CN201492540U - Appliance used for transplanting pig embryo - Google Patents
Appliance used for transplanting pig embryo Download PDFInfo
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- CN201492540U CN201492540U CN2009202277278U CN200920227727U CN201492540U CN 201492540 U CN201492540 U CN 201492540U CN 2009202277278 U CN2009202277278 U CN 2009202277278U CN 200920227727 U CN200920227727 U CN 200920227727U CN 201492540 U CN201492540 U CN 201492540U
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Abstract
The utility model relates to an appliance used for transplanting pig embryos. The appliance comprises ovum transplanting tubes, outer cannulas used for transplanting the oviduct or outer cannulas used for transplanting the uterine horn, wherein the ovum transplanting tube are all arranged inside each outer cannula, each outer cannula is made of hard flexible plastic tubules with length of 65 mm to 70 mm and diameter of tube cavity of 3 mm to 4 mm and thickness of pipe wall of 0.1 mm to 0.2 mm, two ends of the outer cannula used for transplanting the oviduct are polished smooth, one end of the outer cannula used for transplanting the uterine horn is sheared to a slope of 15 DEG to 20 DEG, and the other end thereof is polished smooth. When in transplanting embryos, the outer cannulas are inserted into the ampulla part of the oviduct of a sow or the an appropriate part of the uterine horn of the sow; then the ovum transplanting tubes attached with an embryo are inserted into the ampulla part of the oviduct of the sow or the appropriate part of the uterine horn through the outer cannulas, and then the transplantation is carried out. The appliance overcomes the defect of the prior art that the ovum transplanting tubes are directly inserted into the oviduct or the uterine horn, thereby remarkably improving the success rate and the efficiency of transplanting pig embryos.
Description
Technical field
This utility model relates to a kind of apparatus that is used for the pig embryo transfer, belongs to the embryo transfer field.
Background technology
Pig embryo transfer success of the test is reported 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 embryo transfer technology is applied to blood and international introducing a fine variety outside the locking swinery is introduced.The eighties transgenic animal technology appearance, promoted the application and the development of embryo transfer technology.People such as Hammer in 1985 at first use human growth hormone gene the germ cell that microinjection technique is injected pig, use embryo transfer technology then with its fallopian tube that moves into the receptor sow, obtain transgenic pig.After this, various in the world genetically modified embryos comprise the transgenic embryo that obtains with microinjection, semen transformation, body-cell neucleus transplanting method and other method, produce transgenic pig by embryo transfer technology; The embryo that various embryo engineerings obtain, the reconstruct embryo as embryo, embryonic cell nuclear transplantation and the body-cell neucleus transplanting of external fertilization obtains " test tube pig " and " clone pig " by embryo transfer technology.
China sets up the pig embryo transfer technology the earliest is that the Wei Qing of academy of agricultural sciences, Hubei Province letter waits the people, and they moved into the cornua uteri of receptor sow with the blastula embryo of pig in 1989, obtained the offspring of embryo transfer; 1992, with the embryo of external fertilization, be transplanted to the fallopian tube of sow, obtain " test tube pig "; 1997, with the reconstruct embryo of embryonic cell nuclear transplantation, be transplanted to the fallopian tube of sow, obtain " clone pig "; Successively seven kinds of exogenous genes are imported the germ cell of pig, obtained the transgenic pig of corresponding integration and expression by embryo transfer technology.People such as herding institute Feng of the Chinese Academy of Agricultural Sciences book hall have set up the embryo transfer technology of pig 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 successively use embryo transfer technology, and the reproductive tract with clone embryos or genetically modified embryo immigration sow obtain clone pig or transgenic pig.
The feature of pig embryo transfer prior art is both at home and abroad: when carrying out common oviduct transplantation, directly insert fallopian tube from fimbriae of uterine tube portion with ovum shifting tube; When carrying out the cornua uteri transplanting, directly insert cornua uteri with ovum shifting tube.The defective of this technology has 3 points:
1, ovum shifting tube is difficult to insert darker position, when especially carrying out common oviduct transplantation, be difficult to insert oviducal ampulla, to making the embryo can not move on to only position, thereby influences the success rate of embryo transfer.
2, ovum shifting tube directly inserts fallopian tube from fimbriae of uterine tube portion, or directly inserts cornua uteri, in the process of inserting, because mucous adsorption on endosalpinx or the cornua uteri inner membrance is easy to cause losing of embryo, thereby influences the efficient of embryo transfer.
3, ovum shifting tube directly inserts fallopian tube from fimbriae of uterine tube portion, or directly insert cornua uteri, careless slightly in the process of operation, or the receptor sow has disturbance slightly, because ovum shifting tube is the glass quality, be easy to fracture or fragmentation, thereby cause graft failure, the serious damage that also can cause receptor sow reproductive tract.
What patent CN200520103507.6 related to is people's embryo transfer, and what CN200510105236.2 related to is the embryo transfer of cattle, but they are non-operation transplantation, and trocar sheath wherein just is used for protecting its inner core.Simultaneously because the morphosis of various animal reproductive tract is inequality, apparatus that embryo transfer is used and method are also inequality, if the technical scheme of using above two pieces of patents can not be carried out the embryo transfer of pig, also just can not overcome three point defects of existing pig embryo transfer technology.
Summary of the invention
The purpose of this utility model is to provide a kind of apparatus that is used for the pig embryo transfer, adopts operation transplantation, has overcome three point defects of existing pig embryo transfer technology effectively, has improved the success rate of embryo transfer greatly, and is easy to operate.
Be used for the apparatus of pig embryo transfer, comprise ovum shifting tube, it is characterized in that: comprising also that common oviduct transplantation is transplanted with trocar sheath or cornua uteri uses trocar sheath, and ovum shifting tube is in each trocar sheath inside; The common oviduct transplantation trocar sheath is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw, and two ends polish cunning; It is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw that cornua uteri is transplanted with trocar sheath, and the one end is cut into 15 ° of-20 ° of inclined-planes, and the other end polishes cunning.
The beneficial effects of the utility model are: when this utility model is used for 1-4 cell stage embryo's transplanting, at first common oviduct transplantation is inserted the oviducal ampulla of receptor sow with trocar sheath; When being used for the above embryo's of 4 cell stages transplanting, at first cornua uteri is transplanted the proper site of inserting receptor sow cornua uteri with trocar sheath; The ovum shifting tube that then suction is had the embryo is inserted into the proper site of oviducal ampulla of receptor sow or cornua uteri by trocar sheath, implements to transplant again.This utility model has overcome three point defects of existing pig embryo transfer technology effectively:
(1) because trocar sheath is the plastics quality, it can be smoothed out with the fingers the into very dark position of fallopian tube, make the embryo can move on to only position, thereby improve the success rate of embryo transfer greatly.
(2) because ovum shifting tube is to insert transplantation site by trocar sheath, ovum shifting tube does not contact with endosalpinx or cornua uteri inner membrance in the process of inserting, thereby overcome mucus on endosalpinx or the cornua uteri inner membrance to the adsorption of embryo in the ovum shifting tube, can not cause losing of embryo, effectively improve the efficient of embryo transfer.
(3) because trocar sheath is the plastics quality, in the process of inserting fallopian tube or cornua uteri, even the operator have a little accidentally or the receptor sow disturbance is arranged, can not cause yet trocar sheath fracture or broken, the not graft failure that can therefore cause or the damage of receptor sow reproductive tract.
Description of drawings
Fig. 1 is this utility model embodiment common oviduct transplantation trocar sheath sketch map.
Fig. 2 is angle, a this utility model embodiment uterus transplanting trocar sheath sketch map.
The specific embodiment
Below in conjunction with drawings and Examples this utility model is described further.
The apparatus that is used for the pig embryo transfer comprises that ovum shifting tube, common oviduct transplantation are transplanted with trocar sheath or cornua uteri and uses trocar sheath that ovum shifting tube is in each trocar sheath inside; The common oviduct transplantation trocar sheath is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw, and two ends polish cunning; It is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw that cornua uteri is transplanted with trocar sheath, is cut into 15 ° of-20 ° of inclined-planes at the one end with shears, and the other end polishes cunning.Trocar sheath is rinsed the back natural drying well, sterilizes under uviol lamp with preceding.
When being used for 1-4 cell stage embryo's transplanting, at first the common oviduct transplantation with this utility model embodiment inserts the oviducal ampulla of receptor sow with trocar sheath; When being used for the above embryo's of 4 cell stages transplanting, at first the cornua uteri of this utility model embodiment is transplanted the proper site of inserting receptor sow cornua uteri with trocar sheath; The ovum shifting tube that then suction is had the embryo is inserted into the proper site of oviducal ampulla of receptor sow or cornua uteri by trocar sheath, implements to transplant again.
Concrete steps are as follows:
(1) common oviduct transplantation method: operative incision behind receptor sow anesthesia Baoding, fallopian tube and ovary are drawn outside the otch; Earlier common oviduct transplantation is inserted fallopian tube with trocar sheath from uterine tube umbrella mouth, with hands fallopian tube is smoothed out with the fingers forward along trocar sheath, until ampulla; Then suction there is embryo's ovum shifting tube to insert trocar sheath,, again trocar sheath is withdrawed from the 1-2 centimetre until feeling that with hands ovum shifting tube has passed 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 fimbriae of uterine tube, sews up wound routinely;
(2) cornua uteri implantation method: operative incision behind receptor sow anesthesia Baoding, cornua uteri is drawn outside the otch; Earlier cornua uteri is transplanted with 15 ° of-20 ° of beveled end of trocar sheath and avoided blood vessel insertion cornua uteri inner chamber from the proper site of cornua uteri, trocar sheath enters the degree of depth 4-5 centimetre of cornua uteri inner chamber, confirms that with feel trocar sheath is in cavity of uterus; Then suction there is embryo's ovum shifting tube to insert trocar sheath, until feeling that with hands ovum shifting tube has passed trocar sheath 0.5-1 centimetre; At last embryo in the ovum shifting tube and culture fluid are moved in the cornua uteri; Transplanting finishes, and carefully ovum shifting tube and trocar sheath is withdrawed from, and restores cornua uteri, sews up wound routinely.
Described embryo comprises that the embryo of the internal fertilization of pig, the formed reconstruct embryo of embryo, embryonic cell nuclear transplantation or body-cell neucleus transplanting of external fertilization, genetically modified embryo or blastaea inject formed chimeric embryo.
The embryo transfer 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) PMSG 1000IU 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
Behind the donor insemination of sows 3-6 days, 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 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 uterus or cornua uteri at the front and back position with the pelvic cavity boundary, touches the back and refers to clampings with two, draws the surface to wound.
(4) embryo collection
According to difference towards the ovum time, near or far away, there is not the position of blood vessel to cut an aperture with eye scissors at the cornua uteri back side of distance Utero-fallopian tube junction surface 30-70cm, insert cornua uteri towards oviduct, opening is to palace pipe jointing portion direction, and other end opening connects surface plate or plate.Inject 30-50ml towards ovum liquid from palace pipe jointing portion to cornua uteri with syringe.Through cornua uteri, the embryo is brought into surface plate or the plate that connects ovum towards the ovum liquid stream.
Again the opposite side cornua uteri is drawn outside the otch, use the same method to dash and get the embryo.
4, embryo's collection and quality examination
The surface plate that connects ovum is placed under the stero microscope, in ovum liquid, finding 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 earlier a little phosphate buffer PBS and inhale people's ovum again, on the diverse location flushing ovum of surface plate 3-5 time.Repeat flushing successively in second surface plate, then whole ovum are focused on a surface plate, 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.
Be divided into two kinds of modus operandi and non-modus operandis towards the method for ovum.Non-modus operandi only is applied to cattle at present, and collects after the embryo enters cornua uteri.And laboratory animals such as hog and mice, rat, rabbit are still used modus operandi in the pig, sheep etc.
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 cornua uteri drawn outside the wound.To be used for the trocar sheath beveled end that cornua uteri is transplanted earlier, and avoid blood vessel from the proper site of cornua uteri and insert the cornua uteri inner chamber, trocar sheath enters 4 centimeters of the degree of depth of cornua uteri inner chamber, confirms that with feel trocar sheath is in cavity of uterus; 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 cornua uteri.Transplanting finishes, and carefully ovum shifting tube and trocar sheath is withdrawed from, and restores cornua uteri, sews up wound routinely.It is the hard of long 65mm, lumen diameter 3mm, thickness of pipe wall 0.1mm, resilient igelite tubule that cornua uteri is transplanted with trocar sheath, and the one end is cut into 15 ° of inclined-planes, and the other end polishes cunning.
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 omega-fatty acid gene pig embryo's transplanting
1, the preparation of omega-fatty acid gene
The omega-fatty acid gene plasmid enzyme action 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.The tractive ovary of can not exerting oneself can not directly be pinched ovary with hands, more can not touch ovulation point and congested follicle.Observe ovary surface ovulation point and follicular development, itemized record.
To insert towards the horn mouth of oviduct one end by fimbriae of uterine tube portion, about 1-2cm is dark, holds to become to go in ring with thumb and forefinger and fixes another termination collection ovum ware.With syringe draw 37 ℃ dash ovum liquid 15-20ml,, near oviducal position syringe needle is thrust towards the fallopian tube direction at cornua uteri, the finger of a hands is clutched cornua uteri at the syringe needle rear. another manual injection device, flow into fallopian tube towards ovum liquid by palace pipe jointing portion, flow to collection ovum ware through fallopian tube.The opposite side fallopian tube is drawn outside the otch, embryo collection uses the same method again.
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 omega-fatty acid cdna 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, that is: the embryo who gathers from which sow retracts again in the fallopian tube of this sow behind gene injection.
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.Earlier trocar sheath is inserted fallopian tube from uterine tube umbrella mouth, with hands fallopian tube is smoothed out with the fingers forward along trocar sheath, until ampulla; Then suction there is embryo's ovum shifting tube to insert trocar sheath,, trocar sheath is withdrawed from 1 centimeter again until feeling that with hands ovum shifting tube has passed 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 fimbriae of uterine tube, sews up wound routinely.The common oviduct transplantation trocar sheath is the hard of long 70mm, lumen diameter 4mm, thickness of pipe wall 0.2mm, resilient igelite tubule, and two ends polish cunning.
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.
Claims (1)
1. be used for the apparatus of pig embryo transfer, comprise ovum shifting tube, it is characterized in that: comprising also that common oviduct transplantation is transplanted with trocar sheath or cornua uteri uses trocar sheath, and ovum shifting tube is in each trocar sheath inside; The common oviduct transplantation trocar sheath is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw, and two ends polish cunning; It is the hard of long 65-70mm, lumen diameter 3-4mm, thickness of pipe wall 0.1-0.2mm, resilient plastic straw that cornua uteri is transplanted with trocar sheath, and the one end is cut into 15 ° of-20 ° of inclined-planes, and the other end polishes cunning.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009202277278U CN201492540U (en) | 2009-08-28 | 2009-08-28 | Appliance used for transplanting pig embryo |
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| CN2009202277278U CN201492540U (en) | 2009-08-28 | 2009-08-28 | Appliance used for transplanting pig embryo |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409021A (en) * | 2011-12-19 | 2012-04-11 | 浙江大学 | Establishment and culture method of Jinhua pig fibroblast cell line |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409021A (en) * | 2011-12-19 | 2012-04-11 | 浙江大学 | Establishment and culture method of Jinhua pig fibroblast cell line |
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Granted publication date: 20100602 Termination date: 20140828 |
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