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 PDFInfo
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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
Technical field
The present invention relates to a kind of apparatus and acquisition method that is used to gather pig body early embryo or oocyte, belong to the animal embryo engineering field.
Background technology
The collection of pig body early embryo and transplant and report by Kvashickii early than nineteen fifty-one.To the sixties in 20th century, set up the technology of surgical method collection and transplanting embryo substantially.After the seventies, North America and Europe begin the technology of collection and transplanting embryo is applied to blood and international introducing a fine variety outside the locking swinery is introduced.The eighties transgenic animal technology appearance, this The Application of Technology is expanded.People such as Hammer in 1985 at first with the germ cell of the method collection pig of performing the operation, use human growth hormone gene the germ cell protokaryon that microinjection technique is injected pig then, with its fallopian tube that moves into sow, obtain transgenic pig again.After this, in germ cell, inject exogenous gene, just become one of main method of preparation transgenic pig.In addition, be equipped with transgenic pig with the viral infection legal system, also need at first to gather the body early embryo of pig, the viral vector that will be connected with exogenous gene then injects body early embryo, produces transgenic pig.
The collection of porcine oocytes is mainly used in by external fertilization technology preparation " test tube pig ", by nuclear transfer technology preparation " clone pig ", by the combine pig of preparation " transgene clone pig " and other genetic modification of nuclear transfer technology and transgenic technology.
Use the external fertilization technology and produce " test tube pig " and reported in nineteen eighty-three by Cheng and Polge the earliest, the pig embryo vitro of the setting up technology of producing is used by other research and production mechanism in the world thus, and is developed.Implement pig in-vitro fertilization, at first will obtain the oocyte of pig.The source of its oocyte has two: one, and the oocyte of maturation in vitro is gathered immature oocyte from ovary, after the In vitro culture maturation, is used for external fertilization; The 2nd, the oocyte of cylinder mature.The present invention is used for the collection of pig cylinder mature oocyte.Sophisticated oocyte combines with the pig sperm of capacitation under external control environment, is ivf zygote; Cultivate into body early embryo again under external control environment, transplant and give the receptor sow, the farrowing of conceived back is " test tube pig ".
The application embryonic cell obtains " clone pig " as nuclear donor and is reported in 1989 by Polejaeva and Onishi the earliest, uses somatic cell and is then reported in 2000 by Prather and Onishi the earliest as nuclear donor acquisition " clone pig ".The preparation of " clone pig ", at first sophisticated oocyte is carried out enucleation, then isolating embryonic cell or somatic cell are moved into non-nucleus egg mother cell, merging (or fusion of other method) through electricity handles, In vitro culture, move in the reproductive tract of sow, the piglet of producing is " clone pig " again.The preparation of transgene clone pig needs at first gene transformation to be carried out in somatic cell, uses genetically modified cell as nuclear donor then, obtains the transgene clone pig.People such as Park (2001,2002) do nuclear donor with the fibroblast and the ear epithelial cell of transfection green fluorescence protein gene, obtain the transgene clone pig respectively.Clone technology and transgene clone technology are the means of the tool prospect of swine improvement.Preparation " clone pig " and " transgene clone pig ", the source of its oocyte is the same with external fertilization, and two kinds of cylinder mature and maturation in vitro are arranged.Experiment shows, does nuclear receptor with the porcine oocytes of cylinder mature, than the oocyte of maturation in vitro higher success rate is arranged.
China sets up pig body early embryo and oocyte acquisition technique the earliest is people such as the Wei Qing of academy of agricultural sciences, Hubei Province letter, and they moved into the pig body early embryo of gathering in the fallopian tube of receptor sow in 1989, obtained the offspring of embryo transfer; After this, surplus ten kinds of exogenous genes are imported the germ cell of pig, obtained the transgenic pig of corresponding integration and expression; People such as Zhao Haobin blastomere with body early embryo in 1997 moves into enucleation oocyte, obtains the nuclear transplantation offspring.People such as herding institute Feng of the Chinese Academy of Agricultural Sciences book hall have set up the collection and the implantation technique of pig body early embryo in nineteen ninety, and have obtained the piglet of embryo transfer.Entered since 21 century, units such as China Agricultural University, Northeast Agricultural University, academy of agricultural sciences, Shanghai animal and veterinary institute use nuclear transplantation and transgenic technology, obtain clone pig or transgene clone pig.
The feature of gathering pig body early embryo and cylinder mature oocyte prior art both at home and abroad is: during from fallopian tube towards ovum, syringe directly links to each other with syringe needle (centre does not have hose connection), and syringe needle inserts fallopian tube from the tubal extremity of palace pipe jointing portion; Ovum collector generally be straight, do not have a bending.The defective of this technology mainly contains 3 points:
1, in the ovum process, during pushing syringe, because syringe is rigid the connection with syringe needle, the operator has shake or pig that disturbance is arranged slightly slightly, and syringe needle scratches easily, stroke wear or deviate from fallopian tube, thereby causes tubal hemorrhage or fail towards ovum.
2, because syringe needle inserts fallopian tube from the tubal extremity of palace pipe jointing portion, the one, hemorrhage easily; The 2nd, pierce through fallopian tube easily; The 3rd, if, can not insert syringe needle for the second time from identical position again, need reselect a position contact pin, thereby increase oviducal wound for the first time towards the ovum failure, also be not easy to find suitable position.
3, because ovum collector is straight, the position that collection ovum ware is put is subjected to the narrow and small limitation of art portion, and is careless slightly when connecing ovum, or pig has disturbance slightly, flow to easily outside the collection ovum ware towards ovum liquid, or the ovum liquid that dashes in the collection ovum ware splashes, and causes towards the ovum rate and reduces or fail towards ovum.
Summary of the invention
The object of the present invention is to provide a kind of apparatus and acquisition method that is used to gather pig body early embryo or oocyte, can overcome the defective of prior art effectively, improve the success rate of gathering greatly, obtaining the ovum rate can reach more than 95%, and easy to operate, safety.
Technical scheme of the present invention is: gather the apparatus of pig body early embryo or oocyte, it is characterized in that: comprise towards egg apparatus and ovum collector; Towards egg apparatus is the colloid flexible pipe of long 200-300mm, external diameter 1.0-1.1mm, tube wall 0.08-0.1mm, and an end of flexible pipe is the 9-12 syringe needle, and the other end of flexible pipe is the interface that is connected with ordinary syringe;
Ovum collector is the pipe of long 130-140mm, wall thickness 0.8-1.1mm, internal diameter 3-4mm, and the centre curves 120 ° of-140 ° of angles (flexible pipe can be crooked), and two ends are fired, grind level and smooth.
The apparatus of aforesaid collection pig body early embryo or oocyte, it is characterized in that: ovum collector is the pipe (as plastic tube, sebific duct etc.) of glass tubing or other quality, two ends are fired, grind level and smooth.
Gather the method for pig body early embryo or oocyte, operative incision behind sow anesthesia Baoding is drawn fallopian tube and ovary outside the otch; It is characterized in that: at first the end with ovum collector inserts fallopian tube 10-15mm, another termination collection ovum ware from uterine tube umbrella mouth; Then suction is had 15-20ml towards the syringe of ovum liquid, 5-10ml air be connected towards egg apparatus, insert fallopian tube 5-8mm towards the syringe needle of egg apparatus from palace pipe jointing portion cornua uteri end; Implement towards ovum again.
The invention has the beneficial effects as follows: the present invention has overcome the defective of existing collection pig body early embryo and cylinder mature oocyte technology effectively:
(1) owing to being the colloid flexible pipe towards egg apparatus, when fallopian tube inserts syringe needle, overcome and the direct-connected limitation of syringe, operation can be inserted oviducal inner chamber really with syringe needle freely; In the ovum process,, because the cushioning effect of flexible pipe, can not occur syringe needle yet and scratch, draw and wear or deviate from oviducal phenomenon, thereby improve success rate greatly towards ovum even the operator has shake or pig that disturbance is arranged slightly slightly.
(2) owing to the syringe needle towards egg apparatus inserts fallopian tube from palace pipe jointing portion cornua uteri end, can avoid blood vessel effectively, not cause tubal hemorrhage; Can not pierce through fallopian tube yet; If for the first time towards the ovum failure,, be easy to reselect the position of contact pin, do not cause oviducal wound because the cornua uteri end area is bigger.
(3) because ovum collector has bending, the position that collection ovum ware can be put increases, and limitation is little, though connect sometimes ovum accidentally or pig disturbance is arranged slightly, collection ovum ware can move thereupon, can not cause towards the ovum liquid stream outside collection ovum ware, or dash the phenomenon that ovum liquid is splashed in the collection ovum ware.
Description of drawings
Fig. 1 is that the embodiment of the invention is towards the egg apparatus sketch map.
Fig. 2 is an embodiment of the invention ovum collector sketch map.
The specific embodiment
The present invention is described further below in conjunction with drawings and Examples.
The explanation of labelling among Fig. 1: 1-syringe needle, 2-flexible pipe, 3-interface.
As depicted in figs. 1 and 2, the embodiment of the invention is used to gather the apparatus of pig body early embryo or oocyte, comprises towards egg apparatus and ovum collector; Towards egg apparatus is the colloid flexible pipe 2 of long 200-300mm, external diameter 1.0-1.1mm, tube wall 0.08-0.1mm, and an end of flexible pipe 2 is a 9-12 syringe needle 1, and the other end of flexible pipe 2 is the interface 3 that is connected with ordinary syringe.Ovum collector is to curve 120 ° of-140 ° of angles in the middle of the glass tubing (or the pipe of other quality, as plastic tube, sebific duct etc.) of long 130-140mm, wall thickness 0.8-1.1mm, internal diameter 3-4mm, and two ends are fired, grind level and smooth.Rinse the back natural drying well towards egg apparatus, under uviol lamp, sterilize with preceding.Ovum collector is rinsed the back well and is sterilized in high-pressure sterilizing pot.
When the embodiment of the invention was used to gather pig body early embryo or oocyte, at first the end with the ovum collector of the embodiment of the invention inserted fallopian tube 10-15mm, another termination collection ovum ware from uterine tube umbrella mouth; Then suction there is 15-20ml to be connected with the egg apparatus that dashes of the embodiment of the invention, inserts fallopian tube 5-8mm from palace pipe jointing portion cornua uteri end towards the syringe needle 1 of egg apparatus towards the syringe of ovum liquid, 5-10ml air; Implement towards ovum again.
Concrete steps are as follows:
(1) method of collection pig body early embryo: within the 24-48 behind the insemination of sows hour, operative incision behind anesthesia Baoding is drawn fallopian tube and ovary outside the otch; End with ovum collector inserts fallopian tube 10-15mm, another termination collection ovum ware from uterine tube umbrella mouth earlier; Then suction is had 15-20ml towards the syringe of ovum liquid, 5-10ml air be connected towards egg apparatus, insert fallopian tube 5-8mm towards the syringe needle 1 of egg apparatus from palace pipe jointing portion cornua uteri end; Pushing syringe, intratubal embryo flows into collection ovum ware with the fallopian tube of flowing through towards ovum liquid from ovum collector.Finish towards ovum, carefully will withdraw from, restore fimbriae of uterine tube, sew up wound routinely towards egg apparatus and ovum collector.
(2) method of collection porcine oocytes: do not breed behind the oestrus of sow, within waiting to oestrus 12 hours that finish, operative incision behind anesthesia Baoding is drawn fallopian tube and ovary outside the otch; End with ovum collector inserts fallopian tube 10-15mm, another termination collection ovum ware from uterine tube umbrella mouth earlier; Then suction is had 15-20ml towards the syringe of ovum liquid, 5-10ml air be connected towards egg apparatus, insert fallopian tube 5-8mm towards the syringe needle 1 of egg apparatus from palace pipe jointing portion cornua uteri end; Pushing syringe, intratubal oocyte flow into collection ovum ware with the fallopian tube of flowing through towards ovum liquid from ovum collector.Finish towards ovum, carefully will withdraw from, restore fimbriae of uterine tube, sew up wound routinely towards egg apparatus and ovum collector.
Embryo's collection and the transplanting of embodiment 1 good boar
Do donor with good purebred sow, do receptor with the crossbreeding sow that can not do kind of usefulness, give the crossbreeding sow receptor with the embryo transfer of donor sow, purpose is to produce good purebred pig with crossbreeding sow.Its operating procedure is as follows:
1, superovulation of donor sow and breeding
At 15-18 days of the donor oestrus of sow cycle, intramuscular injection pregnant mare serum gonadotrop(h)in (PMSG) PMSG1000IU injected chorionic-gonadotropin hormone HCG 800IU after 72 hours, do superovulation and handle.Breed with this product breeding boar after oestrusing.
2, the estrus synchronization of receptor sow
15-18 days of receptor oestrus of sow cycle, when the superovulation of donor sow is handled, intramuscular injection PMSG 800IU, HCG injection 600IU after 72 hours.
The sow of perhaps selecting to oestrus on the same day with the donor sow in jumpbogroup is cooked receptor.
3, donor sow embryo's collection
Within the 24-48 behind the donor insemination of sows hour, embryo collection.
(1) anesthesia of donor pig and Baoding
Donor pig palate tractive, restraint is pressed the injection of 20-30mg/kg (per kilogram of body weight milligram) pentobarbital sodium (2.5%) ear vein, anaesthetizes sb. generally.Anesthesia puts in place to swing back and is placed on the operation Shelf for keeing, and extremity are fixed.For preventing in the operation process, the struggle of pig, springing, with rope that extremity and head binding are firm.For abdominal viscera is turned forward, convenient operation, operating-table low before wanting (low) back high (tail height).Operation process is muscle or an amount of ketalar of intravenous drip as required.
(2) operative site and sterilization thereof
Scrub abdominal part and femoribus internus portion with suds and hairbrush, washing back towel wipe up.Operative site is typically chosen between the 2nd, 3 nipple reciprocal, cut cropping with electric scissors or hair earlier in art portion, should cut clean hair stubble, clean art portion with clear water, be coated with iodine tincture then with 2-4%, wait that the alcohol swab of doing back reuse 70%-75% takes off iodine, disinfectant program is: iodine tincture cotton balls → cotton ball soaked in alcohol, from inside to outside; Lid wound towel Rhizoma Atractylodis Macrocephalae portion makes predetermined otch be exposed to the middle part of wound towel opening.
(3) operation method
Along ventrimeson, avoid blood vessel as far as possible, cut about skin 5-8cm, along otch left and right sides passivity fractionation of fatty and Musclar layer, peritoneum is exposed after, cut off peritoneum with blunt nosed shears.Cut peritoneum and should avoid damaging internal organs in the abdomen, mention peritoneum with tweezers earlier, make a kerf mentioning the position, go deep into peritoneum guiding scalpel (outwards otch) with the forefinger of another hands then or with clipper for surgical use the peritoneum incision or cut off.The patient stretches the people abdominal cavity with forefinger and middle finger by otch, touches cornua uteri at the front and back position with the pelvic cavity boundary, touches the back and refers to clampings with two, and fallopian tube and ovary are drawn outside the otch.
(4) embryo collection
End with ovum collector inserts fallopian tube 15mm, another termination collection ovum ware from uterine tube umbrella mouth earlier; Then suction is had 15ml towards the syringe of ovum liquid, 5ml air be connected towards egg apparatus, insert fallopian tube 5mm towards the syringe needle 1 of egg apparatus from palace pipe jointing portion cornua uteri end; Pushing syringe, intratubal embryo flows into collection ovum ware with the fallopian tube of flowing through towards ovum liquid from ovum collector.Finish towards ovum, carefully will withdraw from, restore fimbriae of uterine tube, send the abdominal cavity back to towards egg apparatus and ovum collector.Again opposite side fallopian tube and ovary are drawn outside the otch, use the same method to dash and get the embryo.The internal organs arrangement resets, and the cleaning otch is sewed up wound routinely.
Towards the long 250mm of egg apparatus, external diameter 1.0mm, tube wall 0.1mm; Ovum collector is the glass tubing of long 135mm, wall thickness 1mm, internal diameter 3mm, and the centre curves 135 ° of angles, and two ends are fired, grind level and smooth.
4, embryo's collection and quality examination
To collect the ovum ware and place under the stero microscope, in ovum liquid, find the embryo with the big visual field of low power lens.Pig embryo diameter is that perusal has only the needle point size about 150 μ m, is a spheroplast, and rounded under mirror, its skin is a zona pellucida, and it is stronger than other irregular fragment of tissue refractivities in the refractivity in ovum liquid, and tone is a Lycoperdon polymorphum Vitt.Choose oviduct and suck a little phosphate buffer PBS resorb ovum earlier, wash ovum 3-5 time at the diverse location that collects the ovum ware.Repeat flushing successively in second collection ovum ware, then whole ovum are focused on a collection ovum ware, the ovum of a donor is placed in the ware.The operation room temperature is 20-25 ℃.
The amplification of stereoscope is transferred to 80-100 doubly, the form, blastomere size of observing the embryo and the uniformity, color and luster, cell density, with situations such as zona pellucida gap and cytopathy.The embryo of normal development, its blastomere general appearance is neatly clear, and size is consistent, is evenly distributed and tight, and rate of development is consistent with embryo's age in days.Normotrophic embryo for transplanting, is eliminated unfertilized egg and dysplastic embryo.
5, embryo transfer
According to the method same with the donor sow, to the receptor sow of synchronization of estrus anaesthetize, Baoding, enforcement operation, and fallopian tube and ovary drawn outside the wound.To be used for the trocar sheath beveled end of common oviduct transplantation earlier, insert oviducal ampulla from fimbriae of uterine tube portion; Then suction there is embryo's ovum shifting tube to insert trocar sheath, until feeling that with hands ovum shifting tube has passed 1 centimeter of trocar sheath; At last embryo in the ovum shifting tube and culture fluid are moved in the fallopian tube.Transplanting finishes, and carefully ovum shifting tube and trocar sheath is withdrawed from, and restores fallopian tube and ovary, sends into the abdominal cavity, sews up wound routinely.
6, the management of receptor sow
Receptor sow after the transplanting, initial several days, note double injection antibiotics every day, prevent traumatic infection.Simultaneously, single hurdle to raise, fight between pig only preventing, stave wound or miscarriage.The trophic level of receptor sow, the daily ration cooperation of pressing the breeding station farrowing sow gets final product.Conceived middle and late stage is noted replenishing greenfeed, carries out observation, waits to produce.
Embodiment 2 changes the preparation of human serum albumin gene pig
1, the preparation of human serum albumin gene
The human serum albumin gene plasmid enzyme restriction that builds is linear, be that the cdna solution of 5 μ g/ml is standby with the TE diluted.
2, embryo's collection
With the same method of embodiment 1, sow is carried out superovulation handle, the back breeding of oestrusing; After the breeding 24 hours undergo surgery the last time, anesthesia, Baoding, abdominal part opening, and its process is with embodiment 1.The patient stretches the people abdominal cavity with forefinger and middle finger by otch, touch uterus or cornua uteri at front and back position, touch the back and refer to clampings, draw surface to wound with two with the pelvic cavity boundary, follow a side cornua uteri and derive fallopian tube, find this side ovary at fallopian tube tip turn place.End with ovum collector inserts fallopian tube 12mm, another termination collection ovum ware from uterine tube umbrella mouth earlier; Then suction is had 18ml towards the syringe of ovum liquid, 6ml air be connected towards egg apparatus, insert fallopian tube 6mm towards the syringe needle 1 of egg apparatus from palace pipe jointing portion cornua uteri end; Pushing syringe, intratubal embryo flows into collection ovum ware with the fallopian tube of flowing through towards ovum liquid from ovum collector.Finish towards ovum, carefully will withdraw from, restore fimbriae of uterine tube, send the abdominal cavity back to towards egg apparatus and ovum collector.Again the opposite side cornua uteri is drawn outside the otch, use the same method to dash and get the embryo.
Towards the long 200mm of egg apparatus, external diameter 1.0mm, tube wall 0.08mm; Ovum collector is the glass tubing of long 130mm, wall thickness 0.8mm, internal diameter 3.5mm, and the centre curves 130 ° of angles, and two ends are fired, grind level and smooth.
3, embryo's collection and quality examination
Method is with embodiment 1.Collect the protokaryon embryo of 1 cell stage.
4, the microinjection of gene
The protokaryon embryo can clearly be recognized protokaryon with the centrifugal 3-5min of 10000-15000r/min.Protokaryon embryo after centrifugal and ready human serum albumin gene solution place under the micrurgy instrument, have the entry needle of gene to thrust fast in the protokaryon suction, and injection 2pl cdna solution withdraws from entry needle rapidly.Per injection 15-20 piece ovum is cultivated, and waits to transplant.
5, injection gene embryo's transplanting
Adopt autoplastic mode.After this sow embryo collection, still place on the operating-table, ear vein drop pentobarbital sodium (2.5%) or hydrochloric acid chlore-ammonia ketone are kept its narcotism.During transplanting, fallopian tube and ovary are drawn outside the otch.To be used for the trocar sheath beveled end of common oviduct transplantation earlier, insert oviducal ampulla from fimbriae of uterine tube portion; Then suction there is embryo's ovum shifting tube to insert trocar sheath, until feeling that with hands ovum shifting tube has passed 1 centimeter of trocar sheath; At last embryo in the ovum shifting tube and culture fluid are moved in the fallopian tube.Transplanting finishes, and carefully ovum shifting tube and trocar sheath is withdrawed from, and restores fallopian tube and ovary, sends into the abdominal cavity, sews up wound routinely.
6, transplant the management of back sow
Sow after the transplanting, initial several days, note double injection antibiotics every day, prevent traumatic infection.Simultaneously, single hurdle to raise, fight between pig only preventing, stave wound or miscarriage.The trophic level of receptor sow, the daily ration cooperation of pressing the breeding station farrowing sow gets final product.Conceived middle and late stage is noted replenishing greenfeed, carries out observation, waits to produce.
7, the integration of transgenic pig detects
Newborn piglet is cut overbit sampling, extracts DNA with phenol chloroform method, and PCR detects, and consistent big or small band person with positive control is arranged, tentatively be defined as the transgenic piglet for three times.Further confirm with Southern hybridization.
Embodiment 3 changes the structure of EGFP gene clone pig
1, supplies the preparation of cell
To change the frozen pipe of EGFP gene pig fetal fibroblast and from liquid nitrogen, take out, put into 37 ℃ of medium and small commotio cordises of water-bath, it is thawed rapidly; Centrifugal removal cryopreserving liquid adds 2mL culture fluid, suspension cell gently with pipettor.Adjust cell density to 10
6About individual/mL, in the DMEM that contains 0.5% hyclone, carry out the serum starvation of 48-72 h and cultivate.With trypsinization and collection, the NCSU-23 phosphate culture fluid of putting people's improvement before being used for nuclear donor suspends for 38.5 ℃ and stores 20-120min afterwards.
2, the preparation of oocyte
Do not breed behind the oestrus of sow, within waiting to oestrus 12 hours that finish, operative incision behind anesthesia Baoding is drawn fallopian tube and ovary outside the otch; End with ovum collector inserts fallopian tube 10mm, another termination collection ovum ware from uterine tube umbrella mouth earlier; Then suction is had 20ml towards the syringe of ovum liquid, 8ml air be connected towards egg apparatus, insert fallopian tube 6mm towards the syringe needle 1 of egg apparatus from palace pipe jointing portion cornua uteri end; Pushing syringe, intratubal oocyte flow into collection ovum ware with the fallopian tube of flowing through towards ovum liquid from ovum collector.Finish towards ovum, carefully will withdraw from, restore fimbriae of uterine tube, sew up wound routinely towards egg apparatus and ovum collector.To collect the ovum ware and place under the stero microscope, in ovum liquid, find oocyte with the big visual field of low power lens.Choose a little the phosphate buffer PBS of suction of oviduct elder generation and inhale people's ovum again, wash ovum 3-5 time at the diverse location that collects the ovum ware.Repeat flushing successively in second collection ovum ware, then whole ovum are focused on a collection ovum ware, the ovum of a donor is placed in the ware.The operation room temperature is 20-25 ℃.
Towards the long 300mm of egg apparatus, external diameter 1.0mm, tube wall 0.09mm; Ovum collector is the glass tubing of long 140mm, wall thickness 1.1mm, internal diameter 4mm, and the centre curves 140 ° of angles, and two ends are fired, grind level and smooth.
3, the enucleation of oocyte, move nuclear and activate
The oocyte of collecting is washed several times with the PBS (38 ℃) that contains 4g/L BSA, moves then in 38 ℃ of not calcareous NCSU-23 phosphate buffers of people and cultivates.Under inverted microscope, 1 thirty or 4 thirty the position, with the punching of laser rupture of membranes instrument, draw nuclear together with polar body in 1/3rd interior kytoplasms (blind suction enucleation).
Change the porcine fetus fibroblasts of EGFP gene, after separating under the micrurgy instrument, moves into non-nucleus egg mother cell.To move nucleus and place in the cell fusion instrument, carry out 2 subpulses, place the NCSU-23 phosphate buffer, put into 39 ℃, 5%CO again with the 1.5KV/cm pulse strength
2, cultivate the reconstruct embryo who obtains growing in the 100% humidity incubator.
4, the transplanting of reconstructed embryo
Reconstructed embryo is moved in the receptor sow fallopian tube of synchronization of estrus.The management of implantation method and transplanting back sow is with embodiment 1.
5, the detection of piglet
Behind the sows farrowing, the green fluorescence is sent in positions such as the ear of piglet, tail under uviol lamp person, being changes EGFP gene clone pig.Further confirm with PCR or Southern hybridization.
Claims (3)
1. gather the apparatus of pig body early embryo or oocyte, it is characterized in that: comprise towards egg apparatus and ovum collector; Towards egg apparatus is the colloid flexible pipe of long 200-300mm, external diameter 1.0-1.1mm, tube wall 0.08-0.1mm, and an end of flexible pipe is the 9-12 syringe needle, and the other end of flexible pipe is the interface that is connected with ordinary syringe;
Ovum collector is the pipe of long 130-140mm, wall thickness 0.8-1.1mm, internal diameter 3-4mm, and the centre curves 120 ° of-140 ° of angles.
2. the apparatus of collection pig body early embryo according to claim 1 or oocyte, it is characterized in that: ovum collector is glass tubing, plastic tube or sebific duct, two ends are fired, grind level and smooth.
3. gather the method for pig body early embryo or oocyte, operative incision behind sow anesthesia Baoding is drawn fallopian tube and ovary outside the otch; It is characterized in that: at first the end with ovum collector inserts fallopian tube 10-15mm, another termination collection ovum ware from uterine tube umbrella mouth; Then suction is had 15-20ml towards the syringe of ovum liquid, 5-10ml air be connected towards egg apparatus, insert fallopian tube 5-8mm towards the syringe needle of egg apparatus from palace pipe jointing portion cornua uteri end; Implement towards ovum again.
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Cited By (3)
| 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 |
| CN106479961A (en) * | 2016-11-14 | 2017-03-08 | 哈尔滨医科大学 | A kind of method for collecting pig complexity form embryo |
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| 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 (5)
| 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 | A kind of animal ovary egg mother cell separates and collects device |
| CN106479961A (en) * | 2016-11-14 | 2017-03-08 | 哈尔滨医科大学 | A kind of method for collecting pig complexity form embryo |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101797189B (en) | 2011-08-17 |
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