USE OF THE BIO-FREQUENCY SPECTRUM IN ANIMAL EMBRYO-ENGINEERING

Abstract: The invention discloses the use of bio-frequency spectrum in embryogenesis and development of animal embryo, and the use of bio-frequency spectrum in promoting propagation and development of animals. The mature rate of oocyte, the fertility of fertilized egg, and the quality of embryo can be improved by exposure oocyte, egg cell, sperm and embryo to the minic bio-frequency spectrum irradiation in the course of production of embryo in vitro .

Description: The present invention relates to biological engineering, particularly to the application of bio-spectrum non-human embryonic engineering. The term "embryonic engineering" as used in the following description relates to non-human animal embryos only. The fundamental researches and technical development on animal embryonic engineering have achieved great progress in recent years. The researches on embryonic engineering are moving quickly from the stage of laboratory test to practicalization and commercialization. The animal embryonic engineering mainly includes production of embryos in vitro, cryo-preservation of embryos and micro-manipulation of embryos, etc. The development of the technique for production of embryos in vitro can make full use of animal genetic resources, accelerate improvement of animal gene. The technique can overcome infertility of some rare animals and preserve resources of rare animals. The technique can also supply embryos for production of gene transfer in animals and embryos' sexing. However, the production efficiency and the qualities of embryos produced in vitro are lower. The rate of pregnancy is also lower by embryo transfer. Cryo-preservation of animal embryos is the key technique, which affect whether or not the technique of embryo transfer can be put into practical use. Through the cryo-preserving technique, the animal embryos reservoir can be established to facilitate the transportation and exchange of animal breed resources internationally. Currently there are two internationally acceptable methods of cryo-preserving embryos. One is the slow freezing method, the other is the cryo-preservation. However, the survival rate of frozen/thawed embryos produced in vitro is about 65%, and the pregnant rate of the frozen/thawed embryos is decreased 20% than the fresh embryos. Micro-manipulation of embryos consists of sexing of embryos, embryos'cloning including embryo bisection and nuclear transfer, and gene transfer. The aim of sexing of embryos is to produce defined sexual offspring. Cloning of embryos can produce many identical offspring from an outstanding animal embryo, improving the reproduction efficiency and speeding the animal breeding. Gene transfer animals can be produced by microinjection and sperm mediated method. The aim of gene transfer technique is to speed up the growth rate and increase resistance to diseases of animals, improve quality of animal production, and therefore supply many valuable medicines for human beings. However, the efficiency of micro-manipulation is very low because the embryos will definitely be injured. To solve the above problems, people tried to use laser or magnetic irradiation. For example, SU-A-1724204 discloses the use of infra-red laser radiation having a particular wavelength during the processing of animal reproduction sperm ; SU-A-731 976 discloses a method for improving fertility of lab rodents by subjecting the females to the irradiation of cm band ; SU-A-1757676 discloses a method of irradiating the chicken embryos with He-Ne laser having a particular wavelength of 632.8 nm ; SU-A-1152583 discloses a method of treatment using pure magnetic field ; SU-A-1 402 343 discloses an animal sperm treatment using pure magnetic field and US 493 0505 discloses a therapeutic method using a laser having a particular wavelength. The invention is based on the following understandings. Very few people have conducted research on the regulation of reproductive and growth abilities of animals with physical method, especially application of physical method to embryos' engineering for resolving technical problems. The inventor recognized that all living matter has chemical and physical characteristics at the same time. The living matter has special physical characteristics such as electric charge in cells, etc. The interactions between the substances in living matter which have electric charges and the electromagnetic field in environment can be produced when electromagnetic field in environment and some main physical characteristics of living matter have the same characteristics. The interactions can influence the molecules, atoms and electrons at the same time to produce significant biologic effects. For example, tissues and cells can grow and develop normally in the chemical and physical environment of a living body. The growth and development of tissues and cells are decreased significantly when they are separated from the living body, although many kinds of chemical protecting materials and nutritive materials are used for improving the culture conditions. The inventor further recognized that the growth ability of tissues and cells in culture condition will be improved by applying a simulated bio-spectrum which is a weak electromagnetic field. So, it has significant meanings to apply the simulated bio-spectrum to embryos' engineering. The bio-spectrum will help improve the reproductive ability, growth speed and resistance to diseases of the animals. The object of the invention is to induce irradiation bio-effects by applying the bio-spectrum to animal embryos engineering and animals, which includes: improving the rate of maturing oocytes, fertility in vitro and embryos by bio-spectrum irradiation in production of embryos in vitro. The simulated bio-spectrum mentioned above is described in Chinese patent application No. 91109014.2 which is a wide band synthesized physical field. Its wavelength is 0.2 µm to 10cm. The irradiation signal is very weak in the wave band of 30 µm - 10cm. Some parts of the physical field can produce certain effects. The object of the present invention is realized by the following technical solutions. Methods of applying bio-spectrum to in vitro production of animal embryos. 1. In vitro maturation of collected oocytes: oocytes deposited in a standard or defined medium are irradiated with bio-spectrum generator for 3 to 20 minutes. In this period, the medium temperature is kept not higher than 40 °C. 2. Capacitation of spermatozoa: semen diluted in a standard or defined medium are irradiated with BIO-SPECTRUM generator for 3 to 20 minutes. In this period, the medium temperature is kept not higher than 40 °C. 3. In vitro fertilization of oocytes: both matured oocytes and capacitated sperms are co-cultured in one test tube containing a standard or defined medium and irradiated with bio-spectrum generator for 1-3 times, 3-25 minutes each time. During irradiation, the medium temperature is not higher than 40 °C. 4. In vitro culture of embryos: zygotes transferred in a standard or defined medium are irradiated with bio-spectrum generator for 3-30 minutes. During irradiation the medium temperature is not higher than 40 °C. By using irradiation with bio-spectrum in micro-manipulation of embryos in vitro, the development rate of hemi-morulae and hemi-blastocysts and the rate of embryos stained success could be significantly improved. By using irradiation with bio-spectrum in animal disease control, the diseases could be alleviated and cured. EXAMPLE 1 The oocytes are collected from donor cows by ordinary methods, and than put in common medium or special medium for maturation. During maturation, oocytes are irradiated with bio-spectrum device (model WS-101D) for 15 minutes (at weak level). The temperature should be kept at 38-40 °C by adjusting the distance between embryo container and irradiation device. The special medium is made according to the procedure of Brackett et al. The receipt of the special medium is as follows: components g/l NaCl 6.55 KCl 0.30 CaCl 2 · 2H 2 O 0.33 NaH 2 PO 4 · H 2 O 0.11 MgCl 2 · 6H 2 O 0.11 NaHCO 3 3.10 Glucose 2.25 Bovine Serum Albumin 3.00 Sodium Pyruvate (or Pyruvate) 0.14(or 0.11) Na-Penicillin 0.031(50 IU/ml) The medium needs to be sterilized by filtration after the components have dissolved completely in 1000 ml water. This medium is required to equilibrate in the incubator at 38 °C in 59% CO 2 in the air. The osmolality of this medium is about 300 mOsm/kg H 2 O. The special medium is made by addition of 84 mg NaHCO 3 to 100 ml so-prepared sulotion. The m-HIS is made by addition of 34 mg NaCI to 10 ml medium. Sperm capacitation The fresh semen is firstly treated following the procedure of Bracktt et al. Semen is pre-diluted with special medium (350g). After centrifugation, the sperm pellet is re-suspended with m-HIS medium and put in water bath of 38 °C for 15 minutes. After another centrifugation, the sperm pellet is re-suspended with special medium in a tube for irradiation with bio-spectrum device at weak level for 10 minutes. During irradiation, the temperature of medium should be kept at 38-40 °C by adjusting the distance between the tube and irradiation device. In vitro fertilization of oocytes The in-vitro matured oocytes and in-vitro capacitated sperm are put in the special medium in a tube for irradiation with bio-spectrum device for 20 minutes. The tube is put in CO 2 incubator for one hour culture at 38 °C. Afterwards, the tube is irradiated with bio-spectrum again for 18 minutes and again put in incubator for 5-hour culture. In irradiation, weak level is used and the temperature of medium is kept at 38-40 °C by adjusting the distance between the tube and irradiation device. In vitro culture of embryos Before culture in incubator, the in-vitro fertilized eggs are put in the special medium for irradiation with BIO-SPECTRUM for 25 minutes. Weak level is used. During irradiation the temperature of medium is kept at 38-40 °C by adjusting the distance between the medium container and irradiation device.

Claims: A method of applying biofrequency spectrum irradiation to non-human animal embryonic engineering, said bio-frequency spectrum having wavelength ranging from 0.2 µm to 10 cm, said non-human animal embryonic engineering including : (1) in vitro maturation of collected oocytes ; (2) capacitation of spermatozoa ; (3) in vitro culture of embryos wherein during the in vitro maturation, oocytes cultured in a standard or defined medium are irradiated with a bio-spectrum generator for 3 to 20 minutes, the temperature of the medium during irradiation being kept not higher than 40°C ; during capacitation, semen diluted with a standard or defined medium is irradiated with bio-spectrum generator for 3 to 20 minutes, the temperature of the medium during irradiation being not higher than 40°C ; during in vitro fertilization, both oocytes and spermatozoa capacited in vitro are co-cultured in a test tube containing a standard or defined medium and irradiated with bio spectrum generator for 1 to 3 times, from 3 to 25 minutes each time ; the medium temperature during irradiation being not higher than 40°C ;" during in vitro culture of embryos, potential zygotes are transferred into a standard or defined medium and irradiated with bio-spectrum generator for 3 to 20 minutes ; the medium temperature in this period being not higher than 40°C. A method according to claim 1, wherein the components of said defined medium are NaCI 6.55 g/l, KCI 0.30 g/l, CaCl 2 .2H 2 O 0.33 g/l, NaH 2 PO 4 H 2 O 0.11 g/l, MgCl 2 .6H 2 O 0.11 g/l, NaHCO 3 3.10 g/l, glucose 2.5 g/l, sodium pyruvate 0.14 g/l (pyruvate 0.11 g/l), bovine serum albumin 3.00 g/l, sodium salt penicillin 0.031 g/l (50 IU/ml) and 84 mg NaHCO 3 is added to 100 ml of said defined medium.

Inventors: ZHOU LIN, ZHOU, LIN
Applicants: ZHOU LIN, ZHOU, LIN
Application year: 1995
Main classification: A61D19
IPC codes: A61D19