CN103184189A - Cultivation method of cross-bred wagy - Google Patents
Cultivation method of cross-bred wagy Download PDFInfo
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- CN103184189A CN103184189A CN2012103696576A CN201210369657A CN103184189A CN 103184189 A CN103184189 A CN 103184189A CN 2012103696576 A CN2012103696576 A CN 2012103696576A CN 201210369657 A CN201210369657 A CN 201210369657A CN 103184189 A CN103184189 A CN 103184189A
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Abstract
The invention provides a cultivation method of cross-bred wagy. The method comprises the steps of culturing cross-bred wagy embryo in vitro and transplanting the cultured embryo into uterus of receptor cow to breed, wherein the embryo in-vitro culture method comprises the following steps: (1) taking an ox with 99.9% of Japanese wagy hereditary characters as a paternal line, and Holstein cow as a maternal line, respectively extracting semen of the paternal line and ovocyte of the maternal line; (2) culturing the ovocyte in ovocyte follicle fluid; (3) performing in-vitro fertilization on mature ovocyte in an in-vitro fertilization culture solution; (4) culturing the fertilized ovum in the embryo culture solution; and (5) vitrifying the in-vitro embryo. The in-vitro cultured embryo is transplanted into the uterus of receptor cow by a bilateral uterine lining embryo transplantation method. At last, the cross-bred first filial generation wagy new strain with characters of Japanese wagy and Holstein cow is cultivated, and has high table purpose value and good production performance.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to the method for cultivation of a kind of hybridization and ox.
Background technology
Japan and ox are that Japan Kobe is hedged off from the outer world by several centuries, the long-term beef breed of cultivating.Japan and ox are the outstanding beef breeds of generally acknowledging in the world, the beef quality that utilizes this kind to produce also is unique, its characteristics are: quality softness, delicate succulence, have dense beef-flavouring, the beef outward appearance shows has extremely strong marbling, and how starlikely being there be being distributed in the beef of causing as snowflake is even, fresh and tender good to eat, be rich in iron and unsaturated fatty acids, can significantly improve energy and endurance, and can delay body aging.Deserve to be called the superfine product in the beef product.Japanese government has been regarded as Japanese and ox the ox of national treasure level; Japanese government in last century the nineties promulgate that ban " forbids the outlet with ox and genetic material thereof "; comprising Japanese and ox cattle on the hoof, seminal fluid, embryo, make Japan and the best hereditary beef cattle product in the Niu Zuowei world tie up to the Japanese development of country in addition and obtain limiting.
Review that north America region is created pure lines Japan and ox history is known, 1976 because Japanese government and United States Government's bilateral agreements regulation at that time, and it is that the bull import of 4 Japan of purpose agreement and ox is to the U.S. that Japanese government exchanges with scientific development research with agrotechnique; To introduced 200 bull bull and cows again respectively for twice in the nineties in last century.Because Japanese government takes to forbid Japan and ox genetic material to export, because this twice introduction of and ox Japanese in last century allows the U.S. and Canada seize the opportunity, multiplies pure Japan and ox strain, becomes except Japan, really has the country of Japan and ox strain.The U.S. and Canadian government and related tissue pay much attention to the development of Japan and ox, make careful Japan and ox and follow the trail of and assessment method.The offspring of these pure lines and ox is distributed in U.S.A and adds as number few area and farm at present, has complete pedigree also can carry out the tracking of DNA paternity test.Nowadays reach more than 99.9% with the similar purity of the gene of ox in North America Japan, really have except Japan, have the country of Japan and ox strain.
Yet though Japan and ox are to generally acknowledge one of best beef cattle in the world, because it is small, dressing percentage is low, and the later stage is fattened factors such as cost height.Except Japan, Japan and ox are squeezed all the time outside main flow world beef cattle industry.In order to utilize the U.S. and Canadian Japan and Niu Ziyuan, and the low and high problem of feeding cost of meat yield of small-sized Japan and ox can be solved, Japan and the most significant marbling meat characteristic that is rich in of ox can be guaranteed again.By analyzing the various cattle breeds that U.S. Canada abounds with, comprising Angus, Hareford, Xia Luolai, sharp wood praise, the every index of meat of Xi Mentaer and He Sitan finds, though He Sitan beef matter grade is low, is rich in marbling, it is the ox strain of Japan and ox only time.Holstein cow is very abundant in the U.S. and Canadian population, because characteristics such as its milk yield height, milk quality height, is that world's industry generally acknowledges to have world's the best gene population that gives milk.
Exploitation and the research of technology in vitro fertilization start from the sixties, at the seventies achievement in research is just arranged, the external embryo of early eighties research theoretically, method is able to improve fully and develop rapidly, becomes the important research branch of a fundamental sum in animal embryo engineering and the cell engineering field.Nineteen eighty-two U.S. scientist (people such as Bracket) takes the lead in cultivating the first in the world carcass calf that is fertilized outward, makes domestic animal Study on Technology in vitro fertilization obtain important breakthrough.People (1985) such as Japan scientist Hua Tianzhang are from butchering the ovocyte of cow ovary, cultivate through artificial ageing, in vitro fertilization and cell cultures is transplanted in recipient cattle after becoming the embryo, institute successfully gives birth to the test tube calf in Japanese national herding, really starts the beginning that external Embryo Production technology is used.
Carry out the livestock products breed improvement with embryo transfer, being the non-operation embryo transfer of the first ox in the world in 1964 succeeds and after Britain scientist in 1974 invented the embryo cryopreservation method, embryo transfer just began to prevail in developed country as breeding stock breed improvement and the good heredity means that go down to posterity in Japanese national herding institute.About the embryo vitrifying freeze technology, Scheffen in 1986 etc. have at first reported and have utilized the supper-fast freezing ox mulberry of 25% glycerine+25%1,2 one propylene glycol vitrifyings to bear embryo one blastula embryo.The back single stage method of thawing removes cryoprotectant, has obtained 53% transplant pregnancy rate, has reduced the toxicity of vitrification solution.Utilize the outer embryo of method for vitrification Frozen Body have simple to operate, cost is low, hatching rate advantages of higher behind the embryo thawing, and shortcoming to be the requirement operator have the skill ability that can grasp this method.
Summary of the invention
The object of the present invention is to provide the method for cultivation of a kind of hybridization and ox.
The present invention is to be maternal side with Canadian holstein cow, utilize Canadian slaughterhouse holstein cow ovary resource and extract and obtain ovocyte, be paternal line to have 99.9% Japan and the breeding oxen of ox inherited character, obtain paternal seminal fluid, cultivate the filial generation beef cattle strain with Japan and ox and holstein cow hereditary property by external embryo culture method.Japan of the present invention and ox seminal fluid available from Canadian BC province this that than (the Jingjing Genetic Inc. of the smart hereditary company in city; Burnaby, BC, Canada).
The method of cultivation of hybridization provided by the invention and ox comprises that hybridization and ox embryo's vitro culture and embryo transfer goes into the recipient cattle intrauterine and breed two steps.
Wherein, the outer cultural method of hybridization and bovine embryo comprises the steps:
1) be paternal line with the bull with 99.9% Japan and ox inherited character, holstein cow is maternal, extracts paternal seminal fluid and maternal ovocyte respectively;
2) in oocyte maturation liquid, cultivate ovocyte;
3) ovocyte of maturation is fertilized in nutrient solution in vitro fertilization;
4) zygote is cultivated in embryo medium.
The outer cultural method of hybridization of the present invention and bovine embryo also comprises the external embryo's of step 5) glass freezing.
Step 2 in this cultural method) preferably the ovocyte that cleans up is moved in the tapered tube that 2mL oocyte maturation liquid is housed and cultivates with 100 pieces/pipe.
The compound method of oocyte maturation liquid step 2 described in this cultural method) is:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) oocyte maturation liquid: add hydroxyethyl piperazine second thiosulfonic acid 20-40mL respectively in the cultivation mother liquor, follitropin 0.1-0.3mL, bSA 5-15g, phenol red 0.01-0.02g, penicillin 40-60IU, Streptomycin sulphate 40-60IU adds behind the mixing again and cultivates mother liquor to 1000mL, and regulating the pH value is 7.2.
Step 2 described in this cultural method) culture condition is 5%CO
2, 38.5-39 ℃, cultivate 22-24h.
The in vitro fertilization of step 3) described in this cultural method comprises:
A) sperm quantity is about 2,000 ten thousand seminal fluid is put into the centrifuge tube that is added with 3mL nutrient solution in vitro fertilization, and is centrifugal with whizzer 1500g rotating speed, with the centrifugal sperm that obtains of 400mL nutrient solution dilution in vitro fertilization;
If frozen semen, working method is to take out a frozen semen tubule (0.22cc from liquid nitrogen, wherein sperm quantity is about 2,000 ten thousand) in 30 ℃ of water-baths, thawed 10 seconds after, after cutting off with the sterilization scissors, seminal fluid in the tubule is put into the centrifuge tube that is added with 3mL nutrient solution in vitro fertilization, centrifugal with whizzer 1500g rotating speed, with the centrifugal sperm that obtains of 400mL nutrient solution dilution in vitro fertilization;
B) ovocyte with maturation contains in the fertilization dish of nutrient solution in vitro fertilization with the immigration of 100 pieces/hole, adds the sperm liquid after diluting, and making every hole sperm concentration is 1-2 * 10
6Individual/mL.
The compound method of the nutrient solution in vitro fertilization of step 3) is described in this cultural method:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) nutrient solution in vitro fertilization: cultivate adding hydroxyethyl piperazine second thiosulfonic acid 40-60mL in the mother liquor, heparin 0.4-0.8mL, fructose 0.8-1.5g, lactic acid 5-15mL, bSA 5-8g, phenol red 0.01-0.02g, penicillin 40-60IU, Streptomycin sulphate 40-60IU adds behind the mixing again and cultivates mother liquor to 1000mL, and regulating the pH value is 7.2.
The culture condition of step 3) described in this cultural method is 5%CO
2, 38.5-39 ℃, cultivate 20-22h.
The compound method of the embryo medium of step 4) is described in this cultural method:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) embryo medium: add non-essential amino acid 10-15g respectively, indispensable amino acid 15-25g, fetal bovine serum albumin 50-70g in the cultivation mother liquor, inositol 0.2-0.4g, penicillin 40-60IU, Streptomycin sulphate 40-60IU, add behind the mixing again and cultivate mother liquor to 1000mL, regulating the pH value is 7.2.
The culture condition of step 4 described in this cultural method is 5%CO
2, 38.5-39.2 ℃, embryoplastic incubation time is the 7th and 8 day of 168-200h(after fertilization from the oocyte fertilization to the blastaea).
Above-mentioned steps 5) described embryo's balance liquid prescription is:
A) embryo's balance liquid: bSA 10-12g, ethylene glycol 180-220mL, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL;
B) embryo vitrifying liquid: bSA 10-12g, ethylene glycol 300-400mL, ficoll 200-250g, sucrose 0.2-0.3mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL;
C) embryo vitrifying freeze liquid: according to volume ratio 60% embryo's balance liquid+40% embryo vitrifying liquid, the mixing configuration forms.
The embryo vitrifying freeze method of this step 5) is specially, get four porose discs, the embryo's balance liquid and the embryo vitrifying liquid that in each hole, add inequality, be configured to the solution of different permeability, 39 ℃ of preheating 10min, the blastaea embryo is put into each hole solution respectively carry out balance, starting time 1-2 minute, get Glass tubing (diameter 0.5mm), utilize its siphon with in embryo's tail pipe, loading embryo quantity in every Glass tubing is no more than 10 pieces, the height of refrigerating fulid should be controlled at 2-3mm in the Glass tubing, to smoke about 3 seconds on the liquid nitrogen surface by the Glass tubing of embryo's one end then, and treat to preserve until use in the whole Glass tubing input liquid nitrogen again after refrigerating fulid freezes in the glass-tube.
The present invention also provides the method for cultivation of a kind of hybridization and ox, take uterus bilateral inwall embryo transfer method, the outer cultural method of above-mentioned hybridization and bovine embryo cultivated obtain hybridization and the recipient cattle intrauterine is gone in the ox embryo transfer, breed the new born bovine that obtains and be and hybridize and ox.
The method of cultivation of above-mentioned hybridization and ox also comprises the glass freezing embryo of thawing before the embryo transfer, defreezing method is: respectively with embryo vitrifying thawing solution and embryo thawing balance liquid at 39 ℃ of preheating 10min, take out the glass freezing embryo and preserve glass-tube, in air, stop and to freeze after 3 seconds in the end immersion vitrifying thawing solution that external embryo is arranged, liquid in pipe is discharged in the thawing solution together with the embryo; The embryo is moved into balance 5min in the new embryo vitrifying thawing solution, move into balance 5min taking-up in the embryo thawing balance liquid again, move in the embryo medium that balance is good until use;
Wherein embryo vitrifying thawing solution prescription is: bSA 8-10g, and ethylene glycol 300-350mL, ficoll 150-200g, sucrose 0.4-0.5mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL;
Embryo thawing balance liquid prescription is: bSA 8-12g, and ethylene glycol 180-220mL, sucrose 0.07-0.1mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL.
Select for use the Chinese Cattle catalo as recipient cattle in one embodiment of the present of invention, take recipient cattle uterus bilateral inwall embryo transplantation method, cultivate hybridization and ox new lines.
Requirement as the Chinese Cattle catalo of recipient cattle be healthy no reproductive disease, balanced in nutrition, body condition is moderate, 18-24 monthly age, the cow more than the body weight 450kg.
Oestrusing for inducing the synchronization of estrus method of recipient cattle, its concrete scheme is: put vaginal suppository+2 milliliter R551(estradiol: interstitialcellstimulating hormone (ICSH)=1:1 on the 0th day, volume ratio), beat 0.4 milligram of PG on the 5th day, got bolt on the 7th day, made a call to 1 milliliter of R550(estradiol: progesterone=1:2, volume ratio on the 8th day); The observation of oestrusing of continual recipient cattle should be taked in behind vaginal suppository synchronization of estrus scheme implementation the 9th day.
The recipient cattle embryo transfer time is that the recipient cattle feature of oestrusing is obvious, if any mounting; Corpus luteum on the ox ovary reaches more than 1.5 centimetres, as yet not ovulation.
The embryo thawing method is: get one or four hole culture plates, add embryo medium, be placed on 5%CO
2, standby in the 38.8-39.2 ℃ of incubator.Get the culture plate of 6 cm diameters, add embryo vitrifying thawing solution and embryo thawing balance liquid (table 10 and table 11) respectively, 39 ℃ of preheating 10min.Embryo vitrifying thawing solution dish is placed under the stereomicroscope good burnt, from liquid nitrogen container, take out a glass freezing embryo's preservation pipe, clamp glass-tube with thumb and forefinger, in air, stop after 3 seconds, to freeze the end that external embryo is arranged immerses in the vitrifying thawing solution dish, when seeing that little glass-tube intercrystalline thaws, seal the suitable for reading of glass-tube with forefinger at once when liquid level rises in the glass-tube, liquid in pipe is discharged in the thawing solution together with the embryo.At once the embryo is moved in the new embryo vitrifying thawing solution balance and take out after 5 minutes, move in the embryo thawing balance liquid dish balance again and took out in 5 minutes, move in the embryo medium dish that balance is good until use.
Recipient cattle uterus bilateral inwall embryo transplantation method is that the good glass freezing embryo of will thawing puts into the embryo transfer rifle, and the embryo is put into definite recipient cattle uterus bilateral inwall of having oestrused, and finally cultivates hybridization and ox new lines.
The present invention implements vitrification method to external embryo, among the present invention, the concept of " vitrifying " refers to that water or solution fast cooling reach or when being lower than-100 ℃～-110 ℃ temperature range, form a kind of full-bodied between liquid and solid-state between, non-crystal, transparent vitreous state.Vitrifying freeze process is according to physics principle; the cryoprotective agent of high density is solidified under ultra-low temperature surroundings; form random vitrifying solid, normal molecular and ion distribution when this solids mass-energy keeps liquid, thereby the generation vitrifying plays a protective role in cell.This method has not only been simplified refrigerating process greatly, and has reduced because the cell ice crystal forms caused a series of physics and chemical damage.Molecular motion stops fully in the cell, thereby can reach the purpose of permanent preservation.
The hybridization that the inventive method is cultivated and the beef of ox strain are rich in marbling and quality softness, fresh and tender good to eat, improve the meat standard of holstein cow greatly, rise to B4 level level from original C level beef, increased the meat productivity effect of He Sitan catalo.In method of cultivation provided by the invention, oocyte maturation tapered tube culture technique and nutrient solution thereof reach more than 99% the maturing rate of ovocyte; Nutrient solution in vitro fertilization provided by the invention can increase substantially the in vitro fertilization of mature oocyte, makes mass-producing ovocyte spilting of an egg rate in vitro fertilization up to more than 86%; Use zygote extracorporeal culturing method and the embryo medium of invention, by improving fertilized egg cell's division and vigor and the health in each stage of developing embryo, A and B level embryo's mass-producing germ extraction rate is increased to more than 36%; External love moral provided by the invention and ox embryo's glass freezing technology makes external embryo's survival and hatching rate improve 20% than conventional freezing method, and hatching rate reaches more than 96%.The present invention has reached the meaning that possesses the industrialization practice.Take among the present invention the selection of recipient cattle and the requirement of oestrusing, and the outer embryo transfer of recipient cattle uterus bilateral inner wall, make external embryo's pregnancy rate reach 54%, and calving rate reach 48%, wherein twinning rate accounts for 25% of calving rate.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment, used biochemical reagents are commercially available among the embodiment.
Obtaining with maturation of embodiment 1 ovocyte cultivated
Collect Canadian holstein cow ovary from Canadian slaughterhouse, after handle in the laboratory, extract the ovocyte that obtains.Its working specification is as follows: the apparatus of (a) gathering and load and transport ovary must clean up in advance; (b) prepare physiological saline after high pressure sterile in advance, add 1% antibiotic before using, and insulation in 28 ℃ water bath until use; (c) gather and not have obvious pathology and not through the cow ovary of fecal pollution, ovary is put into the vacuum flask of pre-packed physiological saline; (d) exsomatize to the time that is utilized from the ovary collection and be no more than 10 hours.
The extraction operation program of ovocyte is: (a) ovary is transformed in the wide-mouth vacuum flask, pours about 30 ℃ into and be added with 1% antibiotic physiological saline to the submergence ovary, use behind the cleaned ovary; (b) 18G injection needles on the 10mL syringe jacket, (prescription sees Table 1 to draw a small amount of 601 liquid, the concrete prescription that present embodiment adopts is: sodium-chlor 6g, Repone K 0.5g, potassium primary phosphate 0.2g, magnesium chloride 0.1g, Sodium.alpha.-hydroxypropionate 0.5mL, glucose 0.3g, sodium bicarbonate 2g, Sodium.alpha.-ketopropionate 0.03g, calcium chloride 0.3g, glutamine 0.15g, adding distil water is to 1000mL, and regulating the pH value is 6.8, and sterilising filtration obtains) lubricated pin, insert syringe needle in the about 3mm of distance ovarian follicle position then, extract the ovarian cumulus ovocyte complex body (COCs) in the ovarian follicle of 2 ~ 5 millimeters of ovary surface diameters one by one; (c) liquor folliculi of extracting out is expelled in the 60mm culture dish, with sediments microscope inspection picking ovocyte.
(abbreviate oocyte maturation liquid as 602 liquid among the present invention extracting preceding 2 hours of ovocyte, prescription sees Table 2, the concrete prescription that adopts in the present embodiment is: add hydroxyethyl piperazine second thiosulfonic acid 30mL respectively in the cultivation mother liquor of 800mL, follitropin 0.2mL, bSA 10g, phenol red 0.01g, penicillin 50IU, Streptomycin sulphate 50IU adds behind the mixing again and cultivates mother liquor to 1000mL, regulating the pH value is 7.2), put into CO
2Pre-thermal equilibrium in the incubator.In the oocyte maturation mother liquor, add 1% antibiotic before the ovoscopy, get 2 milliliters of above-mentioned oocyte maturation liquid behind the mixing and put into 15 milliliters of tapered tubes, put back to CO then
2Incubator is stand-by.Satisfactory 100 ovocytes that will find at microscopically are one group, putting into the 60mm culture dish that is added with 602 ripe liquid cleans, 100 ovocytes that will clean then move into and are equipped with in 15 milliliters of tapered tubes of 2 milliliters of above-mentioned 602 ripe liquid, put into 5%CO
2Incubator carries out maturation and cultivates.Culture condition is that 38.5 ℃, the air of 5% carbonic acid gas, incubation time are 23 hours.The configuration of 602 oocyte maturation liquid is to cultivate mother liquor (prescription sees Table 1) basis 601 to prepare, and is placed on after sterile filtration in 4 ℃ and preserves, and is standby.
The present invention utilizes tapered tube that ovocyte is closely gathered naturally, because the maturation in vitro that closely helps ovocyte of cumulus cell and in vitro fertilization.And in promoting 602 ripe liquid of oocyte maturation composition make ovocyte be in more near animal body in envrionment conditions, gonad-stimulating hormone is conducive to change the metabolism of cumulus cell, and blocking-up is connected to the approach of ovum secretion meiosis inhibitory factor by intercellular by cumulus cell, thereby make ovocyte recover ripe fission process, improve the maturing rate of ovocyte greatly.This example cultivation results shows, adopts method of the present invention can make its oocyte maturation rate arrive 98%, and this result exceeds 10% than the oocyte maturation rate of usual way.
Table 1 is cultivated the prescription (pH6.8) of mother liquor (601)
The prescription (pH7.2) of table 2 oocyte maturation liquid (602)
Embodiment 2 is in vitro fertilization
(be called 603 nutrient solutions, its prescription sees Table 3 to nutrient solution in vitro fertilization of the present invention.The concrete prescription of 603 nutrient solutions is in the present embodiment: add hydroxyethyl piperazine second thiosulfonic acid 50mL in the cultivation mother liquor of 800mL respectively, heparin 0.6mL, fructose 1.2g, lactic acid 10mL, bSA 6g, phenol red 0.02g, penicillin 50IU, Streptomycin sulphate 50IU, add again behind the mixing and cultivate mother liquor to 1000mL, regulating the pH value is 7.2) be conventional fertilization nutrient solution (TALP) modified version, can change ovocyte matter by increase and meet more outside the sperm penetrance, and emphasis is considered the capacitation efficient of sperm.When external embryo carried out preceding 2 hours in vitro fertilization, get one or four porose discs, and in each hole, add 600 microlitres, 603 nutrient solutions, put into 39 ℃, 5%CO then
2Incubator is cultivated in advance.The precision processing of freezing in the method for cultivation of the present invention is, to directly put into 30 ℃ of water-baths from the frozen semen taking-up that liquid nitrogen is preserved thawed in 10 seconds, after drying with thieving paper, along the very long 15 milliliters of centrifuge tubes putting into the nutrient solution in vitro fertilization (603 nutrient solution) that is ready to be added with 3 milliliters in advance of test tube wall.After twice centrifugal 5 minutes of speed of 1600 rev/mins were handled, with the dilution of 400 microlitres, 603 nutrient solutions, with microscopic examination sperm activity, sperm activity should reach (sperm activity is lower than 40% and will be used) more than 40% at this moment.During external embryo fertilization, reached ripe ovocyte after maturation cultivated and put in the 603 good liquid of pre-thermal equilibrium and wash once, the good every hole that moves into pre-thermal equilibrium then respectively is added with in 1 milliliter of 603 nutrient solution.Add the sperm liquid after 25 microlitres are handled, make sperm concentration reach 2 * 10
6Individual/mL.Put into incubator then, about 39 ℃, cultivated 21 hours under the air conditions of 5% carbonic acid gas.Adopt external fertilization method of the present invention and cultivation, because serum albumin and cholesterol and calcium ion have very strong binding ability, the content of reducing cholesterol in plasma membrane causes the stability of plasmalemmae of sperms to change, and reaches thus and improves frozen sperm capacitation state.The result makes ripe ovocyte spilting of an egg rate reach 86%, such result exceeds 8 percentage points than the rate in vitro fertilization of using usual way, the results are shown in Table 4, wherein TCM199+TALP-1/-2/-3 is respectively 3 test group results of usual way, and 602 liquid+603 liquid-1/-2/-3 is respectively 3 test group results that adopt nutrient solution prescription of the present invention and cultural method.
The prescription (pH7.2) of table 3 nutrient solution in vitro fertilization (603)
Ripe and the spilting of an egg experimental result in vitro fertilization of table 4 ovocyte
Embodiment 3 extracorporeal culturing embryos
The configuration of embryo medium of the present invention is to cultivate mother liquor (prescription sees Table 1) basis 601 to add.Embryo medium abbreviates 604 nutrient solutions as among the present invention, and prescription sees Table 5.In the present embodiment, the concrete prescription of 604 nutrient solutions is: add glycine 3g respectively in the cultivation mother liquor of 800mL, L-Ala 2g, arginine 3g, L-glutamic acid 2g, Gelucystine 2g, Methionin 5g, methionine(Met) 5g, leucine 5g, phenylalanine 5g, fetal bovine serum albumin 60g, inositol 0.3g, penicillin 50IU, Streptomycin sulphate 40IU, add behind the mixing again and cultivate mother liquor to 1000mL, regulating the pH value is 7.2.Preserve finally by being placed on after the sterile filtration in 4 ℃, standby.Implementing external embryo culture preceding 2 hours, get four porose discs, and add 680 microlitres, 604 nutrient solutions in every hole, add 320 microlitres sterilization paraffin oil at its nutrient solution then, put into the CO about 39 ℃
2Stand-by in the incubator.
Mature oocyte after learning from else's experience 21 hours in vitro fertilization, ovocyte (about 400) is moved into 15 milliliters of centrifuge tubes that are added with 2 milliliter of 604 nutrient solution, centrifuge tube is placed on the vortex mixed instrument, with the 80% speed concussion of the fastest rotating speed after 3 minutes, with solution in the centrifuge tube to going in the 30mm culture dish, draw 604 liquid with the 1mL pipettor and wash around the centrifugal scale tube wall, the liquid sucking-off is incorporated in the same culture dish, repeated washing is one time in case of necessity.Choose at the ovum that microscopically is undesired and dead with form, other normal zygotes are cleaned 3 times with fresh 604.The zygote that cleans up is cleaned 1 time in 604 liquid, move into then in pre-incubated 604 culture plates, every hole is put into 100 pieces, requires the zygote in every hole to flock together during immigration.Observed ovocyte spilting of an egg situation (the normal fertilization ovum should be grown to more than 8 cells), and abandoned 4 cells and following zygote thereof in the 3rd day after being subjected to precision processing.If stay in the dish 8 cell count very little, then can and the dish, 8 cells or above zygote about 75 pieces are put in general every hole.Put back to 39 ℃, 5%CO then
2Continue in the incubator to cultivate, collect the embryo who has formed of after fertilization the 7th and 8, the oozy glass embryo cryopreservation of going forward side by side.
The prescription (pH7.2) of table 5 embryo medium (604)
Extracorporeal embryo development is cultivated because external embryo's growth will be passed through the early stage spilting of an egg, breaks through the stages such as 8～16 cell stage retardances, morula, blastaea and ovum blastaea, becomes very difficult.Because external embryo not only will be subjected to restriction and the influence of ectogenesis culture systems itself, but also to be subjected to fertilization process and the fertilization influence of oocyte maturation degree in the past.Broken away from uterine tube-intrauterine environment fully, required numerous nutritive ingredients, hormone, protein, the polypeptide factor of external embryo's further growth all will manage to satisfy.The embryo medium of Shi Yonging takes into full account the synthetic required nutritional needs of a large amount of albumen in external embryo's the cellular process in the present invention, to satisfy and ovocyte divides beyond its nutritional need, the present invention also will mix granulosa cell and grow the co-cultivation environmental system of the ovocyte that the germinal layer cell construction divided in culture systems, impel external embryo's spilting of an egg, break through the retardance of 8～16 cells.Present embodiment has added 6% foetal calf serum in cultivating mother liquor (601 mother liquor), because contained anchoring factor is again to the obvious promoter action of being formed with of granular cell layer in the serum; And the utilization of inositol makes that the quality of blastaea is more healthy.Compare the result with utilizing SOF (synthetic uterine tube liquid) nutrient solution usually, the germ extraction rate of zygote reaches 20%-25% than SOF nutrient solution commonly used and exceeds more than 10% and (the results are shown in Table 6) up to 35% in the inventive method.
Table 6 cleavage-cell grows the comparative experiments result who forms blastaea
Embodiment 4 external embryos' glass freezing
The glass freezing prescription that the present invention uses is: embryo's balance liquid (605), embryo vitrifying liquid (606) and embryo vitrifying freeze liquid (607), its prescription see Table 7, table 8, table 9.The concrete prescription of embryo's balance liquid (605) is in the present embodiment: bSA 10g, and ethylene glycol 200mL, penicillin 60IU, Streptomycin sulphate 80IU adds phosphate buffered saline buffer and transfers to 1000mL; The concrete prescription of embryo vitrifying liquid (606) is in the present embodiment: bSA 11g, and ethylene glycol 300mL, ficoll 250g, sucrose 0.2mol/L, penicillin 80IU, Streptomycin sulphate 60IU adds phosphate buffered saline buffer and transfers to 1000mL; The concrete prescription of embryo vitrifying freeze liquid (607) is in the present embodiment: according to volume ratio 60% embryo's balance liquid+40% embryo vitrifying liquid, the mixing configuration forms.
External embryo cryopreservation program is, at first making the solution that is used for embryo's adjusting seepage water pressure is: past hole 1, hole 2, hole 3 and hole 4 add 605 embryo's balance liquids 800,800,800 and 600 microlitres respectively to get four porose discs, past hole 3, hole 4 add 606 embryo vitrifying liquid 200,400 microlitres respectively then, mixing, preheating is 10 minutes on 39 ℃ of hot plates.
The prescription (pH7.0) of table 7 embryo balance liquid (605)
The prescription (pH7.0) of table 8 embryo vitrifying liquid (606)
The prescription (pH7.0) of table 9 embryo vitrifying freeze liquid (607)
The embryo vitrifying freeze Glass tubing is the common blood sampling Glass tubing that adopts 0.5 millimeter internal diameter, runs label information with marker pen well at glass before freezing.Among the external embryo in the solution in four different holes the residence time be respectively, hole 1 stops 2 minutes, hole 2 and stops 2 minutes, hole 3 to stop 1 minute, 4 residence time of embryo hole more short more good.When the embryo 3 when stopping, from hole 4 gets 20 microlitre liquid with transfer pipet in the hole, be placed on four porose discs and cover and make droplet.The embryo who rests on Kong Si is moved into the droplet for preparing, by syphonic effect the embryo is drawn in little glass-tube with the good Glass tubing of mark that (the loading embryo quantity in every Glass tubing had better not be above 10 pieces, the glass freezing liquid level is easy at 2-3mm in little glass-tube), to freeze then has the little glass-tube of embryo's one end to immerse in the liquid nitrogen after smoked about 3 seconds on the liquid nitrogen surface, treat again whole glass-tube to be dropped in the liquid nitrogen about 5.5min of whole embryo vitrifying freeze operating time after refrigerating fulid freezes in the glass-tube.External embryo finally reaches cell surface vitrifying state by the asymptotic change of seepage water pressure, forever preserves thereby can directly put into-196 ℃ of liquid nitrogen.
The glass freezing prescription that the present invention uses is: embryo vitrifying thawing solution (608) and embryo thawing balance liquid (609), its prescription see Table 10, table 11.
The concrete prescription of embryo vitrifying thawing solution (608) is in the present embodiment: bSA 9g, and ethylene glycol 300mL, ficoll 180g, sucrose 0.4mol/L, penicillin 70IU, Streptomycin sulphate 70IU adds phosphate buffered saline buffer and transfers to 1000mL.
The concrete prescription of embryo thawing balance liquid (609) is in the present embodiment: bSA 10g, and ethylene glycol 200mL, sucrose 0.08mol/L, penicillin 60IU, Streptomycin sulphate 80IU adds phosphate buffered saline buffer and transfers to 1000mL.
The prescription (pH7.0) of table 10 embryo vitrifying thawing solution (608)
The prescription (pH7.0) of table 11 embryo thawing balance liquid (609)
Glass freezing embryo's the program of dissolving is: embryo vitrifying thawing solution dish is placed under the stereomicroscope good burnt, from liquid nitrogen container, take out a glass freezing embryo's preservation pipe, clamp glass-tube with thumb and forefinger, in air, stop after 3 seconds, to freeze the end that external embryo is arranged immerses in the vitrifying thawing solution dish, when seeing that little glass-tube intercrystalline thaws, seal the suitable for reading of glass-tube with forefinger at once when liquid level rises in the glass-tube, liquid in pipe is discharged in the thawing solution together with the embryo.At once the embryo is moved in the new embryo vitrifying thawing solution balance and take out after 5 minutes, move in the embryo thawing balance liquid dish balance again and took out in 5 minutes, move in the embryo medium dish that balance is good until use.
The present invention uses thawing of external embryo experimental results show that by the external embryo of glass freezing with conventional, compare and freeze the dehydration of preservatives gradient with adopting lower concentration, through the program frigorimeter slowly after the thawing of external embryo cryopreservation technology of the adjective law of cooling embryonic hatching be 79%, adopt the external embryo cryopreservation of vitrifying and deforst technique, external embryo's hatching rate is increased to more than 96% (the results are shown in Table 12).
Hatching rate after table 12 vitrifying freeze process and conventional external embryo cryopreservation method are thawed relatively
Embodiment 5 external embryos' transplanting
External embryo transfer of the present invention is as recipient cattle with Chinese Cattle catalo (the Luxi Yellow cattle catalo of Chinese Qingdao Pingdu purchase), induce recipient cattle to oestrus at one time by the synchronization of estrus method, then the recipient cattle of confirming to oestrus is implemented recipient cattle uterus bilateral inwall embryo transplantation method, finally cultivate hybridization and ox new lines.Carry out embryo transfer, cultivate filial generation and ox new lines.
External embryo transfer of the present invention is, select the Chinese Cattle catalo (condition be healthy no reproductive disease, balanced in nutrition, body condition is moderate, the 18-24 monthly age is more than the body weight 450kg).Enforcement induces the synchronization of estrus program to be: put vaginal suppository (progestogen delayed release device)+2 milliliters of R551(estradiol: interstitialcellstimulating hormone (ICSH)=1:1 on the 0th day, volume ratio), beats 0.4 milligram of PG on the 5th day, got bolt on the 7th day, made a call to 1 milliliter of R550(estradiol: progesterone=1:2, volume ratio on the 8th day).Should take continual recipient cattle to oestrus in the 9th day behind vaginal suppository synchronization of estrus scheme implementation.When finding that the recipient cattle feature of oestrusing is obvious, if any mounting; Corpus luteum on the ox ovary reaches more than 1.5 centimetres, as yet not ovulation.Implement external embryo transfer scheme at once.
Transplanting the external frozen embryo thaw routine of using is: get one or four hole culture plates, add embryo medium (604), be placed on 5%CO
2, standby in 39 ℃ of incubators.Get 3 of the culture plates of 6 cm diameters, wherein 2 culture plates are added with 2 milliliters of embryo vitrifying thawing solutions (608) respectively, and another culture plate is added with 2 milliliters of embryo thawing balance liquids (609), 39 ℃ of preheating 10min.Embryo vitrifying thawing solution (608) dish will be housed to be placed under the stereomicroscope good burnt, from liquid nitrogen container, take out a glass freezing embryo's preservation pipe, clamp glass-tube with thumb and forefinger, in air, stop after 3 seconds, to freeze the end that external embryo is arranged immerses in the vitrifying thawing solution dish, when seeing that little glass-tube intercrystalline thaws, seal the suitable for reading of glass-tube with forefinger at once when liquid level rises in the glass-tube, liquid in pipe is discharged in the thawing solution together with the embryo.At once the embryo is moved into the middle balance of new embryo vitrifying thawing solution (608 liquid) and take out after 5 minutes, move into again balance taking-up in 5 minutes in embryo thawing balance liquid (609) dish is housed, move in the embryo medium dish that balance is good until use.
Recipient cattle embryo transfer testing sequence is: the experiment ox is totally 200 of Chinese Cattle catalos (the Luxi Yellow cattle catalo of Chinese Qingdao Pingdu purchase).100 is that one group of ox is implemented uterus bilateral inwall embryo transfer; 100 is that one group of ox is implemented the one-sided inwall embryo transfer in uterus.Its program is: the good glass freezing embryo of will thawing puts into the embryo transfer rifle, the embryo is put into definite recipient cattle uterus bilateral inwall of having oestrused, with the examination per rectum method ginseng is joined cow in 60 days after the external embryo transfer and carry out pregnancy check, finally cultivate hybridization and ox new lines, and calculate experimental result (seeing Table 13) such as calving rate respectively.
Experimental result shows, takes among the present invention the selection of recipient cattle and the requirement of oestrusing, and the outer embryo transfer of recipient cattle uterus bilateral inner wall, pregnancy rate is 54%, and calving rate is 48%, and wherein twinning rate accounts for 25% of calving number; And the pregnancy rate of the outer embryo transfer of the one-sided inner wall in recipient cattle uterus is 42%, and calving rate reaches 36%.Per hundred outer embryo transfer results of recipient cattle uterus bilateral inner wall are Duoed 24 than the calving number of the outer embryo transfer of the one-sided inner wall in recipient cattle uterus.Its embryo transfer efficient is remarkable, has commercial application and is worth.
The external embryo transfer experimental result of two kinds of different methods of table 13
Hybridization and Niu Tichong more than 800 kilograms, average marbling grade were 4-5 when the hybridization that the present invention is cultivated and ox new-born calve were delivered for sale through raising 24 monthly ages, comprehensively the meat grade is the B4 level.
Claims (13)
1. the outer cultural method of a hybridization and bovine embryo comprises the steps:
1) be paternal line with the bull with 99.9% Japan and ox inherited character, holstein cow is maternal, extracts paternal seminal fluid and maternal ovocyte respectively;
2) in oocyte maturation liquid, cultivate ovocyte;
3) ovocyte of maturation is fertilized in nutrient solution in vitro fertilization;
4) zygote is cultivated in embryo medium.
2. method according to claim 1 is characterized in that, also comprises the external embryo's of step 5) glass freezing.
3. method according to claim 1 is characterized in that, described step 2) be that the ovocyte that will clean up moves in the tapered tube that 2mL oocyte maturation liquid is housed and cultivates with 100 pieces/pipe.
4. method according to claim 1 is characterized in that, described step 2) the compound method of oocyte maturation liquid be:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) oocyte maturation liquid: add hydroxyethyl piperazine second thiosulfonic acid 20-40mL respectively in the cultivation mother liquor, follitropin 0.1-0.3mL, bSA 5-15g, phenol red 0.01-0.02g, penicillin 40-60IU, Streptomycin sulphate 40-60IU adds behind the mixing again and cultivates mother liquor to 1000mL, and regulating the pH value is 7.2.
5. method according to claim 1 is characterized in that, described step 2) culture condition be 5%CO
2, 38.5-39 ℃, cultivate 22-24h.
6. method according to claim 1 is characterized in that, the in vitro fertilization of described step 3) comprises:
A) sperm quantity is about 2,000 ten thousand seminal fluid is put into the centrifuge tube that is added with 3mL nutrient solution in vitro fertilization, and is centrifugal with whizzer 1500g rotating speed, with the centrifugal sperm that obtains of 400mL nutrient solution dilution in vitro fertilization;
B) ovocyte with maturation contains in the fertilization dish of nutrient solution in vitro fertilization with the immigration of 100 pieces/hole, adds the sperm liquid after diluting, and making every hole sperm concentration is 1-2 * 10
6Individual/mL.
7. method according to claim 1 is characterized in that, the compound method of the nutrient solution in vitro fertilization of described step 3) is:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) nutrient solution in vitro fertilization: add hydroxyethyl piperazine second thiosulfonic acid 40-60mL respectively in the cultivation mother liquor, heparin 0.4-0.8mL, fructose 0.8-1.5g, lactic acid 5-15mL, bSA 5-8g, phenol red 0.01-0.02g, penicillin 40-60IU, Streptomycin sulphate 40-60IU adds behind the mixing again and cultivates mother liquor to 1000mL, and regulating the pH value is 7.2.
8. method according to claim 1 is characterized in that, the culture condition of described step 3) is 5%CO
2, 38.5-39 ℃, cultivate 20-22h.
9. method according to claim 1 is characterized in that, the compound method of the embryo medium of described step 4) is:
A) mother liquor is cultivated in preparation: sodium-chlor 5-7g, Repone K 0.4-0.6g, potassium primary phosphate 0.1-0.3g, magnesium chloride 0.08-0.14g, Sodium.alpha.-hydroxypropionate 0.4-0.6mL, glucose 0.1-0.5g, sodium bicarbonate 1-4g, Sodium.alpha.-ketopropionate 0.01-0.06g, calcium chloride 0.1-0.5g, glutamine 0.05-0.25g, adding distil water is to 1000mL, regulating the pH value is 6.8, and sterilising filtration obtains cultivating mother liquor;
B) embryo medium: add non-essential amino acid 10-15g respectively, indispensable amino acid 15-25g, fetal bovine serum albumin 50-70g in the cultivation mother liquor, inositol 0.2-0.4g, penicillin 40-60IU, Streptomycin sulphate 40-60IU, add behind the mixing again and cultivate mother liquor to 1000mL, regulating the pH value is 7.2.
10. method according to claim 1 is characterized in that, the culture condition of described step 4) is 5%CO
2, 38.8-39.2 ℃, forming incubation time from oocyte fertilization to the blastaea embryo is 168-200h.
11. method according to claim 2 is characterized in that, the embryo vitrifying freeze method of described step 5) is:
A) embryo's balance liquid: bSA 10-12g, ethylene glycol 180-220mL, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL;
B) embryo vitrifying liquid: bSA 10-12g, ethylene glycol 300-400mL, ficoll 200-250g, sucrose 0.2-0.3mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphate buffered saline buffer and transfers to 1000mL;
C) embryo vitrifying freeze liquid: according to volume ratio 60% embryo's balance liquid+40% embryo vitrifying liquid, the mixing configuration forms.
12. the method for cultivation of a hybridization and ox, it is characterized in that, application rights takes uterus bilateral inwall embryo transfer method that the recipient cattle intrauterine is gone in embryo transfer after requiring the arbitrary described method vitro culture hybridization of 1-11 and ox embryo, breeds the new born bovine that obtains and is hybridization and ox.
13. method as claimed in claim 12, it is characterized in that, also comprise the glass freezing embryo of thawing before the embryo transfer, defreezing method is: respectively with embryo vitrifying thawing solution and embryo thawing balance liquid at 39 ℃ of preheating 10min, take out the glass freezing embryo and preserve glass-tube, in air, stop and to freeze after 3 seconds in the end immersion vitrifying thawing solution that external embryo is arranged, liquid in pipe is discharged in the thawing solution together with the embryo; The embryo is moved into balance 5min in the new embryo vitrifying thawing solution, move into balance 5min taking-up in the embryo thawing balance liquid again, move in the embryo medium that balance is good until use;
Described embryo vitrifying thawing solution prescription is: bSA 8-10g, and ethylene glycol 300-350mL, ficoll 150-200g, sucrose 0.4-0.5mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphoric acid buffer and transfers to 1000mL;
Described embryo thawing balance liquid prescription is: bSA 8-12g, and ethylene glycol 180-220mL, sucrose 0.07-0.1mol/L, penicillin 60-80IU, Streptomycin sulphate 60-80IU adds phosphoric acid buffer and transfers to 1000mL;
Described embryo medium 1L prescription is: add non-essential amino acid 10-15g respectively in the cultivation mother liquor, indispensable amino acid 15-25g, fetal bovine serum albumin 50-70g, inositol 0.2-0.4g, penicillin 40-60IU, Streptomycin sulphate 40-60IU adds behind the mixing again and cultivates mother liquor to 1000mL, and regulating the pH value is 7.2.
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| CN109414008A (en) * | 2016-05-13 | 2019-03-01 | 密苏里大学管理机构 | Prevent the freezen protective culture medium and method of recrystallization |
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| CN109064469B (en) * | 2018-10-31 | 2023-08-15 | 北京新网视信传媒科技有限公司 | Sperm quality detector and sperm quality detection system |
| CN109673623A (en) * | 2019-02-21 | 2019-04-26 | 白晓红 | A kind of glass freezing reagent and vitrifying defrosting reagent and its application and application method |
| CN111937812A (en) * | 2020-09-15 | 2020-11-17 | 广西华胥水牛生物科技有限公司 | Method for improving breeding efficiency of buffalo |
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