AU704690B2

AU704690B2 - Use of the bio-spectrum in animal embryo-engineering

Info

Publication number: AU704690B2
Application number: AU38386/95A
Authority: AU
Inventors: Lin Zhou
Original assignee: Individual
Current assignee: Individual
Priority date: 1994-11-24
Filing date: 1995-11-02
Publication date: 1999-04-29
Anticipated expiration: 2015-11-02
Also published as: CA2207013A1; EP0872219A1; EP0872219A4; WO1996015732A1; CN1068195C; AU3838695A; CA2207013C; DE69528295D1; EP0872219B1; CN1123131A; DE69528295T2

Description

gene transfer. The aim of embryos sexing is to produce the offspring of animals with expected sexuality. Embryos cloning is to produce many identical offspring from an outstanding animal embryo, improving the reproduction efficiency and speeding up animal breeding. Gene transfer, mainly, micro-injection and sperm mediated method is to speed up the growth rate and strengthen disease resistance of the animals, improve quality of animal production, and therefore supply valuable medicines for human beings. However, the efficiency of micro-manipulation currently is very low because the embryos will definitely be injured during manipulation.

The invention is based on the following understandings. Very few people have conducted research on the regulation of reproductive and S growth abilities of animals with physical method, especially use of physical method to embryos' engineering to resolve technical problems.

The inventor recognized that all living things have chemical and physical characteristics at the same time. The living things have special physical characteristics such as electric charge in their cells, etc. The substances in living things that have electric charges and the external electromagnetic field may interact when the external electromagnetic .field and certain main physical characteristics of living things have the similar characteristics. The interactions can influence the molecules, atoms and electrons at the same time to produce significant biologic effects. For example, the isolated tissues and cell lines can grow and develop normally in the chemical and physical environment of 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 protective 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 is of significance to apply the simulated biospectrum to embryos' engineering. The bio-spectrum will help improve the reproductive ability, growth speed and disease resistance of the animals.

Thus the preferred object of the invention is to induce bio-effects by the applying a bio-spectrum to animal embryos in the course of biological <rls engineering which includes: improving the mature rate of oocytes, fertility in vitro and embryos by irradiating simulated bio-spectrum in production of embryos in vitro; improving the viability and pregnant rate of frozen/thawed embryos by irradiating simulated bio-spectrum after freeze-preservation of embryos; improving the efficiency of micro-manipulation and repairing the injury of embryos by irradiating simulated bio-spectrum in the progress of micro-manipulation; improving the rate of embryos survival, ovulation, fertilization and the development of uterus by irradiating simulated bio-spectrum to live female animals; *o Simproving qualities of sperms by irradiating simulated biospectrum to live male animals.

The simulated bio-spectrum mentioned above was described in Chinese patent application No. 91109014.2 which was a synthesized physical field with wide band. Its wavelength is 0.2 tpm to 10 cm. The irradiation signal is very weak in the wave band of 30 [tm -10 cm. Some parts of the physical field can also produce certain effects.

The preferred object of the present invention is realized by the ring following technical solutions.

The method of applying bio-spectrum to the production of animal embryos in vitro includes: 1. Collecting coccyges; 2. In vitro maturation of oocytes, in which oocytes deposited in a standard or defined medium are irradiated with a bio-spectrum generator for 3 to 20 minutes while the medium temperature is kept not higher than 40 "C; sT3. Capacitation of spermatozoa, in which semen diluted in a Sstandard or defined medium are irradiated with a bio-spectrum generator for 3 to 20 minutes while the medium temperature is kept not higher than 40 "C; 4. In vitro fertilization of oocytes in which both matured oocytes and capacitated sperms are co-cultured in one test tube containing a standard or defined medium and irradiated with a bio-spectrum generator for 1-3 times, 3-25 minutes each time while the medium temperature is not higher than 40 "C; In vitro culture of embryos in which zygotes transferred in a standard or defined medium are irradiated with a bio-spectrum generator for 3-30 minutes while the medium temperature is not higher than 40 "C.

ft Method of applying bio-spectrum to freeze-preservation of animal embryos includes irradiating thawed embryos deposited in a culture medium are irradiated with a bio-spectrum generator for 3-20 minutes while keeping the medium temperature not higher than 40 "C.

Method of applying bio-spectrum to micro-manipulation of animal embryos includes depositing embryos after micro-manipulation in a culture medium and irradiating the same with a bio-spectrum generator for 3-25 minutes or irradiating embryos before micro-manipulation with a bio-spectrum generator for 3-25 minutes to achieve stronger effect while keeping the medium temperature not higher than 40 "C.

Method of applying bio-spectrum to animal reproduction, development and growth includes irradiating animals with a biospectrum generator once or twice a day for 30-60 minutes each time while keeping the surface temperature of the animals not higher than "C To have better results, conventional techniques of animal reproduction, development and growth can be used such as injection of gonadotrophin..

Method of using bio-spectrum in preventing and curing animal diseases includes irradiating parts or whole body of an animal with a bio-spectrum generator 1-3 times every day, for 6-60 minutes each time P while keeping the temperature of the irradiated part not higher than °oC.

By bio-spectrum irradiation in the production of embryos in vitro, the fertility in vitro is increased by at least 18% and the development rate of in vitro fertilized ova is increased by least 19%.

By bio-spectrum irradiation in freeze-preservation of embryos, the viability of embryos after frozen/thawed increases by at least 16% and the pregnant rate of embryos could be also significantly improved.

By bio-spectrum irradiation in micro-manipulation of embryos, the development rate of demi-morulae and demi-blastocysts in vitro and the rate of embryos stained success could be significantly improved.

-S

By bio-spectrum irradiation in the reproduction of animal; the uterus development, ovulation rate, fertilization rate of oocytes, survival and developmental rate of fertilized eggs in female animals and the sperm quality in male animals could be improved.

By bio-spectrum irradiation in animal disease control, the diseases could be alleviated and cured.

Bio-Spectrum is the expression of physical information correlated with biological progress of a living body itself. It is the faint, quasicontinuous, wide electromagnetic wave emitted by a living body. Its wavelength ranges between visible light and millimeter wave, with the main-energy band concentrated in the infrared region.

A bio-spectrum can also be produced by a bio-spectrum generator.

One generator of choice used in following non-limiting examples is the commercially available WS-101D. The WS-101D is one type of BFS health care series bio-spectrum generators produced by ZhouLin Bio- Spectrum Co. where WS is the series of the production, 1 is the main type, 01 is the secondary type, and D is the design sequence.

The main parameters of the "WS-101D" model bio-spectrum -generator are as follows: Spectrum Range 5-13500pm Voltage 220V,50HZ-60HZ Power 90-350W Environment Temperature -10+40 °C Humidity Embodiment 1 S. .0* a S 0

ID

*1 0 0

S

0 em 0@ 0 0 t 0 00 0

S

5* *e Oe 0 e @000 0* em em 6

S

The oocytes are collected from donor cows by ordinary methods, and then put in common medium or special medium for maturation.

During maturation, oocytes are irradiated with a (commercially available) bio-spectrum generator (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 the generator. The special medium is made according to the procedure of Benjamin G. Brackett, et al (In Vitro Fertilizing Ability of Testicular, Epididymal, and Ejaculated Rabbit Spermatozoa, Fertility and Sterility, The American Fertility Society, 1978). The receipt of the special medium is as follows: NaCI 6.55 g/l KCl 0.30 g/1 CaCI 2 2H20 0.33 g/l NaH 2

PO

4

H

2 0 0.11 g/l MgCl 2 6H 2 0 0.11 g/l NaHCO 3 3.10 g/l Glucose 2.50 g/1 Bovine Serum Albumin 3.00 g/1 Sodium Pyruvate (or Pyruvate) 0.14 (or 0.11) g/1 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 5.9% CO 2 in the air.

The osmolality of this medium is bout 300 mOsm/kg H 2 0. The special medium is made by addition of 84 ml NaCHO 3 to 100 ml so-prepared solution. The m-HIS is made by addition of 34 mg NaCl to 10 ml medium.

In the process of sperm capacitation, the fresh semen is firstly treated following the above-mentioned procedure of Benjamin G.Bracktt et al. Semen is pre-diluted with special medium (350g). After centrifugation, the supernatant is removed, and the sperm pellet is resuspended with m-HIS medium and put in water bath of 38 "C for minutes. Then centrifugation again, the sperm pellet is re-suspended with special medium in a tube for irradiation with a bio-spectrum generator at weak level for 10 minutes. During irradiation, the temperature of the medium should be kept at 38-40 "C by adjusting the 0 distance between the tube and the generator.

o*oooo* In the process of fertilization of oocytes in-vitro, the matured oocytes in-vitro and capacitated sperm in-vitro are put in the special medium in a tube for irradiation with a bio-spectrum generator for minutes. The tube is put in CO 2 incubator for one hour culture at 38 "C.

Afterwards, the tube is irradiated with a bio-spectrum generator again for 18 minutes and put in incubator again for 5 hour's culture. In irradiation, weak level is used and the temperature of the medium is kept at 38-40 °C by adjusting the distance between the tube and the generator.

V q, In the process of culture of embryos in-vitro, the in-vitro fertilized eggs are put in the special medium for irradiation with a bio-spectrum S 6 generator for 25 minutes before culture in incubator. During irradiation, weak level is used and the temperature of the medium is kept at 38-40 °C by adjusting the distance between the medium container and the generator.

Embodiment 2 Bovine embryos are put in a special medium and irradiated with a bio-spectrum generator for 15 minutes. During irradiation, weak level is used and the temperature of the medium is kept at 38-40 0 C by adjusting the distance between the medium container and the generator.

The freeze-protective agent is added following the procedure of Inoue Tadahiro, et al (Japanese Animal Reproduction, volume 28 PP 153, 1982). The embryos are transferred step by step in PBS CS in 0.18, 0.33, 0.75, 0.88, 1.OM Glycerol, respectively, each step for 5 minutes. The embryos are put in a freezing medium (PBS plus CS in 1.OM Glycerol) for 30 minutes and then placed in a 0.5 ml fine tube. The tube is cooled in a freezer to -7 °C at the speed of 1 *C/min., then at this temperature crystallization is induced by adding artificial crystal nucleus, meanwhile the tube is cooled down to -35 °C slowly at 0.3 °C/min. and further down to -36 0 C at -0.1 °C/min., finally plunged into liquid nitrogen (-196 and stored.

In the process of embryo thawing, the tube containg embryos is placed into a water bath of21 °C immediately after being taken out from the liquid nitrogen and slightly shaken. The speed of thawing is 360 °C /min. After thawing, the freeze-protective agent is removed from ":00 embryos step by step in a way that is in reverse of adding freezeprotective agent. Then embryos are placed in the special medium and irradiated with a bio-spectrum generator for 20 minutes. During irradiation, weak level is used and the temperature of the medium is kept at 38-40 0 C by adjusting the distance between the medium container and the generator.

Embodiment 3 -Freeze-reservation of embryos was made by nitrification (Kasai et.

Sa. J. Reproduction Fert. 89: 91-97). The bovine embryos are put in a special medium and irradiated with a bio-spectrum generator for minutes. During irradiation, weak level is used and the temperature of the medium is kept at 38-40 "C by adjusting the distance between the medium container and the generator. A 0.25 ml tube is used taking in successively 100 [tl of S-PBS medium, 20 pl of air, 6tl of EFS medium, 6 pl of air, 40 pl of EFS medium. The embryos are equilibrated in EFS medium at the room temperature for 3 minutes, and then immediately inhaled into the section of the tube that has 40 pl of EFS medium. Then successively 6 pl of air, 6 ut of EFS medium, 15 tl of air and 20 ll of S- PBS medium are inhaled into the tube. Finally, the end of the tube is sealed using pre-heated hot forceps.

In the process of thawing, the tube containg embryos is placed into a water bath of 20 °C immediately after being taken out from the liquid nitrogen and slightly shaken. The embryos are quickly flushed out from the tube using 0.5 ml of S-PBS medium, then transferred to S-PBS medium for 5 minutes before transferred to m-PBS medium. After washing in the special medium for three times, the embryos are irradiated with a bio-spectrum generator for 10 minutes at weak level.

The temperature of the medium is kept at 38-40 "C by adjusting the distance between the medium container and the generator.

The receipt of S-PBS medium is as follows: NaCl 8 g/l KCI 0.2 g/1 NaH 2

PO

4 1.15 g/1

KH

2 P0 4 0.2 g/1 CaCl 2 0.1 g/1 MgCl 2 6H 2 0 0.1 g/1 Na pyruvate 0.036 g/1 Glucose 1 g/1 Penicillin 100 IU/ml Streptomycin 0.05 g/1 Glycerol EFS medium is 0.5M sucrose solution (EF medium) which contains 30% poly-sucrose. EFS medium is made by mixture of ethylene and EF medium Embodiment 4 In the process of embryo splitting, the rat, goat and bovine embryos are split by metal knife following the procedure of Kazuya Matsumoto et al. (Japanese Journal of Animal Reproduction Volume 33, 1-5, 1987).

The micro-surgical blade made from razor is fixed with a micromanipulator. Before splitting, the embryos are put in the culture medium and irradiated with a bio-spectrum generator for 15 minutes. After irradiation, the embryos are held in a droplet of 0.5 ml of PBS plus FCS on the center of the plastic dish (diameter 8 cm, hight 1 cm). The micro-surgical bisection is performed using a micro-manipulation unit consisting of an inverted microscope.

After splitting and treatment, demi-embryos are put in the culture medium and irradiated with a bio-spectrum generator for 30 minutes.

During irradiation, weak level is used and the temperature of the medium is kept at 38-40 °C by adjusting the distance between the medium container and the generator.

o Embodiment In the process of sex identification, the sex of the embryos can be accurately determined through sampling several cells from the embryos.

After sampling, the embryos is partly damaged, and the viability decreases. The viability of the embryo can be raised with the treatment of a bio-spectrum generator for 20 minutes (Model WS- 101 D).

Embodiment 6 Reproduction, development and growth of animals can be promoted by using bio-spectrum.

Mice were used as experimental animals. Female mice are randomly divided into 2 groups, A and B, 20 in each group. Group A is irradiated with bio-spectrum, and group B is used as control group without irradiation of bio-spectrum. Both group A and group B are under the same experimental conditions. Each treatment had 2 TRA/ replicates, 10 mice per replicate. The mice in group A are irradiated with a bio-spectrum generator (model WS-101 manufactured by Zhoulin Bio-Spectrum Company) at strong level for 20 minutes a day at a fixed time for 10 consecutive days. The temperature over the mouse back is controlled under 38 On the 4th day, every mouse in group A and group B are injected with PMSG101U, on the 6th day, further injected with HCG101U. After injection, one male mouse is put into each cage for mating. On the 7th day, eggs are collected from the oviducts of 10 mice in each group, the comparative results showed that bio-spectrum irradiation could significantly protect the fertilized eggs.

On the 10th day, blastocysts are collected from the rest 10 mice in each group. The results showed that bio-spectrum could improve the ovulation and fertilization ability of eggs, and significantly improve the development of blastocyst. The comparison between group A and group B also shows that bio-spectrum irradiation can strongly stimulate the development of uterus in female mouse. This experiment shows-'that bio-spectrum irradiation can improve the reproduction, development and growth of animals.

Embodiment 7 9 The abdomen of lambs with diarrhea are irradiated with a biospectrum generator (model WS-101 mamufactured by Zhoulin Bio- Spectrum Company) at strong level for 20 minutes a day for 2-4 consecutive days, while the surface temperature of the abdomen is kept not higher than 45 the diarrhea could be controlled and the results are obvious.

The inventor believes the aplication of bio-spectrum in bio- S engineering is not limited to the above embodiments and bio-spectrum Scan be applied to many other objects according to the main aspects of the invention. It can not only solve many buffing problems in the fields of both embryo-engineering and bio-engineering, but also simplify the complex and difficult techniques used in these fields. So there will be other embodiments in the field of bio-engineering and all of these embodiments will fall into the protection scope of this invention.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" or the term "includes" or variations thereof, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of the claims is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.

p a p p -11- The Claims defining the Invention are as follows: 1. Application of a bio-spectrum in promoting maturation of oocytes in vitro comprising the steps of: culturing oocytes in a standard or defined medium; and irradiating said oocytes with a bio-spectrum generator for 3 to minutes while keeping the temperature of the medium not higher than

"C.

2. Application of a bio-spectrum according to Claim 1 wherein said defined medium is formulated by dissolving in 1000 ml distilled water NaCl 6.55 g/l, KC1 0.30 g/l, CaCl 2 2H 2 0 0.33 g/l, NaH 2 P0 4 0.11 g/1, MgCl 2 6H 2 0 0.11 g/l, NaHCO 3 3.10 g/l, glucose 2.5 g/l, Na pyruviate 0.14 g/1 (Pyruvate 0.11 bovine serum albumin 3.00 g/1, sodium salt penicillin 0.031 g/l (50 Iv/ml) and then adding 840 mg NaHCO 3 into the dissolved solution.

3. Application of a bio-spectrum in promoting capacitation of Sspermatozoa comprising the steps of: diluting spermatozoa with a standard or defined medium; and irradiating said spermatozoa with a bio-spectrum generator for 3 to minutes while keeping the temperature of the medium not higher than 40 0

C;

a 4. Application of a bio-spectrum according to Claim 3 wherein said defined medium is formulated by dissolving in 1000 ml distilled water NaCl 6.55 g/l, KC1 0.30 g/l, CaC1 2 2H 2 0 0.33 g/l, NaH 2

PO

4 0.11 g/l, MgCl 2 6H 2 0 0.11 g/l, NaHCO 3 3.10 g/l, glucose 2.5 g/l, Na pyruviate 0.14 g/1 (Pyruvate 0.11 bovine serum albumin 3.00 g/l, sodium salt penicillin 0.031 g/1 (50 Iv/ml) and then adding 840 mg NaHCO 3 into the dissolved solution.

Application of a bio-spectrum in promoting fertilization in vitro M comprising the steps of:

Claims

6. Application of a bio-spectrum according to Claim 5 wherein said defined medium is formulated by dissolving in 1000 ml distilled water NaC1 6.55 g/l, KC1 0.30 g/l, CaCl 2 2H 2 0 0.33 g/l, NaH 2 PO 4 H 2 0 0.11 g/1, MgCl 2 6H20 0.11 g/l, NaHCO 3 3.10 g/l, glucose 2.5 g/l, Na pyruviate 0.14 g/l (Pyruvate 0.11 bovine serum albumin 3.00 g/l, sodium salt penicillin 0.031 g/1 (50 Iv/ml) and then adding 840 mg NaHCO 3 into the dissolved solution.
7. Application of a bio-spectrum in promoting culture of embryos in vitro comprising the steps of transferring potential fertilized eggs into a standard or defined medium; and irradiating said fertilized eggs with bio-spectrum generator for 3 to 20 minutes while keeping the medium temperature not higher than C. 0.
8. Application of a bio-spectrum according to Claim 7 wherein said defined medium is formulated by dissolving in 1000 ml distilled water NaC 6.55 g/l, KC1 0.30 g/l, CaC 2 2H 2 0 0.33 g/l, NaH 2 PO 4 0.11 g/l, MgC12, 6H20 0.11 g/l, NaHCO 3 3.10 g/l, glucose 2.5 g/l, Na pyruviate 0.14 g/1 (Pyruvate 0.11 bovine serum albumin 3.00 g/l, sodium salt penicillin 0.031 g/1 (50 Iv/ml) and then adding 840 mg NaHCO 3 into the dissolved solution.
9. Application of a bio-spectrum substantially as hereinbefore described with reference to Embodiments 1 to 7. DATED this 11th day of September, 1998 LIN ZHOU By His Patent Attorneys SDAVIES COLLISON CAVE Abstract The invention concerns application of bio-spectrum in animal embryonic engineering which includes: improving the rate of maturing oocytes, fertility in vitro and embryos by bio-spectrum irradiation in production of embryos in vitro improving the survival and pregnancy rate of frozen/thawed embryos by bio-spectrum irradiation in the process of cryo- preservetion of embryos improving the efficiency of micro-manipulation and repairing the injury of embryos by bio-spectrum irradiation in the process of micro-manipulation improving the rate of embryos survival, ovulation, fertilization and the development of uterus by bio-spectrum irradiation to live female animals improving qualities of sperms by bio- spectrum irradiation to live male animals.