] (1)采集猪早期胚胎的方法:母猪配种后的24-48小时之内,麻醉保定后手术切口,将输卵管和卵巢引出切口之外;先将集卵管的一端从输卵管伞口插入输卵管10-15mm, 另一端接集卵皿;然后将吸有15_20ml冲卵液、5-10ml空气的注射器与冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管5-8mm ;推动注射器,输卵管内的胚胎与冲卵液一起流经输卵管,从集卵管流入集卵皿。 Method [0027] (1) Acquisition of embryonic swine: within 24-48 hours after the breeding sows were anesthetized Baoding surgical incision, the incision outside the fallopian tubes and ovaries lead; fallopian tube first from one end of the set tubal 10-15mm port into the fallopian tube, the other end of the egg collection dish; then blotted 15_20ml red liquid egg, 5-10 ml of air with a syringe connected egg punch, punch needle 1 is inserted into the egg from the uterine horns pipe end coupling portion Palace tubal 5-8mm; push syringe, tubal embryo flowing through the oviduct was washed with eggs, egg dishes from the incoming current collector fallopian tube. 冲卵完毕,小心将冲卵器和集卵管退出,复原输卵管伞,按常规缝合创口。 Complete egg punch, carefully washed and egg collection exit tubal, tubal rehabilitation, according to conventional suturing wounds.

[0028] (2)采集猪卵母细胞的方法:母猪发情后不配种,待发情结束的12小时之内,麻醉保定后手术切口,将输卵管和卵巢引出切口之外;先将集卵管的一端从输卵管伞口插入输卵管10-15mm,另一端接集卵皿;然后将吸有15_20ml冲卵液、5_10ml空气的注射器与冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管5-8mm ;推动注射器,输卵管内的卵母细胞与冲卵液一起流经输卵管,从集卵管流入集卵皿。 Method [0028] (2) Acquisition porcine oocytes: no estrus after breeding, the outgoing end of the case 12 hours after the surgical incision Baoding anesthesia, the fallopian tubes and ovaries lead outside the incision; first set tubal insert the end of the fallopian tubal opening 10-15mm, set the other end of the egg dish; then washed eggs were blotted 15_20ml, 5_10ml air syringe connected egg punch, punch needle 1 from the egg's house junction pipe uterus 5-8mm angle end is inserted into the fallopian tube; push syringe, and flows through the oviduct oocytes eggs were washed with the oviduct, current flows from the collector tubal egg dish. 冲卵完毕,小心将冲卵器和集卵管退出,复原输卵管伞,按常规缝合创口。 Complete egg punch, carefully washed and egg collection exit tubal, tubal rehabilitation, according to conventional suturing wounds.

[0029] 实施例1优良种猪的胚胎采集和移植 [0029] Example 1 excellent pig embryo collection and transplantation

[0030] 以优良纯种母猪做供体,以不能做种用的杂交母猪做受体,将供体母猪的胚胎移植给杂交母猪受体,目的在于用杂交母猪生产优良纯种猪。 [0030] In doing fine purebred sows donor and hybrid breeding sows do not make the receptor, the donor transplant to the embryo body sows hybrid receptor sows, the pure object of producing excellent hybridization sows pigs. 其操作步骤如下: The steps are as follows:

[0031] 1、供体母猪的超数排卵和配种[0032] 在供体母猪发情周期的第15-18天,肌肉注射孕马血清促性腺激素PMSG1000IU, 72小时后注射绒毛膜促性腺激素HCG 800IU,做超数排卵处理。 [0031] 1, superovulation and mating [0032] Donor sows at 15-18 days of the estrous cycle donor sows, intramuscular pregnant mare serum gonadotropin PMSG1000IU, 72 hours after the injection of human chorionic gonadotropin hormone HCG 800IU, do superovulation. 发情后用本品种公猪配种。 Estrus after breeding boars with this species.

[0033] 2、受体母猪的同期发情 [0033] 2 receptor sows estrus synchronization

[0034] 在受体母猪发情周期的第15-18天,在供体母猪超数排卵处理的同时,肌肉注射PMSG 800IU,72 小时后注射HCG 600IU。 [0034] At 15-18 day estrus cycle receptor, while the number of donor sow super ovulation process, intramuscular PMSG 800IU, 72 hours after injection of HCG 600IU.

[0035] 或者在大群内选择与供体母猪同一天发情的母猪做受体。 [0035] or selected within a large donor sows do the same day estrus sows receptor.

[0036] 3、供体母猪胚胎的采集 [0036] 3, donor embryo collection sows

[0037] 在供体母猪配种后的24-48小时之内,采集胚胎。 [0037] 24-48 hours after breeding sows donor, the collected embryos.

[0038] (1)供体猪的麻醉和保定 Baoding anesthesia and [0038] (1) donor pigs

[0039] 供体猪上腭牵拉,限制活动,按20-30mg/kg (每公斤体重毫克)戊巴比妥钠(2.5%)耳静脉注射,进行全身麻醉。 A [0039] pulling the donor pig palate, restricted activity, according to 20-30mg / kg (milligram per kilogram of body weight) of sodium pentobarbital (2.5%) ear vein injection, general anesthesia. 麻醉到位后仰放在手术保定架上,四肢固定。 Anesthesia place back on the shelves Baoding surgery, limbs fixed. 为防止手术过程中,猪的挣扎、弹动,要用绳索将四肢和头部捆绑牢靠。 To avoid the surgery, the pigs struggling, bouncing, head and limbs to use rope tied securely. 为使腹腔内脏向前倾斜,方便手术,手术台要前低(头低)后高(尾高)。 For abdominal organs tilted forward, easy operation, low operating table to the front (lower head) after high (high tail). 手术过程根据需要肌肉或静脉滴注适量的盐酸氯胺酮。 Ketamine surgical procedures as needed intramuscular or intravenous infusion of an appropriate amount of hydrochloric acid.

[0040] (2)手术部位及其消毒 [0040] (2) and sterilized surgical site

[0041] 用肥皂水和毛刷洗刷腹部和大腿内侧部,水洗后用毛巾揩干净。 [0041] abdomen and thigh portions washing with soap and brush, after washing with a towel wipe clean. 手术部位一般选择在倒数第2、3个乳头之间,先用电剪或毛剪在术部剪毛,应剪净毛茬,用清水洗净术部, 然后涂以2-4%的碘酒,待干后再用70% -75%的酒精棉脱碘,消毒程序是:碘酊棉球一酒精棉球,由内向外;盖创巾于术部,使预定的切口暴露在创巾开口的中部。 Surgical site is generally selected between 2,3 penultimate nipple, the first electric scissors or shearing Clipper intraoperatively portion should cut stubble hair net, washed with water operation unit, and then coated with 2-4% of iodine , until dry and then with 70% -75% alcohol cotton deiodination, disinfection procedures are: an alcohol swab iodine cotton, from the inside out; lid invasive surgical towel section in the predetermined invasive incision exposed towel opening central.

[0042] (3)手术方法 [0042] (3) surgical methods

[0043] 沿腹中线,尽量避开血管,切开皮肤5_8cm左右,沿着切口左右钝性分离脂肪和肌肉层,使腹膜露出后,用钝头剪刀剪开腹膜。 [0043] along the midline, try to avoid blood vessels, the skin incision approximately 5_8cm, blunt separating fat and muscle layer in the lateral cutout, so that the exposed peritoneum, the peritoneum using a blunt scissors. 切开腹膜应避免损伤腹内脏器,先用镊子提起腹膜,在提起部位作一切口,然后用另一只手的食指深入腹膜引导手术刀(向外切口)或用外科剪把腹膜切开或剪开。 Peritoneal incision to avoid damage to intra-abdominal organ, tweezers to lift the peritoneum for lifting cutout portion, and then further guided surgical intraperitoneally with the index finger of the other hand (cut-out), or by surgical shears to cut or intraperitoneal cut. 术者将食指及中指由切口伸人腹腔,在与骨盆腔交界的前后位置触摸子宫角,摸到后用二指夹持,将输卵管和卵巢引出切口之外。 The surgeon by the index and middle fingers extending incision into the abdominal cavity, the uterine horn in touch with the front and rear positions pelvic junction, a finger touch after clamping, the fallopian tubes and ovaries lead outside the incision.

[0044] (4)采集胚胎 [0044] (4) Embryo collection

[0045] 先将集卵管的一端从输卵管伞口插入输卵管15mm,另一端接集卵皿;然后将吸有15ml冲卵液、5ml空气的注射器与冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管5mm ;推动注射器,输卵管内的胚胎与冲卵液一起流经输卵管,从集卵管流入集卵皿。 [0045] The first end is inserted into the fallopian tube set from the fallopian tubal opening 15mm, set the other end of the egg dish; then washed eggs were blotted 15ml, 5ml syringe with an air connection egg punch, needle punching device 1 egg uterine horn from the end of the pipe coupling portion is inserted into the fallopian tube Palace 5mm; push syringe, tubal embryo flowing through the oviduct was washed with eggs, egg dishes from the incoming current collector fallopian tube. 冲卵完毕,小心将冲卵器和集卵管退出,复原输卵管伞,送回腹腔。 Complete egg punch, carefully washed and egg collection exit tubal, tubal recovery, back to the abdominal cavity. 再将另一侧输卵管和卵巢引出切口之外,用同样的方法冲取胚胎。 Addition to the other side of the incision and then lead fallopian tubes and ovaries, in the same manner punch taken embryos. 脏器整理复位,清理切口,按常规缝合创口。 Reset organs finishing, clean incision wound conventionally sutured.

[0046] 冲卵器长250mm、外径1. 0mm、管壁0. Imm ;集卵管是长135mm、壁厚1mm、内径3mm的玻璃管,中间弯成135°角,两端烧制、研磨平滑。 [0046] Chong archegonium length 250mm, an outer diameter of 1. 0mm, wall 0. Imm; tubal set is 135mm long, a wall thickness of 1mm, 3mm inner diameter of the glass tube, bent at an intermediate angle of 135 °, the firing ends, polishing smooth.

[0047] 4、胚胎的收集及质量检查 [0047] 4, and embryos collected QA

[0048] 将集卵皿置于实体显微镜下,用低倍镜的大视野在冲卵液中找到胚胎。 [0048] The set eggs dish was placed under a stereoscopic microscope, a large field low magnification to find the egg embryos in the red liquid. 猪胚胎直径为150 μ m左右,肉眼观察只有针尖大小,为一球形体,在镜下呈圆形,其外层是透明带, 它在冲卵液内的折光性比其他不规则组织碎片折光性强,色调为灰色。 Porcine Embryonic diameter of about 150 μ m, only the tip size was visually observed as a spherical body, a circular shape in the microscope, with the outer layer is transparent, it is within the egg punch refraction than the other irregular tissue fragments liquid refractive strong tone gray. 拣卵管先吸入少许磷酸缓冲液PBS再吸入卵,在集卵皿的不同位置冲洗卵3-5次。 Picking tubal first few suction phosphate buffer PBS rebreathing egg, egg rinsed 3-5 times at different positions set egg dish. 依次在第二个集卵皿内重复冲洗,然后把全部卵集中到一个集卵皿,一只供体的卵放在一个皿内。 Sequentially rinsed repeatedly in the second set of eggs dish, and then concentrated to a set of all eggs egg dish, an egg donor body is placed in a dish. 操作室温为20-25°C。 Operation at room temperature is 20-25 ° C. [0049] 将体视镜的放大倍数调至80-100倍,观察胚胎的形态、卵裂球大小与均勻度、色泽、细胞密度、与透明带间隙以及细胞变性等情况。 [0049] The stereoscope is adjusted to 80-100 times magnification, observation of the embryo morphology, blastomere size uniformity, color, cell density, and the transparent band gap and cell degeneration and so on. 正常发育的胚胎,其卵裂球一般外形整齐清晰,大小一致,分布均勻而紧密,发育速度与胚胎日龄相一致。 Normal embryonic development, blastomere which generally neat appearance clear, uniform size, distribution and close, the embryo development rate consistent with age. 对发育正常的胚胎供移植,淘汰未受精卵及发育异常的胚胎。 Normal embryonic development for transplantation, developmental abnormalities and out of unfertilized embryo.

[0050] 5、胚胎移植 [0050] 5, embryo transfer

[0051] 按照与供体母猪同样的方法,对同步发情的受体母猪进行麻醉、保定、实施手术, 并将输卵管和卵巢引出创口之外。 [0051] According to the same method sows donor, receptor synchronized estrus sows were anesthetized Baoding, surgery, fallopian tubes and ovaries and lead outside the wound. 先将用于输卵管移植的外套管斜面端,从输卵管伞部插入输卵管的壶腹部;然后将吸有胚胎的移卵管插入外套管,直至用手能感觉到移卵管已穿过外套管1公分;最后将移卵管内的胚胎和培养液移入输卵管内。 A first beveled end overtube intrafallopian transfer inserted from the oviduct ampulla of the oviduct umbrella portion; then suction tubal embryo shift overtube inserted, the outer sleeve 1 until the shift feel by hand passed through the tubal cm; Finally, the embryos in the culture solution into the fallopian tubal shift. 移植完毕,小心将移卵管和外套管退出,复原输卵管和卵巢,送入腹腔,按常规缝合创口。 Been ported, and fallopian tube will be moved carefully withdraw the outer sleeve, fallopian tubes and ovaries restoration, into the abdominal cavity, conventionally sutured wound.

[0052] 6、受体母猪的管理 [0052] 6 receptor management sows

[0053] 移植后的受体母猪,最初几天,要注意每天两次注射抗菌素,防止创口感染。 [0053] receptor sows after the transplant, the first few days, pay attention to antibiotics by injection twice a day, to prevent wound infection. 同时, 要单栏饲养,以防止猪只之间打架,弄破伤口或流产。 At the same time, keeping to a single column, in order to prevent a fight between pigs break wound or abortion. 受体母猪的营养水平,按育种场怀孕母猪的日粮配合即可。 Nutrient levels receptor sows, according to breeding farms pregnant sows with diets can be. 怀孕中后期,注意补充青饲料,做好观察,待产。 In late pregnancy, pay attention to add green fodder, good observation, to be produced.

[0054] 实施例2转人血清白蛋白基因猪的制备 [0054] Example 2 Preparation of gene transfected human serum albumin pigs

[0055] 1、人血清白蛋白基因的准备 [0055] 1, human serum albumin gene preparation

[0056] 将构建好的人血清白蛋白基因质粒酶切成线性,用TE稀释液稀释为5 μ g/ml的基因溶液备用。 [0056] The constructed plasmid was digested linear good human serum albumin solution was diluted gene standby 5 μ g / ml dilution with TE.

[0057] 2、胚胎的采集 [0057] 2, embryo collection

[0058] 用实施例1同样的方法,对母猪进行超数排卵处理,发情后配种;在最后一次配种之后的24小时,进行手术,麻醉、保定、腹部开口,其过程同实施例1。 [0058] the same manner as in Example 1, superovulation sows after estrus; 24 hours after the last breeding, surgery, anesthesia, Baoding, opening the abdomen, the process described in Example 1. 术者将食指及中指由切口伸人腹腔,在与骨盆腔交界的前后位置触摸子宫或子宫角,摸到后用二指夹持,牵引至创口的表面,循一侧子宫角导出输卵管,在输卵管末端转弯处找到该侧卵巢。 The surgeon by the index and middle fingers extending incision into the abdominal cavity, pelvic touch with uterine junction of the uterine horn or front and rear positions, the clamping finger touch after use, pulling to the surface of the wound, through the side of the uterine horn oviduct derived, in found at the end of the fallopian tube bend side ovary. 先将集卵管的一端从输卵管伞口插入输卵管12mm,另一端接集卵皿;然后将吸有18ml冲卵液、6ml空气的注射器与冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管6mm ;推动注射器,输卵管内的胚胎与冲卵液一起流经输卵管,从集卵管流入集卵皿。 First set insertion end fallopian tubal fimbriae 12mm from the mouth, the other end of the egg collection dish; then washed eggs were blotted 18ml, washed with 6 ml of air syringe connected egg, the egg is punched from a needle tube Palace binding portion 6mm fallopian tube end into the uterine horn; push syringe, washed with flowing tubal embryo eggs with fluid in the fallopian tube, the incoming current collector tubal egg dish. 冲卵完毕,小心将冲卵器和集卵管退出,复原输卵管伞,送回腹腔。 Complete egg punch, carefully washed and egg collection exit tubal, tubal recovery, back to the abdominal cavity. 再将另一侧子宫角引出切口之外,用同样的方法冲取胚胎。 Then the other side outside the uterine incision lead angle, using the same method to take punch embryo.

[0059] 冲卵器长200mm、外径1. 0mm、管壁0. 08mm ;集卵管是长130mm、壁厚0. 8mm、内径 [0059] Chong archegonium length 200mm, an outer diameter of 1. 0mm, wall 0. 08mm; fallopian tube is set long 130mm, wall thickness of 0. 8mm, an inner diameter

3. 5mm的玻璃管,中间弯成130°角,两端烧制、研磨平滑。 3. 5mm glass tube, the intermediate bent angle of 130 °, the firing ends, polishing smooth.

[0060] 3、胚胎的收集及质量检查 [0060] 3, and embryos collected QA

[0061] 方法同实施例1。 [0061] The same method as in Example 1. 收集1细胞期的原核胚。 1 cells were collected in prokaryotic embryo.

[0062] 4、基因的显微注射 [0062] 4, microinjection of gene

[0063] 原核胚以10000-15000r/min离心3-5min,能清楚地辨认原核。 [0063] Prokaryotic embryos 10000-15000r / min centrifuged 3-5min, can clearly identify a prokaryotic. 离心后的原核胚和准备好的人血清白蛋白基因溶液置于显微操作仪下,将吸有基因的注射针快速刺入原核中,注射2pl基因溶液,迅速退出注射针。 Prokaryotic and the embryo prepared human serum albumin gene was placed micromanipulator after centrifugation, suction rapid injection needle puncturing a prokaryotic gene, the gene 2pl injectable solutions, quickly withdraw the needle. 每次注射15-20枚卵,培养,待移植。 15-20 eggs per injection, culture, to be transplanted.

[0064] 5、注射基因胚胎的移植 [0064] 5, Gene injected embryo transplantation

[0065] 采用自体移植的方式。 [0065] The embodiment autograft. 该头母猪采集胚胎之后,仍置于手术台上,耳静脉点滴戊巴比妥钠(2.5%)或盐酸氯氨酮维持其麻醉状态。 The sows after embryo collection, still placed on the operating table, the ear intravenous pentobarbital sodium (2.5%) or ketamine hydrochloride maintain anesthesia. 移植时,将输卵管和卵巢引出切口之外。 When transplanting, the fallopian tubes and ovaries lead outside the incision. 先将用于输卵管移植的外套管斜面端,从输卵管伞部插入输卵管的壶腹部;然后将吸有胚胎的移卵管插入外套管,直至用手能感觉到移卵管已穿过外套管1公分;最后将移卵管内的胚胎和培养液移入输卵管内。 A first beveled end overtube intrafallopian transfer inserted from the oviduct ampulla of the oviduct umbrella portion; then suction tubal embryo shift overtube inserted, the outer sleeve 1 until the shift feel by hand passed through the tubal cm; Finally, the embryos in the culture solution into the fallopian tubal shift. 移植完毕,小心将移卵管和外套管退出,复原输卵管和卵巢, 送入腹腔,按常规缝合创口。 Been ported, and fallopian tube will be moved carefully withdraw the outer sleeve, fallopian tubes and ovaries restoration, into the abdominal cavity, conventionally sutured wound.

[0066] 6、移植后母猪的管理 [0066] 6, the management of sows after transplantation

[0067] 移植后的母猪,最初几天,要注意每天两次注射抗菌素,防止创口感染。 [0067] sows after the transplant, the first few days, pay attention to twice daily injections of antibiotics to prevent wound infection. 同时,要单栏饲养,以防止猪只之间打架,弄破伤口或流产。 At the same time, keeping to a single column, in order to prevent a fight between pigs break wound or abortion. 受体母猪的营养水平,按育种场怀孕母猪的日粮配合即可。 Nutrient levels receptor sows, according to breeding farms pregnant sows with diets can be. 怀孕中后期,注意补充青饲料,做好观察,待产。 In late pregnancy, pay attention to add green fodder, good observation, to be produced.

[0068] 7、转基因猪的整合检测 [0068] 7, detection of gene transfer to integrate the pig

[0069] 新生仔猪剪耳号取样,用酚氯仿法提取DNA,PCR检测,三次有与阳性对照一致大小的带者,初步确定为转基因仔猪。 [0069] The number of newborn piglets ears cut sample, DNA was extracted with phenol-chloroform, PCR, detection, there are three bands are the same size as the positive control, initially identified as a transgenic pig. 进一步用Southern杂交确认。 Further confirmed by Southern hybridization.

[0070] 实施例3转EGFP基因克隆猪的构建 [0070] Example 3 transfected EGFP construct cloned pigs

[0071] 1、供细胞的准备 [0071] 1, for the preparation of cells

[0072] 将转EGFP基因猪胎儿成纤维细胞冻存管从液氮中取出,放入37°C水浴中小心震荡,使其迅速解冻;离心去除冻存液,用移液器加入2mL培养液,轻轻悬浮细胞。 [0072] The EGFP gene transfected porcine fetal fibroblasts vial was removed from liquid nitrogen and placed in 37 ° C water bath shaking carefully, it quickly thawed; cryopreservation solution is removed by centrifugation, the culture solution was pipetted added 2mL gently resuspend the cells. 调整细胞密度到IO6个/mL左右,在含0. 5%胎牛血清的DMEM中进行48-72 h的血清饥饿培养。 Cell density was adjusted to about IO6 / mL, and cultured for 48-72 h in serum-starved in 0.5% fetal bovine serum-containing DMEM. 之后用胰蛋白酶消化并收集,在用于核供体之前放人改良的NCSU-23磷酸盐培养液中38. 5°C悬浮C存20-120min。 After digestion with trypsin and collected, prior to release for improved nuclear donor is a phosphate NCSU-23 culture solution C 38. 5 ° C suspension kept 20-120min.

[0073] 2、卵母细胞的准备 [0073] 2, prepared oocytes

[0074] 母猪发情后不配种,待发情结束的12小时之内,麻醉保定后手术切口,将输卵管和卵巢引出切口之外;先将集卵管的一端从输卵管伞口插入输卵管10mm,另一端接集卵皿;然后将吸有20ml冲卵液、8ml空气的注射器与冲卵器连接,冲卵器的针头1从宫管结合部子宫角端插入输卵管6mm;推动注射器,输卵管内的卵母细胞与冲卵液一起流经输卵管, 从集卵管流入集卵皿。 [0074] After the breeding sows do not heat, the situation within the outgoing end of 12 hours, after anesthesia Baoding surgical incision, the fallopian tubes and ovaries lead outside the incision; first end is inserted into the fallopian tube set from fallopian tubal opening 10mm, other a terminating set egg dish; then blotted 20ml red egg liquid, 8ml air syringe punch egg connected, red egg's needle 1 is inserted from the uterine horns end Palace pipe joining portion oviduct 6mm; push the syringe, the eggs in the fallopian tube oviduct cells flowing through the mother solution washed with egg, egg dishes from the incoming current collector fallopian tube. 冲卵完毕,小心将冲卵器和集卵管退出,复原输卵管伞,按常规缝合创口。 Complete egg punch, carefully washed and egg collection exit tubal, tubal rehabilitation, according to conventional suturing wounds. 将集卵皿置于实体显微镜下,用低倍镜的大视野在冲卵液中找到卵母细胞。 The set eggs dish was placed under a stereoscopic microscope, a large field of view with low magnification to find the egg in the red oocytes solution. 拣卵管先吸入少许磷酸缓冲液PBS再吸人卵,在集卵皿的不同位置冲洗卵3-5次。 Picking tubal first few suction phosphate buffer and then PBS inhalation egg, egg rinsed 3-5 times at different positions set egg dish. 依次在第二个集卵皿内重复冲洗,然后把全部卵集中到一个集卵皿,一只供体的卵放在一个皿内。 Sequentially rinsed repeatedly in the second set of eggs dish, and then concentrated to a set of all eggs egg dish, an egg donor body is placed in a dish. 操作室温为20-25 °C。 Operation at room temperature is 20-25 ° C.

[0075] 冲卵器长300mm、外径1. 0mm、管壁0. 09mm ;集卵管是长140mm、壁厚1. 1mm、内径 [0075] Chong archegonium length 300mm, an outer diameter of 1. 0mm, wall 0. 09mm; fallopian tube is set long 140mm, wall thickness of 1. 1mm, the inner diameter

4mm的玻璃管,中间弯成140°角,两端烧制、研磨平滑。 4mm glass tube, the intermediate bent angle of 140 °, the firing ends, polishing smooth.

[0076] 3、卵母细胞的去核、移核和激活 [0076] 3, enucleated oocytes, nuclear transfer and activation

[0077] 收集到的卵母细胞用含有4g/L BSA的PBS(38°C )洗几遍,然后移人38°C不含钙的NCSU-23磷酸缓冲液中培养。 [0077] The oocytes collected is washed several times with PBS (38 ° C) containing 4g / L BSA, and then moves NCSU-23 phosphate buffer at 38 ° C human calcium-cultured. 在倒置显微镜下,在1点半或4点半位置,用激光破膜仪打孔,吸取核连同极体在内的三分之一胞质(盲吸去核)。 Under an inverted microscope, at 1:30 or 4:30 position, with the laser perforation apparatus rupture, draw together with polar body including nuclear third cytoplasmic (suction enucleated blind).

[0078] 转EGFP基因的猪胎儿成纤维细胞,分离之后,在显微操作仪下,移入去核的卵母细胞。 Pigs [0078] EGFP gene is transfected fetal fibroblasts, after separation, at a micromanipulator, into enucleated oocytes. 将移核细胞置于细胞融合仪内,用1.5KV/cm脉冲强度进行2次脉冲,置于NCSU-23 磷酸缓冲液中,再放入39°C、5% C02、100%湿度培养箱中培养,得到发育的重构胚胎。 The cells were placed in a nuclear transfer cell fusion apparatus, twice pulses 1.5KV / cm pulse intensity, placed in phosphate buffered NCSU-23 solution, and then placed in 39 ° C, 5% C02,100% humidity incubator culture, to give a reconstructed embryo.

[0079] 4、重构胚的移植 [0079] 4, the reconstructed embryo transplant

[0080] 将重构胚移入同步发情的受体母猪输卵管内。 [0080] The reconstructed embryo into the oviduct receptor sow estrus synchronization. 移植方法及移植后母猪的管理同实施例1。 Transplantation and post-transplant management of sows in Example 1. [0081] 5、仔猪的检测 [0081] 5, detection of piglets

[0082] 母猪产仔后,仔猪的耳、尾等部位在紫外灯下发出绿色荧光者,即为转EGFP基因克隆猪。 After [0082] farrowing, pigs ear portions, such as the end by green fluorescence under UV light, namely EGFP transfected cloned pigs. 进一步用PCR或Southern杂交确认。 Further confirmed by PCR or Southern hybridization.

Claims (3)

  1. 采集猪早期胚胎或卵母细胞的器械,其特征在于:包括冲卵器和集卵管;冲卵器是长200-300mm、外径1.0-1.1mm、管壁0.08-0.1mm的胶质软管,软管的一端为9-12号针头,软管的另一端为与普通注射器连接的接口;集卵管是长130-140mm、壁厚0.8-1.1mm、内径3-4mm的管,中间弯成120°-140°角。 Collected early embryos or oocytes instrument pigs, characterized by: a punch and a set of tubal eggs; red egg is 200-300mm long, an outer diameter of 1.0-1.1mm, soft gelatinous wall of 0.08-0.1mm tube, one end of the hoses 9-12 needle, the other end of the hose is connected to an interface of an ordinary syringe; fallopian tube is set long 130-140mm, the wall thickness of 0.8-1.1mm, 3-4mm tube inner diameter, the intermediate bent angle of 120 ° -140 °.
  2. 2.根据权利要求1所述的采集猪早期胚胎或卵母细胞的器械,其特征在于:集卵管是玻璃管、塑料管或胶管,两端烧制、研磨平滑。 The collected early embryos or oocytes instrument pig according to claim 1, wherein: a set tubal glass, plastic tube or hose, both ends of the fired abrasive smoothing.
  3. 3.采集猪早期胚胎或卵母细胞的方法,母猪麻醉保定后手术切口,将输卵管和卵巢引出切口之外;其特征在于:首先将集卵管的一端从输卵管伞口插入输卵管10-15mm,另一端接集卵皿;然后将吸有15-20ml冲卵液、5-10ml空气的注射器与冲卵器连接,冲卵器的针头从宫管结合部子宫角端插入输卵管5-8mm ;再实施冲卵。 3. The method of addition or early embryos collected pig oocytes, sows after anesthesia Baoding incision, the fallopian tubes and ovaries lead incision; wherein: the first end is inserted into the fallopian tubal set tubal port from 10-15mm , the other end of collection dish eggs; 15-20ml then blotted red liquid egg, 5-10 ml of air with a syringe connected egg punch, punch needle from the egg's house pipe joining end portion is inserted into the uterine horn 5-8mm fallopian tube; then washed eggs embodiment.
CN 201010112198 2010-02-23 2010-02-23 Apparatus for collecting early-stage embryo or early-stage oocyte of pig CN101797189B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102100585A (en) * 2011-03-17 2011-06-22 安徽农业大学 Uterine horn embryo-flushing needle suitable for sheep
CN105838581A (en) * 2016-05-11 2016-08-10 安徽农业大学 Animal ovary oocyte separation and collection device

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5108366A (en) * 1990-09-28 1992-04-28 Ovamed Corporation Delivery catheter
US6610005B1 (en) * 1996-04-26 2003-08-26 Jun Tao Catheter system for implanting embryos
US20040092791A1 (en) * 2002-08-30 2004-05-13 Timothy Bateman Oocyte and embryo handling apparatus
CN2698293Y (en) * 2004-05-28 2005-05-11 胡金岗 Balloon catheter unit for collection and transplantation of sheep embryo
CN101642389A (en) * 2009-08-28 2010-02-10 湖北省农业科学院畜牧兽医研究所 Pig embryo transplantation instrument and transplantation method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5108366A (en) * 1990-09-28 1992-04-28 Ovamed Corporation Delivery catheter
US6610005B1 (en) * 1996-04-26 2003-08-26 Jun Tao Catheter system for implanting embryos
US20040092791A1 (en) * 2002-08-30 2004-05-13 Timothy Bateman Oocyte and embryo handling apparatus
CN2698293Y (en) * 2004-05-28 2005-05-11 胡金岗 Balloon catheter unit for collection and transplantation of sheep embryo
CN101642389A (en) * 2009-08-28 2010-02-10 湖北省农业科学院畜牧兽医研究所 Pig embryo transplantation instrument and transplantation method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102100585A (en) * 2011-03-17 2011-06-22 安徽农业大学 Uterine horn embryo-flushing needle suitable for sheep
CN102100585B (en) 2011-03-17 2013-04-10 安徽农业大学 Uterine horn embryo-flushing needle suitable for sheep
CN105838581A (en) * 2016-05-11 2016-08-10 安徽农业大学 Animal ovary oocyte separation and collection device
CN105838581B (en) * 2016-05-11 2017-11-10 安徽农业大学 An animal oocyte collection separation means

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