In vitro fertilization or external fertilization refers tomammalOfspermandeggIt is completed in an external manually controlled environmentFertilization processThe technology of IVF is called IVF for short.Because it is associated withEmbryo transfer technology(ET) is inseparable, also referred to asIVF-ET。In biology, in vitro fertilizationembryo transferAnimal scales obtained after arriving at the motherTest tube animal（test-tube animal）。This technology was successful in the 1950s and has developed rapidly in the past 20 years. It has become increasingly mature and has become an important and conventional animal reproduction biotechnology.
In June 2024, Sichuan is promoting the work of "including assisted reproductive medical services into the payment scope of basic medical insurance" according to the procedure (assisted reproductive technology includes in vitro fertilization), and striving to achieve medical insurance reimbursement of related projects across the province by the end of 2024.[1]
In vitro fertilization organisms in nature include fish and amphibians, which refer to sperm andeggstayfemaleThe mode of fertilization in which a fertilized egg is produced by combining outside the organism.This form of fertilization is usually accomplished in water.Sperm can use its ability to move in the water to swim to the egg and combine with it.
technological development
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Microoperation diagram of gene injection
From the early 1960s to the mid-1980s, people carried out a lot of basic research with rabbits, mice and rats as experimental materialsSperm capacitationGreat progress has been made in terms of mechanisms and enabling methods.Spermatozoa from the same or different speciesfemaleGenital tractIncubation and capacitation, developed into userUterine fluid、follicular Fluid、endometriumExtract or serum, etcin vitro cultureEnable, and finally use the chemical compositionSolution cultureYes.At the same time, by ejecting sperm andEpididymal spermThe comparative study of the capacitation effect found that the ejaculated semen containedTo be ableAnd realize that the essence of capacitation is to remove the disenergizing factors on the sperm surface.These theoretical and methodological achievements have promoted the development of IVF technology. In vitro mice (Whittingham, 1968), rats (Toyoda and Chang, 1974), infants (Steptoe and Edwards, 1978), cattle (Brackett et al., 1982), goats (Hamda, 1985), sheep (Handa, 1985) and pigs (Chang et al., 1986) were born successively.
Ⅳ F technology
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Since the mid-1980s, the livestock Ⅳ F technology represented by cattle has developed rapidly. In 1987, Parrish et al. treated the frozen semen of cattle with a medium containing heparin, and thenoocyteIn vitro fertilization was successful.This is of great significance for the research and application of cattle Ⅳ F, because this method can use theovaryAnd frozen semenembryoIn vitro production, not only low cost, but also stable effect.After that, bovine oocytes matured in vitro andEmbryo cultureThe system gradually became mature, and the efficiency of embryo production in vitro was greatly improved.useⅣF_ETEach pair of abandoned bovine ovaries can obtain about 3 calves.To make full use of improved cowsgenetic resources In the late 1980s, the technology of bovine ovum pick UP. OPU developed rapidly.The combination of live egg retrieval and Ⅳ F_ET has become an important breeding technology for farmers in Europe, the United States, Oceania and other developed animal husbandry countries to expand the selection of improved breed cows.
In the treatment of infertility, in vitro fertilization is associated with embryo transfer technology.That is to say, when the fertilized egg is divided under the condition of artificial incubation and reaches 8 to 16 cleavage cells, it is manually transferred into the uterus of the woman in secretory stage to implant the fertilized egg.The combination of in vitro fertilization and embryo transfer technology is popularly known“tube baby”Technology, with the development of science, this technology will play a greater role in the treatment of infertility.
Assisted in vitro fertilization (IVF) is the process of taking sperm or eggs out of the body, treating them or cultivating them into embryos, and then implanting them into the human body to overcome the obstacles of traditional treatment. The most familiar treatment is IVF.In fact, the simplest sperm washing combined with in vitro fertilization of uterus is also a kind of in vitro fertilization. For mild infertility diseases, such as mild poor sperm activityAntisperm antibodyAutoimmune diseases, cervical diseases, sexual intercourse andEjaculatory disturbanceThe pregnancy rate of patients treated with IVF is 20% each time.
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Collection culture
1．oocyteCollection of oocytes: There are usually three methods to collect oocytes.
（1）Superovulation: The main methods used for experimental animals such as mice, rabbits, domestic animals such as pigs and sheep are:;useGonadotropinTreatment, so that more eggs are discharged, and thenFallopian tubeMature eggs collected from the middle flush can be directly fertilized with capacitated sperm.In livestock, due to the complex operation procedure and high cost, it is seldom used.The key to this method is to master the time when the egg enters the fallopian tube and the egg maintains the fertilization ability in the fallopian tube. Generally, it is required to flush the egg before it has strong energy.
(2) From livingovaryCollect oocytes from living animals' ovaries by means of ultrasonic detector, endoscope or laparoscope.
In domestic animals, cattle, horses and other domestic animals often use ultrasonic detectors to assist in taking eggs by handrectumGrasping ovary, menstruationVaginal wallPuncture and insert the egg aspirating needle, and absorb theoocyte。According to the technical level, a healthy cow can obtain 5-10 eggs per week.In domestic animals, oocytes collected in vivo can not be fertilized with sperm until they are matured and cultured.This method is of great significance to the expansion of fine female livestock, and has been used for commercial production in some countries.
(3) From slaughtered female livestockovaryCollect oocytes from newly slaughtered female animals: this method is to take ovaries from newly slaughtered female animals, wash them and keep them warm (30 ℃~37 ℃), and then quickly transport them to the laboratory. Under sterile conditions, use a syringe or vacuum pump to aspirate oocytes from follicles with a certain diameter on the ovarian surface (the straight diameter of bovine follicles is required to be 3-10mm), and also slice the ovaries to collect oocytes.The oocytes obtained by this method are mostly in the germinal vesicle stage (GV stage), and can be fertilized with sperm only after in vitro culture and maturation.The key to collecting oocytes from abandoned ovaries is to keep the ovaries warm and prevent bacterial contamination.Therefore, after the ovaries are taken from the livestock, they must be put intonormal salineorPhosphate buffer(PBS)vacuum flaskMedium;Ovaries should be washed with normal saline or PBS several times before egg aspiration;Antibiotics should be added to all solutions used.
The greatest advantage of this method is that the material source is rich and the cost is low, but it is difficult to determine the pedigree of female animals.
2. Selection of mother cells: collectedoocyteMost of them are related toCumulus cellformationCumulus oocyte complex（cumulus oocyte complesx COC）。No matter what method is used, the collected COC requires regular oocyte morphology, uniform cytoplasm, and tightly surrounded by multiple cumulus cells.In the study of in vitro fertilization of domestic animals, immature oocytes are often divided into four grades: A, B, C and D.Grade A oocytes are required to have more than three layers of cumulus cells closely surrounded, with uniform cytoplasm;Level B requires that the oocyte cytoplasm is uniform, and the cumulus cell layer is lower than three layers or partially surrounds the oocyte;Grade C: naked oocytes without cumulus cells;Grade D refers to dead or degenerated oocytes.In IVF practice, only grade A and grade B oocytes are generally cultivated.
3. Maturation and culture of oocytes:SuperovulationThe collected oocytes have matured in vivo and can be directly fertilized with sperm without culturein vitro cultureMaturity.During culture, the collected oocytes are first placed in theSolid microscopeAfter selection and washing, it is put into mature culture medium for culture.livestockoocyteTCM199 is generally used to add pregnant bovine serum, gonadotropin, estrogen and antibiotic ingredients to the mature culture medium of TCM199.Usuallymicrodrop cultureMethod: The volume of microdrops is 50-200 microliters, and the number of oocytes put into each drop is calculated as per 5 microliters.After the oocytes are transferred into the dropletsCO2 incubatorMedium culture under 39 ℃, 100% humidity and 5%carbon dioxideAir.The cattle culture time is 20-24 hours,Cumulus ovaleAfter maturation and culture of oocyte complex, cumulus cell layer diffuses, and cumulus cells around oocytes are arranged radiallyRadial crown, stained with DNA special dye, and observed under the microscope, it can be seen that the oocyte is in the second maturationMetaphase of fission。
Sperm collection
1. False vagina method
2. Hand holding method
3. Electrical stimulation
Detailed introduction
1. SpermatogenicEnableTreatment: There are two methods for mammalian sperm capacitation: culture and chemical induction.Cattle and sheep spermatozoa are commonly used to induce capacitation with chemicals, and commonly used drugs to induce capacitationheparinandCalcium ion carrier。
2. Fertilization: that is, capacitation of sperm and mature eggCocultureIn addition to calcium ion carrier induced capacitation, sperm and eggs generally complete the fertilization process in capacitation fluid.The time of fertilization and culture is related to the method of capacitation.stayB2 liquidIn general, it takes 6-8 hours to use TALP or SF solution can be cultured for 18~24 hours when it is used as receiving semen.Spermatozoa and eggs are often co cultured in droplets, and the sperm density during fertilization is 1~9 × 10 6/ml, every 10Microlitre1~2 eggs are put into semen, and the volume of droplets is generally 50~200 μ l.
Embryo culture
After fertilization of sperm and eggs, the fertilized eggs need to be transferred to the development medium for further culture to check the fertilization status and the development potential of the fertilized eggsembryoIt can be moved into the reproductive tract of the recipient female animal to continue to develop and mature or carry outCryopreservation。
The key factor to improve the development rate of fertilized eggs is to select an ideal culture system.Among livestock,Embryo cultureThe culture medium can be divided into two categories: complex culture medium and clear chemical composition culture medium.There are many components in the complex culture medium, except inorganic andOrganic saltIn addition, vitamins, amino acidsnucleotideandpurineTCM199, B2 and F10 are the most commonly used nutrients and serum.When they are used to culture embryos, somatic cells can be usedCocultureSystem, that is, somatic cells and embryos are co cultured in microdrops, and beneficial factors secreted during somatic cell growth are used to promote embryonic development and overcome development blockage.
The culture of fertilized eggs is widely usedMicrodrop method，embryoThe ratio to culture medium is 3~10 μ L culture medium for one embryo;Generally, 5-10 embryos are cultured in a small drop to make use of theActive factorAnd promote the development of each other.Embryo cultureConditions andOocyte maturationThe culture conditions are the same.Some laboratories use 88% N2, 7% O2 and 5% CO2mixed gas To reduce the concentration of oxygen free radicals in the culture medium and improve the embryo development rate.In the process of embryo culture, it is required to change the culture medium every 48-72 hours, and observe the development of the embryo.When the embryo develops to a certain stageembryo transferorCryopreservationThe fertilized eggs of cattle and sheep are usually cultured to densityMorulaorblastulaTransplantation or cryopreservation shall be carried out.
Livestock information
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Development status
After nearly 20 years of development, in vitro fertilization technology for domestic animals has made great progress, among which cattle have the highest level of Ⅳ FoocyteThe cleavage rate (i.e. entering mature culture) is 80%~90%, the blastocyst development rate on the seventh day after fertilization is 40%~50%, the blastocyst continued development rate after cryopreservation is 80%, and the calving rate after transplantation is 30%~40%.Average eachovaryAbout 10 A-grade oocytes can be obtained, and 3-4 blastocysts can be obtained through in vitro fertilization, and 1-2 calves can be born after transplantation.
Existing problems
(1) The blastocyst development rate is low and the number of cells is small.In vitro fertilized eggs commonly exist in the process of cultureblock of development(developmental block), that is, the embryo stops developing and degenerates after it reaches a certain stage.cattleembryoBlocking occurs in the 8-16 cell stage, which results in the blastocyst development rate of IVF eggs being much lower than that of IVF eggsIn vivo fertilization。In addition, compared with in vivo fertilized blastocysts, the total number of cells of in vitro fertilized blastocysts andInner cell massThe number of cells decreased significantly.
(2) Low calving rate and high birth weight.In vitro fertilized embryos of domestic animals, especially bovine IVF embryos transferred into the recipient, the calving rate was 15%~20% lower than that of in vivo fertilization, but the newborn weight of the fetus was 3~4kg higher than that of the offspring of in vivo fertilization, which led to a high rate of dystocia in the recipient female animals.
development direction
(1) In depth study of the molecular mechanism of oocyte maturation and embryo development The main reason for the low efficiency of IVF is that peopleOogenesisandembryonic developmentThe molecular mechanism is not well understood.The premise of greatly improving the efficiency of Ⅳ F is to find outoocyteAnd the molecular regulation mechanism of early embryonic development, and then study the ideal culture system under the guidance of this theory to promote the stable and orderly expression of embryonic genome.
(2) Strengthen the research of preantral follicle culture and use the genetic resources of excellent female livestock Ⅳ The oocytes used by the technology are insufficient for domestic animalsovaryOne thousandth of the total number of upper oocytes.For this reason, on the one hand, we need to improve the technology of ovum retrieval in vivo, and on the other hand, we need to study the technology of in vitro maturation of preantral follicles and small follicles.In order to ensure the stable source of oocytes and the conservation of improved female animals or endangered animals, the ultralow temperature of follicles and oocytesCryopreservationTechnical research must also be strengthened.
(3) Strengthen the combination of IVF and other biotechnology.In vitro fertilization andtransgene, CloneGender controlandembryonic stem cellThe cultivation of is inseparable.In vitro fertilization can provide sufficientembryoSource;byCloning technologyProvide maturityoocyteAnd the culture system of cloned embryos;Sex control of mammals can be carried out by in vitro fertilization of isolated X and Y sperm and eggs.Similarly, the separation of embryonic stem cells also requires IVF technology to provide embryos and culture systems.The comprehensive development of these biotechnology will have a significant impact on human life.
Included in medical insurance
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In June 2024, Sichuan is promoting the work of "including assisted reproductive medical services into the payment scope of basic medical insurance" according to the procedure (assisted reproductive technology includes in vitro fertilization), and striving to achieve medical insurance reimbursement of related projects across the province by the end of 2024.[1]
