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Cow sex control technology
The identification of the sex of livestock embryos and the control of the proportion of offspring are one of the major issues in modern animal husbandry scientific research and production. It is of great significance to improving the economic benefits of animal husbandry. People have different requirements for cattle of different uses (varieties). Due to different genders, the production performance and characteristics of cattle are not the same, and the economic value between the sexes is also very different. Taking cattle as an example, for dairy cows, the economic benefits of milking cows are higher, so people want it to produce more females. If the male birth rate can be controlled in a planned manner, the number of dairy cows can be increased exponentially for maximum economic benefit. (i) Sex identification of embryos With the development of embryo segmentation and embryo cryogenic technology, it is possible to use one half-embryoid for sex identification; while the other half embryo is used for transplantation after freezing, thus greatly shortening the embryo In vitro culture time. Picand (1984) et al. reported that the time for semi-embryo culture in vitro was shortened from 24 h to 4 h, and the success rate of embryo transfer after identification was increased from 5% to 60%. At present, the identification of embryo sex is mainly through cytological methods and immunological methods. 1. Cytology is the use of microsurgery to remove some of the embryonic cells, in vitro culture to observe X and Y chromosomes. The reliability of sex identification of cattle embryos from 10 to 12 days can reach 70%. In Canada, a 14-day-old bovine embryo was used to obtain a small slice of trophoblast cells for biopsy. Chromosome was used to determine the sex of the embryo. Thirty-two embryos were tested, and 22 embryos of the sex were judged. After the transplant, there were 10 pregnancies. Germany has a higher rate of accuracy in determining gender. Seven or eight calves were born in 1978 and the results are completely accurate. However, the sex identification of 7-day-old cattle embryos was ineffective because the number of chromosomes available for embryos during this period was too small. Hu Mingxin et al. used animal sex chromosomes RSP for sex identification, used animal DNA restriction enzymes to identify specific base sequences, specifically cut DNA into fragments of a certain size, and prepared specific genes with isotopic markers. The probes are molecularly hybridized to allow the identification of genes and to study the lack or abnormality of these fragments and genes. As a result of gene mutations, a certain DNA restriction endonuclease polymorphism (referred to as RSP) was present, and the sexual chromosomal DNA zymogram was used to identify the sex. RSP is of great significance for improving the level of genetic breeding and shortening the determination time of descendants. 2. Immunological Methods This method uses specific antibodies to measure Hg antigen (a histocompatibility antigen) on the surface of male embryonic cells. It is a specific protein antigen that only male cells can produce. This specific protein is translated from the DNA on the Y chromosome, and there are thousands of copies of this translation from each male cell membrane, so measure this Proteins are more sensitive than observational sex chromosomes (DNA) because DNA has only one copy. This method is characterized by the fact that it is not necessary to kill the embryo, or it can be determined without taking live tissue from the embryo. It can be observed by means of a clonal antibody that binds to a fluorescent substance. China's Ma Xiangdong applied the principles and methods of reproductive immunology. The preparation of HY monoclonal antibody for pre-transplant embryo sex identification has been successful. Hu Mingxin et al. synthesized Sry specific primers for cows and directly amplified the Sry-specific DNA sequences of bovine embryos by PCR. The sex identification of bovine embryos was equivalent to the results of childbirth, indicating that controlling sex will soon become a reality. (b) the control of the proportion of offspring's sex The modern research on sex determinants suggests that mammals contain Y chromosome sperm and eggs fertilized to form XY-type zygote and develop into male individuals. X-chromosome-containing sperm and egg fertilization form XX zygote into a female individual. Based on this basic principle, people have conducted a series of studies in controlling the sex ratio of future generations. Can be roughly divided into: 1 treatment of semen, separation of X, Y sperm; 2 control of environmental factors make it suitable for the required sperm, is conducive to fertilization. Divide as follows: Treatment of Semen and Separation of X, Y Sperm (1) Differences between X and Y Sperm According to recent studies, there are certain differences between X and Y sperm in many aspects. For example, the X spermatozoon is larger and rounder than the Y sperm, and the body and nucleus are also larger. The Y spermatozoon has a small and pointed head; the X and Y sperm have different DNA contents, and the difference is 3% to 9% in cattle; the sperm membrane The distribution of Y is also different. Y spermatozoa has a higher membrane charge on the tail membrane and X spermatozoa have a higher membrane charge. Y spermatozoa have HY antigen and X spermatozoa do not; Y sperm responds to HY antigen and X sperm does not. . Caperosn (1968) used the QM method to find strong fluorescent spots at the ends of the long arms of the chromosomes. These bright spots are called F-bodies (fluorescent bodies). Semen can be isolated using this method, and it can also be used to identify sperm separated by other methods. However, sperm treated by this method (Q-M method) has been destroyed without fertilization. X and Y sperm are also different in their ability to exercise. Y sperm has a strong ability to move and it moves straight in the diluent containing serum proteins. In addition, it has been reported that Y sperm is more resistant to acidity than weak alkali, and X sperm is more resistant to weak acidity and alkali resistance. Based on the above circumstances, people conducted a large number of studies on the separation of X and Y sperm. (2) According to the difference in sperm size and specific gravity in the centrifugation method, the rabbit semen is divided into upper and lower layers by a gradient spin column containing egg yolk-sodium citrate liquid. The upper semen has 65% of male rabbits, and the lower semen has female rabbits. %. (3) Sedimentation Separation and separation of bovine semen was achieved by using a liquid with certain density, viscosity, pH, osmotic pressure, and nutrients. After fertilization, the proportion of females was significantly increased (53.9%: 46.8%), but any Does not increase the proportion of males. After Bhattasharga et al. (1977) treated the semen of cattle, the semen was purified by the strong-convection method. 81.1% of males were obtained in the upper part of the stratum and 91.9% of females were found in the bottom part. It is generally believed that the above two methods can only increase the proportion of a certain sex by 70% to 75%. (4) The filtration method utilizes the characteristics of different sperm size of X and Y, and filtration using a G-50 Sephadex column or filter. This method has achieved better results in rabbits and has not been reported in cattle. (5) Fluorescent dye labeling method The fluorescence body of the Y sperm was labeled with a fluorochrome and the X and Y sperms were separated using an electro-optical apparatus. It is reported that the separation rate can reach 80.4%. However, it has not been reported specifically for insemination and offspring sex ratio. (6) Electrophoresis uses electrophoresis to separate the sperm using X, Y spermatozoa, and to collect the anode and cathode to move the sperm. After fertilization, 71.3% of the offspring of anode spermatozoa and 63.8% of cathode males were females. In addition to the above methods, there are immunological methods, flow cytometry, and the like. But summarizing the various separation methods for sperm above have not yet reached the practical stage. There are some methods and theories are also controversial. At present, research at home and abroad is continuing and we expect a major breakthrough. 2. Controlling the sex ratio of offspring using mating insemination environmental conditions (1) Arginine method Nippon Kuroki, Changchun In 1978, arginine was diluted with normal saline to be divided into high, medium, and low concentrations, and a concentration of 20 to 30 minutes before insemination was input to the vagina. A solution of 1 to 2 ml resulted in high male production at high and low concentrations and more females at medium concentrations. China's Inner Mongolia Wengniute Banner Zhan Shuilin According to Japan's Heihe Changchun's use of the arginine method to control the sex ratio of Japanese black cattle, in 1982, 11 heads of cold cattle were allocated to obtain 9 head mites, accounting for 81.8%. However, the test data is small and persuasive. Chen Shounan (1987-1988) of the Animal Husbandry Bureau of Yucheng County, Henan Province (1987-1988) used arginine method in rural areas to co-proportionate 145 female cattle, followed by 86 symbiotic females, accounting for 71.66% of the total. The rate of conception was basically the same (82.76%: 82.06%), while the ratio of male to female was 28.34% (male): 71.66% (female) and 59% (male): 60% (female), the difference was extremely significant (P Zhongwei County, Ningxia Livestock Husbandry Station Zhang Jushun Under the enlightenment of the Japanese Blackwood Experiment, a similar experiment was conducted on cows and cattle in the Zhongwei County Fine Breeding Farm and the Rouyuan Breeding Station in 1986. The experiment was divided into two phases: breeding in 1986 and production in 1987. In the first stage, pupa was a repetitive trial stage from 1987 to 1988. The test cows were cows of the right age who had no genital tract diseases in the dairy breeding group, and were randomly divided into three groups.The first group and the second group were Group 15 were treated with arginine at a concentration of 5% and 10%, respectively (Arginine is a human injection made by the Biochemical Microbiology Factory at Wuhan University, Wuhan University, 20ml per ampule, containing 25% arginine. Diluted to 5% and 10% 2 concentrations with normal saline solution, then dispensed in sterile 2ml ampoules. The 1st and 2nd groups of cows were injected with 5% and 10% arginine at a concentration of 3 cm in the cervix before estrus mating, and then frozen semen was input at intervals of 20-30 min. The test cattle cattle group also took the same method for treatment.Group 3 20 heads were used as control, no arginine treatment.In the trial group, the cows were all house-fed, and the same feed formula was used before and after the experiment. Grazing was carried out at two points, and the test cattle were of medium lyrical degree and were inseminated with the same cow or the frozen sperm of Simmental bull.The results of the experiment showed that: 1 The communist trial group communist trials were conducted during the two phases of the 1986-1988 trial of the dairy cow team. There were 56 females, including 41 males, accounting for 73.2%, and 34 females in the control group, including 14 females, accounting for 41.2% of the females, and 38.01% of females in the 6 years before the trial (1981-1986). In the same period, the proportion of maternal cubs in the control group was 32% higher, which was 35.1% higher than the average value in the 6 years before the experiment.2 In the 1986-1988 two-stage test of the Rouyuan Point Cattle Group, 47 communist calves were found in the experimental group, 33 of which were females. 70.2%; 31 communist pigs in the control group, including females 15 The head, accounting for 48.4%, was 21.8% higher in the test group than in the control group.3 Comparison of the two concentrations.From the results of the two-year test, the 5% of the dairy cows had a total of 26 cockroaches, of which 17 were females. , accounting for 65.4%; 10% of the communist comamos in the concentration group, including 24 females, accounting for 80.0%; the percentage of females in the 10% concentration group is 14.5% higher than that in the 5% group. Twenty-five heads, of which 18 were females, accounted for 72.0%, and the percentage of mothers who gave birth in the 10%-concentration group was 0.7% higher than that of the 5%-concentration group.From the results of the above tests, it was seen that the use of arginine increased the proportion of females and females in cows and cattle. Both have obvious results. Among them, the effect of cows is better than that of cattle. Dairy cows had better results with 10% arginine than 5%, but there were no significant differences between the two concentrations for the cattle, and the reasons for this need further analysis. The arginine method has the advantages of easy material acquisition, simple method, convenient operation, low cost, stable effect and the like, and is convenient for promotion in a large area at the grassroots level. (2) Relationship between pH and sex ratio As described above, it is determined that the male gene is located on the Y chromosome. The spermatozoa containing the Y chromosome are poor in acid resistance, and the spermatozoa live rate drops rapidly in the slightly acidic environment. The spermatozoa containing the X chromosome are relatively resistant to acid. When the pH of the thawing fluid is below 6.8, the sperm containing the Y chromosome loses its vigor and its movement is slow. When it has not reached the site of fertilization and has been fertilized after being capacitated, the egg may have been combined with the sperm containing the X chromosome. Therefore, after the cows are pregnant, they will have more mothers. If the pH of the thawing fluid is higher than 7, the sperm containing the Y chromosome will increase in vigor, move rapidly, reach the site of fertilization faster, and there will be more opportunities to combine with the egg, so the cow will produce more public money after pregnancy. According to Jiang Xi'an et al. (1988), when the thawing solution had a pH of 6.4, the ratio of pupa was the highest, reaching 71.0%, followed by the pH of 7.1 and 6.5, which were 62% and 60%, respectively, and the pH was again 6.3 and 6.6. The proportion of mothers that produce mothers is 55%, the worst is pH 6.2, and the ratio of mothers to females is 47%. It shows that the sperm containing X chromosome has stronger acid resistance than Y chromosome. According to Li Fanglai's (1984) experiment on the relationship between the pH of the vaginal mucus and the conception rate and the sex of the calves of the Wenling peak cow cow, it was determined that 306 peak cows had 289 breedings at a time and the continuation rate was 98.4% (96.1% to 100%). ). When the pH is less than 6.8, the conception rate is 50%, and when the pH is ≤7.6, the fertility rate is 76.7%. The author investigated the sex of the calving of 258 cows, and had 224 calves, including 114 males and 50.89% of them. The higher the vaginal pH at the time of mating, the higher the proportion of males who gave birth to calves after pregnancy. high. When the pH is less than or equal to 7.6, the male ratio is 66.7%; when the pH is less than or equal to 6.8, the male ratio is 37.5%, that is, the calf female is more male than male. At a pH of 7.8, the sex ratio of the yaks produced is greater than that of females. This test result is basically consistent with the conclusion of the above-mentioned Jiang Xi'an test.