Progress and Industrialization of Sexually Controlled IVF Embryos
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1. The status quo of China's dairy industry and its strategic objectives for development The latest statistics show that the total number of dairy cows in China in 2002 reached 6.873 million (of which about 2 million were dairy cows, accounting for 29% of the total number of dairy cows), with a total milk production of 14.004 million tons (of which 12.998 million tons of milk, accounting for total milk production 92.8%). Calculated by 3.78 million adult cows (about 55% of the herd), the yield is only 3400kg, while the United States Israel and other countries is 8400kg, Denmark, France, Japan is more than 6500kg, the world per capita milk consumption is 97.6kg, the development of the country It is 50.9kg, China's 10.9kg, far below the world average.
According to the China Food and Nutrition Development Outline (2001-2010), by 2010, the national average milk consumption level will reach 16kg. Among them, urban residents will reach 32kg, and milk production safety and protection targets will be 26 million tons. After 7 years, milk production will need to increase by 11.794 million tons (including milk about 11.154 million tons), and the average annual increase in milk production will be 1.713 million tons (among which The milk is 1,593,000 tons. Therefore, it is required that the dairy industry in China must develop rapidly.
Dairy cows have insufficient resources and low yields. They have long been the main factor restricting China's development. At present, China's appropriate breeding cows account for about 55% of the total population, the annual reproductive survival rate is about 80%, and the annual average elimination rate is about 20%. The annual mortality rate is 3%, and the ratio of public funds and mother-in-law is 1:1. The objective law defines that the annual growth rate of dairy cows in China is about 10%. Therefore, relying on conventional technology to gradually develop the existing dairy cows or spend high prices to purchase some high-yielding dairy cows from abroad, it is impossible to achieve rapid development beyond the normal speed. Through the crossbreeding and improvement of local yellow cattle with semen cow semen, it has played a certain historical role in the rapid increase of the total number of dairy cows and total milk production in China since the 1980s, but it has caused China's total head Among the 6 million cows, only about 2 million Holstein cows have been improved, and adult cows have only produced more than 3,000 kilograms per year in recent years. The annual growth rate is about 100-200 kilograms per year. This is in contrast to international development in dairy cows. The main growth mode is to increase the level of individual production, rather than the development gap in the increase in the number of dairy cows. In Australia, from 1980 to 2000, the number of heads of dairy cows increased by 18%, the individual production of dairy cows increased from 2977kg to 5088kg, and the total milk production increased from 5.66 million tons to 11.17 million tons, and the milk production doubled.
Therefore, if the dairy cow industry in China is to achieve rapid development, it should speed up the genetic improvement of the elite breeder herds. It must also break the rules, apply the domestic and foreign cow embryo embryo bioengineering techniques and the gender control of segregation of elite bulls as soon as possible. Technical research results in order to achieve the purpose of rapid and healthy growth in the quantity and quality of dairy herds.
2. Research progress of sexually controlled IVF frozen embryos Utilizing the abundant ovarian resources obtained after slaughtering of high-producing dairy cows in North America, oocytes and the resources and technical advantages of in vitro fertilization, sperm separation, and in vitro fertilization of embryos in the United States and Canada , Production-controlled IVF frozen embryos and experimental study of transplantation have made the following progress:
2.1 In Vitro Fertilization Test Using Sperm Separated by Flow Cytometry Separator According to Zhang Ming and Lu Kehuan of Animal Breeding Research Institute of Guangxi University, it was proved in the United States that in vitro fertilization of matured oocytes was performed with isolated spermatozoa and unseparated spermatozoa. Reduced fissure rate (P indicates that: Separation of sperm process and dye Hoechst 33342 have no effect on embryonic development. Separation of spermatozoa by flow cytometry can be effectively used for sexually controlled IVF embryo production. However, according to the Canadian Edel Group’s embryos in Quebec Technical company technicians introduced that a pair of ovary oocytes of a cow, in vitro fertilization with unseparated sperm can obtain an average of four commercial IVF embryos, and in vitro fertilization with isolated X sperm, on average, only one Commercially controlled IVF embryos.
Table I Cleavage rate and blastocyst rate after fertilization of isolated and unseparated spermatozoa Sperm type Oocyte number Cleavage rate Blastocyst rate Isolated 136453.666.0320.42.49 Unseparated 130571.932.6922.141.69 2.2 Different freezing methods Availability of controlled IVF embryos after freezing and thawing According to data provided by researchers in the United States and Canada, microdroplet vitrification is used, and the availability rate after thawing is about 95%, and thawing is performed using a 0.25 ml tube conventional freezing method. After the availability rate is about 85%.
2.3 The Quality of Sexually Controlled IVF Frozen Embryos In laboratory applications of the PCR Sex Sex Identification method, the sexually controlled IVF embryos were tested with female embryo rates ranging from 95% to 96%. It is expected that maternal nevus fertility rates may be higher for pregnant recipients of transplanted pregnancies. More than 90%. After thawing, the available embryo rate is 85%-95%, the hatching rate is more than 60%, and the pregnancy rate for transplant is 35%-45%. It is expected that the first child born after embryo transfer will be able to reach 8000kg under scientific feeding and management conditions. the above.
2.4 Different freezing methods Sexually controlled IVF embryos and post-thaw transplantation tests Table 2 Different freezing methods Sexually-controlled IVF embryo thawing Results of transplantation Cryopreservation Number of transplanted heads 60 days Checking number of gestational heads Pregnancy rate Microdroplet vitrification 139594 2.5% Tubular conventional freezing Method 127247837.6% The effect of transplantation of droplet vitrification on frozen embryos was slightly higher than that of conventional tube freezing.
2.5 7-day transplantation of in vitro fertilized embryos to estrus 5 to 5.5 days in recipient cows 7 days in vivo embryos collected from donor cows need to be transplanted to estrus since the corpus luteum has formed and has a secretory ketone function. 7 days of estrus in the same period fertilized cows, in order to achieve the consistency of the physiological environment of the embryo and the uterus. In in vitro fertilized embryos, during 7 days of in vitro culture development, the embryos were cultured without progesterone added. Therefore, if the same requirements as for 7-day embryos in vivo are required for the recipient cow to be transplanted 7 days after estrus, it is possible that Affecting the success rate of in vitro fertilized embryo transfer, transplanting 7 days of in vitro fertilization blastocyst at 5-5.5 days after estrus of the recipient cows may increase the rate of transplanted pregnancy. The results of domestic unit tests are as follows (Table 3).
Table 3 5-5.5 days after in vitro fertilization blastocyst transfer Recipient cow trials Units Transplant recipients Cow number 60 days Pregnancy test Pregnancy cows Pregnancy rate Remarks Guangxi Buffalo Institute 7571.4% Buffalo Cows in Fengnan District, Tangshan, Hebei A preliminary trial of 4375% dairy cows showed that the rate of pregnancy can reach 70%-75% in in vitro-fertilized blastocysts recipients after 5 to 5.5 days of estrus. Although the number of cows in recipients of transplant trials is small, it can be shown that An in vitro fertilized embryo with 7 days of blastocyst development in a culture medium without the addition of progesterone at a certain concentration, 5 - 5.5 days after transplantation into estrus, corpus luteum, a recipient cow with secretion of progesterone function, may be more in line with the embryo and the uterus The basic principle of consistency in the physiological environment, thereby improving the success rate of IVF frozen embryos.
3. Safety of Sexually Controlled IVF Embryos Sexually Controlled IVF Frozen Embryos imported from Sexually Controlled IVF Embryonic Production Bases in the United States and Canada are strictly in accordance with Protocols for the Production of IVF Embryo Quarantine and Sanitary Conditions, which have been signed by China, Canada, and the U.S. , Ovarian collection from slaughterhouses, oocyte collection in laboratory, oocyte maturation culture, in vitro fertilization, in vitro development and culture of fertilized eggs, veterinary quarantine treatment of in vitro fertilized embryos, embryo freezing and preservation, and other serial technical operations Strict veterinary quarantine requirements, and must pass the on-site acceptance by the national health inspection department, sexually controlled IVF frozen embryos can be exported to China, therefore, sexually controlled IVF frozen embryos are not easy to cause the spread of infectious diseases. 4. Genealogy-controlled IVF embryos of sexually controlled IVF embryos can be produced entirely in the technically well-proven IVF embryos, but the oocyte collected from a pair of ovary of each cow must be subjected to a systematic operation of the in vitro fertilization process alone. As a result, production efficiency is reduced and production costs are increased.
China's current dairy herds, like the dairy herds in some international countries, have both a well-defined herd of breeders with pedigree registration and a large number of commercial herds with less clear pedigrees. Therefore, IVF embryo production units plan to produce the following two kinds of IVF embryos. : (1) The production of semi-lineage IVF embryos with a well-defined bovine gene line can provide reference for the selection and selection of future generations of embryos to increase the number of high-yielding dairy herds in China; (2) strict selection of cows and breeding bulls, production of paternal lines, All lineage IVF embryos with clearly defined maternal pedigrees have their embryos packaged with offspring, females can supplement dairy herds, and males can be used as reserve oxes.
5. The concrete embodiment of the industrialization of IVF embryos in industrialized dairy cattle embryo transfer in China should be: a scaled and standardized embryo production base and capacity; a scaled, intensively-accepted cow breeding base and embryo transfer technology Strength; large-scale, batch-based embryo transfer of offspring.
Canada Aide Modern Niuye Development Group established IVF embryo production base in Quebec, Canada and Pennsylvania, USA. It basically grasps the ovarian resources of North American high-producing dairy cows. Since the establishment in September 2003, it has purchased flow-type sperm separation. Instrument, segregated semen of the top 100 bulls from the United States and Canada each year was selected for isolation. The segregated X sperm was used to produce sexually-controlled IVF embryos, and each month had the capacity to produce 20,000 or more IVF embryos on a daily basis.
In China, Hebei Province, Henan Province, Shandong Province, Hubei Province, Xinjiang Province, Gansu Province, Heilongjiang Province and other provinces have established joint ventures with local government investment agencies or enterprises to establish and establish more than a dozen sex-controlled IVF embryo transfer industries with 3,000 recipient cows. The chemical base plans to raise the total number of accepting cows by the end of 2004 to about 25,000 heads, to transplant approximately 20,000 cows from pregnant recipients, and initially form industrialized IVF embryo transfer to provide development for the dairy industry in China. A technological approach to the rapid development of the dairy industry is based on the combination of embryonic bioengineering technology and gender control technology supplemented by gradually increasing the individual production level of dairy cows and supplementing the total number of dairy cows.