CA2207013C - Application of bio-spectrum in animal embryonic engineering - Google Patents
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Abstract
The present invention relates to a method of applying a bio-spectrum to the field of animal embryonic engineering. The present invention further relates to methods of applying a bio-spectrum to cryopreservation and micro-manipulatio n of animals embryos. The aim of the present invention is to improve the maturation rate of oocytes, the efficiency of fertilized eggs and the qualit y of embryos by irradiating oocytes, egg cells, sperm and embryos with a bio-spectrum throughout in vitro embryo production.
Description
APPLICATION OF BIO SPECTRUM IN ANIMAL EMBRYONIC ENGINEERING
The present invention relates to the field of biological engineering, more particularly to the application of bio spectrum in anima( embryonic engineering.
The initial research and technical developments in the field of animal embryonic engineering have led to great progress in the last few years. Embryonic engineering research has quickly moved from experimental testings to practice and commercialization, Animal embryonic engineering mainly includes in vifro production, cryopreservation and micromanipulation of embryos. The development of in vitro production of embryos can make full use of animal genetic resources as well as accelerate improvements in the animal's genes.
The technique can overcome infertility of some animals reared for breeding and preserve resources thereof. The technique can also supply embryos for production of gene transfer animals and embryo sex determination. However, in vifro embryo production also leads to a lower production efficiency and a lower quality of embryos thereby produced. Moreover, embryo transfer also leads to a lower rate of pregnancy.
In practice, use of embryo transfer largely depends on cryopreservation. In other words, owing to cryopreservation, a pool of animal embryos can be established to facilitate both the transport and the exchange of animal breed resources internationally. Currently, two internationally acceptable methods of embryo cryopreservation exist:
(i) the slow freezing method; and (ii) cryopreservation.
However, the survival rate of frozen/thawed embryos produced in vitro is about 65% and use of frozen/thawed embryos leads to a decreased rate of pregnancy, namely by 20% compared with the use of fresh embryos.
The present invention relates to the field of biological engineering, more particularly to the application of bio spectrum in anima( embryonic engineering.
The initial research and technical developments in the field of animal embryonic engineering have led to great progress in the last few years. Embryonic engineering research has quickly moved from experimental testings to practice and commercialization, Animal embryonic engineering mainly includes in vifro production, cryopreservation and micromanipulation of embryos. The development of in vitro production of embryos can make full use of animal genetic resources as well as accelerate improvements in the animal's genes.
The technique can overcome infertility of some animals reared for breeding and preserve resources thereof. The technique can also supply embryos for production of gene transfer animals and embryo sex determination. However, in vifro embryo production also leads to a lower production efficiency and a lower quality of embryos thereby produced. Moreover, embryo transfer also leads to a lower rate of pregnancy.
In practice, use of embryo transfer largely depends on cryopreservation. In other words, owing to cryopreservation, a pool of animal embryos can be established to facilitate both the transport and the exchange of animal breed resources internationally. Currently, two internationally acceptable methods of embryo cryopreservation exist:
(i) the slow freezing method; and (ii) cryopreservation.
However, the survival rate of frozen/thawed embryos produced in vitro is about 65% and use of frozen/thawed embryos leads to a decreased rate of pregnancy, namely by 20% compared with the use of fresh embryos.
Micro-manipulation of embryos consists of sex determination of the embryos, embryo cloning including embryo bisects and nuclear transfer, and gene transfer. The aim of determining the sex of embryos is to produce offspring of predetermined gender. Cloning of embryos can lead to the production of many identical offsprings from a single animal embryo thus improving the reproduction efficiency as well as speeding the animal breeding process.
Gene transfer animals can be produced by micro-injection and sperm mediated methods. The aim of gene transfer is to speed up the growth rate and increase disease resistance in animals, improve the quality of animal production and therefore supply many valuable medicines for human beings. However, due to embryo injury, the efficiency of micro-manipulation is very low.
Up to now, very few people have conducted research on the regulation of reproduction and growth potential of animals by use of a physical method. More specifically, there is yet to be a study on the application of a physical method to embryo engineering for solving technical problems.
The Applicant noted that all living things share similar chemical and physical characteristics. For instance, the electric charge in cells is among the special physical characteristics of living things.
When these electric charges in living things and the electromagnetic field in the environment share the same characteristics, interactions between the charges and the field can occur. Understandably, these interactions can also influence the molecules, atoms and electrons to induce significant biological effects.
For example, as long as tissues and cells remain in a living body, they can grow and develop normally in this specific chemical and physical environment.
However, when tissues and cells are separated from the living body i.e. away from their normal chemical and physical environment, their growth and development decrease significantly. Although various chemical protecting materials and nutritive materials have been used to alleviate this decrease, the problem remains.
In the context of the present invention, it was surprisingly found that the growth potential of tissues and cells under culture conditions can be improved by applying a simulated bio-spectrum displaying a weak electromagnetic field.
Application of the simulated bio-spectrum to embryo engineering is thus highly important.
The bio-spectrum will contribute to improve the reproductive ability, growth rate and resistance of animals to various diseases.
SUMMARY OF THE INVENTION
An object of the invention is to induce radiation bio-effects by applying a bio-spectrum to animals and animal embryos.
More specifically, an object of the present invention is to provide a method of applying a bio-spectrum to animal embryonic engineering, said method including the steps of:
(1 ) in vitro maturation of oocytes to obtain matured oocytes;
(2) capacitation of spermatozoa to obtain in vitro capacitated spermatozoa;
(3) in vitro fertilization of oocytes to obtain fertilized oocytes; and (4) in vitro culture of embryos;
said method being characterized in that:
- the bio-spectrum is used at each of the steps (1 ) to (4) and has a wavelength spectrum ranging from 0.2 pm to 10 cm;
- in step (1 ), the oocytes are cultured in a standard or special medium, irradiated with a bio-spectrum generator for 3 to 20 minutes, while keeping temperature of the medium at no higher than 40°C;
- in the step (2), semen diluted with the standard or special medium is irradiated with the bio-spectrum generator for 3 to 20 minutes, while the temperature of the medium is kept at no higher than 40°C;
- in step (3), the matured oocytes and in vitro capacitated spermatozoa are co-cultured in a test tube containing the standard or special medium, and irradiated with the bio-spectrum from 1 to 3 times, for 3 to 25 minutes each time, while keeping the temperature of the medium at no higher than 40°C; and - in step (4), the fertilized oocytes are transferred to the standard or special medium and irradiated with the bio-spectrum generator for 3 to 20 minutes, while keeping the temperature of the medium at no higher than 40°C.
Another object of the invention is to provide a method of applying bio-spectrum to cryopreservation of animal embryos method of applying bio-spectrum to cryopreservation of animal embryos, said method comprising the steps of:
(1 ) putting the embryos in a culture medium; and (2) irradiating the embryos of step (1 ) with a bio-spectrum generator for 3 to 35 minutes before cryopreservation, after cryopreservation or after thawing;
said method being characterized in that:
- the bio-spectrum has a wavelength spectrum ranging from 0.2 pm to 10 cm; and - during step (2), temperature of the medium is kept at no higher than 40°C.
Yet another object of the invention is to provide a method of applying bio-spectrum to micro-manipulation of embryos, said method comprising the steps of:
(1 ) placing the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio--spectrum generator for 3 to 35 minutes before or after micro-manipulation, while temperature of the medium is kept at no higher than 40C.
In view of the above-detailed objects, it will be understood that the present invention improves the following factors:
- the rate of maturing oocytes, in vitro fertilization and embryos by bio-spectrum irradiation for in vitro production of embryos;
- the rates of survival and pregnancy following the use of frozenlthawed embryos by bio-spectrum irradiation in the process of cryopreservation of '10 embryos;
- the efficiency of micro-manipulation and repair of embryos' injuries by bio-spectrum irradiation in the process of micro-manipulation;
- the rate of embryos survival, ovulation, and fertilization as well as development of the uterus by bio-spectrum irradiation to live female animals; and - the sperm quality by bio-spectrum irradiation to live male animals.
The simulated bio-spectrum mentioned above is described in Chinese patent application No. 91109014.2. This bio-spectrum has a wide band synthesized physical field. lts wavelength ranges from 0.2 Nm to 10 cm. The irradiation signal 20 is very weak and has a wavelength ranging from 30 pm to 10 cm. Same parts of the physical field can produce certain effects.
The objects of the present irEVention were reached by the following technical solutions, Methods of applying the bio-spectrum to in vitro production of animal embryos 1. Collection of coccyges.
2. In vitro maturation of oocytes: oocytes deposited in a standard or defined medium were irradiated with the bio-spectrum generator for 3 to 20 minutes.
During this time period, the average temperature was kept at no higher than 40°C.
3. Capacitation of spermatozoa: semen diluted in a standard or defined medium was irradiated with the bio-spectrum generator for 3 to 20 minutes.
During this time period, the average temperature was kept at no higher than 40°C.
4. In vitro fertilization of oocytes: both matured oocytes and capacitated sperms were co-cultured in one test tube containing a standard or defined medium and irradiated with the bio-spectrum generator from once to 3 times, for 3 to 25 minutes each time. During irradiation, the average temperature was not higher than 40°C.
In vifro culture of embryos: zygotes transferred to a standard or defined medium were irradiated with the bio-spectrum generator for 3 to 30 minutes. During irradiation, the average temperature was not higher than 40°C.
Methods of applying the bio-spectrum to cryopreservation of animal embryos Thawed embryos deposited in a culture medium were irradiated with the bio-spectrum generator for 3 to 20 minutes. During irradiation, the average temperature was not higher than 40°C.
Methods of applying the bio-spectrum to micro-manipulation of animal embryos After micro-manipulation, embryos were deposited in a culture medium and were irradiated with the bio-spectrum generator for 3 to 25 minutes. In order to get stronger effects, embryos were irradiated with the bio-spectrum generator for 3 to 25 minutes before micro-manipulation. During irradiation, the average temperature was not higher than 40°C.
Methods of applying bio-spectrum to animal reproduction, development and growth Animals were irradiated with the bio-spectrum generator once or twice daily, for 30 to 60 minutes each time. During irradiation, the surface temperature of the animals was kept at no higher than 45°C. The results will be even better when conventional techniques of anima( reproduction, development and growth, will be used together with the irradiation. For example, all kinds of gonadotrophin can be envisioned as potential partners.
Methods of applying the bio-spectrum to prevent and cure animal diseases Parts of or whole animal bodies were irradiated with the bio-spectrum generator from 1 to 3 times daily, for 6 to 60 minutes each time. During irradiation, the temperature of the irradiated part was not higher than 45°C.
By using irradiation with the bio-spectrum in in vitro production of embryos, in vitro fertilization increased by at least 18% and the development rate of in vitro fertilized embryos increased by at least 19%.
By using irradiation with the bio-spectrum in cryopreservation of embryos, the survival rate of embryos, after they were frozen/thawed, increased by at least 16% and the rate of pregnancy induced by transferred embryos was also significantly improved.
By using irradiation with the bio-spectrum in micro-manipulation of embryos, the in vifro development rate of demi-morulae and demi-blastocysts and the rate of embryos sustained success was significantly improved.
By using irradiation with the bio-spectrum in animal reproduction, 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 was improved.
By using irradiation with the bio-spectrum in animal disease control, the diseases were alleviated and cured.
DESCRIPTION OF PREFERRED EMBODIMENTS
Embodiment 1 The oocytes were collected from donor cows by conventional methods, and then put in regular or special medium for maturation. During maturation, oocytes were irradiated with the bio-spectrum device (model WS-101 D) for 15 minutes (at weak level). The temperature was kept at 38-40°C by adjusting the distance between the embryo container and the irradiation device. The special medium was made according to the procedure of Brackett et al. (1978, Fertility and Sterility, 29(5):571-582). The recipe for the special medium was as follows:
Components g/L
NaCI 6.55 KCI 0.30 CaCl2 ~ 2H20 0.33 NaH2P04 ~ H20 0.11 MgCl2 ~ 6H20 0.11 NaHCl3 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 was sterilized by filtration after the components had completely dissolved in 1000 ml water. The sterile medium was then equilibrated in an incubator, at 38°C in 59% C02 in the air. The osmolality of the medium was about 300 mOsm/kg H20. The special medium can also be made by adding 84 mg NaCH03 to 100 ml of medium. The m-HIS (modified high ionic strength solution) was made by adding 34 mg NaCI to 10 ml of the above medium.
Sperm capacitation First, the fresh semen was treated according to the procedure of Brackett et al.
(1978, Fertility and Sterility, 29(5):571-582). Semen was pre-diluted with the special medium. After a centrifugation at 350 g, the sperm pellet was resuspended with m-HIS medium and put in a water bath at 38°C for 15 minutes. After another centrifugation, the sperm pellet was resuspended with the special medium in a tube and irradiated with the bio-spectrum device at a weak irradiation level for 10 minutes. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the tube and the irradiation device.
In vitro fertilization of oocytes The in vitro matured oocytes and in vitro capacitated sperm were put in a tube containing the special medium, and irradiated with the bio-spectrum device for 20 minutes. The tube was put in a C02 incubator for a one-hour culture at 38°C.
The tube was then again irradiated with the bio-spectrum for 18 minutes and put in the incubator for a 5-hour culture. A weak level of irradiation was used and the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the tube and the irradiation device.
In vitro culture of embr)ros Before incubation, the in vitro fertilized eggs were put in the special medium for a 25-minute irradiation with the bio-spectrum. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C
by adjusting the distance k~etween the medium container and the irradiation device.
Embodirrtent 2 Bovine embryos were put in the special medium and irradiated with the bio-spectrum for 15 minutes. A weak irradiation level was used. During irradiation, the temperature of rr~edium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device. The cryoprotectant was added according to the procedure set forth in the Japanese Journal of 10 Animal Reproduction 28: 7 50-153, 1982. llAore specifically, the embryos were transferred in a stepwise fashion to PBS plus 2p% CS and 0.18, 0.33, 0.~5, 0.88, 1.0M Glycerol, respectively, each step for 5 minutes. The embryos were put in freezing medium (PBS plus 20°/o CS and ~.OM Glycerol) for 30 minutes were then placed in a 0.5 rnt straw. The straws were ct~oled in a freezer to -7°C
at a rate of t°ClmirZ., then artificially seeded, slowly cooled at -0.3°Clmin. to -35~C and cooled at -0.1 °Clmin. to -36°C, then plunged into liquid nitrogen (-19~6°C) and stored.
Embryo thawing: the straws were removed from the liquid nitrogen, inserted into a 21 °C water bath, and slightly shaken until the 'tce melted. The rate of thawing was 360°Clmin. After thawing, the cryoprotectant was removed by holding embryos each step in reverse of adding eryoprotectant. Embryos were then suspended in the special medium before their irradiation with the bio-spectrum for 20 minutes. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
Embodiment 3 Cryapreservation of embryos was preferably achieved by vitrification according ko the method disclosed by Kasai et al. in J. Reproduction & Fert. 89:91-97.
More specifically, the bovine embryos were suspended in the special medium before their irradiation with the bio-spectrum for 15 minutes. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C, by adjusting the distance between the medium container and the irradiation device. A 0.25 ml straw was used to take in 100 pl S-PBS medium (Brackett ef al., 1978, Fertility and Sterility, 29(5):571-582), 20 NI air, 6 pl EFS
medium (see Kasai et al., J. Reproduction & Fert. 89:91-97), 6 NI air, 40 NI
EFS
medium with embryos after a 3-minute equilibration in EFS medium at room temperature, 6 pl air, 6 NI EFS medium, 15 pl air and 20 pl S-PBS medium.
Finally, the end of the straw was sealed using hot forceps.
Thawing:
The straws were taken out of the nitrogen, inserted into a 20°C water bath, and slightly shaken until the ice melted. The embryos were then quickly flushed out from the straws with 0.5 ml S-PBS medium (see following recipe), transferred to S-PBS medium for 5 minutes and transferred to m-PBS medium (ie. an S-PBS
medium minus glycerol, see R. Dulbeco & M. Vogt, 1954, J. Exp. Med., 99,167).
After three washes in the special medium, the embryos were irradiated with the bio-spectrum for 10 minutes. A weak irradiation level was used. The temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
The recipe for the S-PBS standard medium was as follows:
Components gIL
NaCI 8 KCI 0.2 NaH2P04 1.15 KH2P04 0.2 CaCl2 0.1 MgCI ~ 6H20 0.1 Napyruvate 0.036 Glucose 1 Penicillin 100 IV/ml Streptomycin 0.05 g/L
Glycerol 0.5 M
The EF medium was a 0.5 M sucrose solution (EF medium) which contained 30% polysucrose. The EFS medium was made by mixing ethylene (40%) with the EF medium (60%).
Embodiment 4 Embryo splitting: Rat, goat and bovine embryos were split with a metal knife according to the procedure of Matsu Moto Katsu Ya et al. (Japanese Journal of Animal Reproduction 33, 1-5, 1987). The micro-surgical razor blade was fixed within a micro-manipulator. Before splitting, the embryos were suspended in the culture medium before the irradiation with the bio-spectrum for 15 minutes.
After irradiation, the embryos were kept within a droplet of 0.5 ml PBS (see R.
Dulbeco & M. Vogt, 1954, J. Exp.Med, 99, 167) plus 20% FCS (see Matsu Moto Katsu Ya et al., Japanese Journal of Animal Reproduction, 33, 1-5, 1987) at the center of a plastic dish (diameter 8 cm, height 1 cm). The micro-surgical bisection was performed using a micro-manipulation unit consisting of an inverted microscope.
After splitting and treatment, demi-embryos were suspended in the culture medium before their irradiation with the bio-spectrum for 30 minutes. A weak irradiation level was used. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
Embodiment 5 Sex identification: The sex of an embryo can be accurately determined through sampling several cells therefrom. After sampling, the embryos were partly damaged, and their viability decreased. The viability of the embryos was increased by treating them with the bio-spectrum for 20 minutes (Model WS-101 D).
Embodiment 6 Reproduction, Development and Growth of the Animals Mice were used as experimental animals. Female mice were randomly divided into 2 groups, A and B with 20 mice in each group. Group A mice were irradiated with the bio-spectrum; group B mice were not irradiated and were thus used as controls. Both group A and group B mice were under the same experimental conditions. Each treatment had 2 replicates, with 10 mice per replicate. The mice in group A were irradiated with the bio-spectrum for 20 minutes once a day at a set time. The WS-101 bio-spectrum model, made by Zhoulin Bio-Spectrum Company, was used. A high level of irradiation was used. The temperature over the mouse back was kept under 38°C. The mice were irradiated 10 times in 10 days. On the 4th day, every mouse in group A and group B were injected with PMSG101U (a hormone produced by Tianjin Experiment Animal Center, Tianjin, China). On the 6th day, they were further injected with HCG101U (a hormone produced by Shanghai Biochemistry Pharmaceutical Factory, Shanghai, China).
After treatment, one male mouse was used for mating in each cage. On the 7th day, fertilized eggs were 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 day 10, blastocysts were collected from the remaining 10 mice in each group. The results showed that bio-spectrum improved the ovulation and fertilization ability of eggs, and significantly improved the development of blastocysts. The comparison between group A and group B
also revealed that bio-spectrum irradiation strongly stimulated uterine development in female mice. This experiment showed that bio-spectrum irradiation improved the reproduction, development and growth of the animals.
Embodiment 7 The AES-101 Bio-Spectrum Health Care Device (the bio-spectrum device) made by Beijing Zhoulin-Bio-Spectrum Company was used for irradiating the abdomen of Iambs afflicted with diarrhea. These Iambs were treated with a high level of irradiation, once a day for 20 minutes each time.
The surface temperature of the area being irradiated was maintained at no more than 45°C. The total treatment lasted for 2 to 4 days. Diarrhea was clearly controlled.
In view of the above, it will be understood that the bio-spectrum can have many applications in bio-engineering in addition to the above examples. The use of the bio-spectrum can understandably solve many problems in areas related to both embryonic engineering and bio-engineering, by simplifying the complex and difficult techniques in such areas, for example.
While the invention has been described in conjunction with example embodiments, it will be understood that it is not intended to limit the scope of the invention to such embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents which may be included as defined by the appended claims.
Gene transfer animals can be produced by micro-injection and sperm mediated methods. The aim of gene transfer is to speed up the growth rate and increase disease resistance in animals, improve the quality of animal production and therefore supply many valuable medicines for human beings. However, due to embryo injury, the efficiency of micro-manipulation is very low.
Up to now, very few people have conducted research on the regulation of reproduction and growth potential of animals by use of a physical method. More specifically, there is yet to be a study on the application of a physical method to embryo engineering for solving technical problems.
The Applicant noted that all living things share similar chemical and physical characteristics. For instance, the electric charge in cells is among the special physical characteristics of living things.
When these electric charges in living things and the electromagnetic field in the environment share the same characteristics, interactions between the charges and the field can occur. Understandably, these interactions can also influence the molecules, atoms and electrons to induce significant biological effects.
For example, as long as tissues and cells remain in a living body, they can grow and develop normally in this specific chemical and physical environment.
However, when tissues and cells are separated from the living body i.e. away from their normal chemical and physical environment, their growth and development decrease significantly. Although various chemical protecting materials and nutritive materials have been used to alleviate this decrease, the problem remains.
In the context of the present invention, it was surprisingly found that the growth potential of tissues and cells under culture conditions can be improved by applying a simulated bio-spectrum displaying a weak electromagnetic field.
Application of the simulated bio-spectrum to embryo engineering is thus highly important.
The bio-spectrum will contribute to improve the reproductive ability, growth rate and resistance of animals to various diseases.
SUMMARY OF THE INVENTION
An object of the invention is to induce radiation bio-effects by applying a bio-spectrum to animals and animal embryos.
More specifically, an object of the present invention is to provide a method of applying a bio-spectrum to animal embryonic engineering, said method including the steps of:
(1 ) in vitro maturation of oocytes to obtain matured oocytes;
(2) capacitation of spermatozoa to obtain in vitro capacitated spermatozoa;
(3) in vitro fertilization of oocytes to obtain fertilized oocytes; and (4) in vitro culture of embryos;
said method being characterized in that:
- the bio-spectrum is used at each of the steps (1 ) to (4) and has a wavelength spectrum ranging from 0.2 pm to 10 cm;
- in step (1 ), the oocytes are cultured in a standard or special medium, irradiated with a bio-spectrum generator for 3 to 20 minutes, while keeping temperature of the medium at no higher than 40°C;
- in the step (2), semen diluted with the standard or special medium is irradiated with the bio-spectrum generator for 3 to 20 minutes, while the temperature of the medium is kept at no higher than 40°C;
- in step (3), the matured oocytes and in vitro capacitated spermatozoa are co-cultured in a test tube containing the standard or special medium, and irradiated with the bio-spectrum from 1 to 3 times, for 3 to 25 minutes each time, while keeping the temperature of the medium at no higher than 40°C; and - in step (4), the fertilized oocytes are transferred to the standard or special medium and irradiated with the bio-spectrum generator for 3 to 20 minutes, while keeping the temperature of the medium at no higher than 40°C.
Another object of the invention is to provide a method of applying bio-spectrum to cryopreservation of animal embryos method of applying bio-spectrum to cryopreservation of animal embryos, said method comprising the steps of:
(1 ) putting the embryos in a culture medium; and (2) irradiating the embryos of step (1 ) with a bio-spectrum generator for 3 to 35 minutes before cryopreservation, after cryopreservation or after thawing;
said method being characterized in that:
- the bio-spectrum has a wavelength spectrum ranging from 0.2 pm to 10 cm; and - during step (2), temperature of the medium is kept at no higher than 40°C.
Yet another object of the invention is to provide a method of applying bio-spectrum to micro-manipulation of embryos, said method comprising the steps of:
(1 ) placing the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio--spectrum generator for 3 to 35 minutes before or after micro-manipulation, while temperature of the medium is kept at no higher than 40C.
In view of the above-detailed objects, it will be understood that the present invention improves the following factors:
- the rate of maturing oocytes, in vitro fertilization and embryos by bio-spectrum irradiation for in vitro production of embryos;
- the rates of survival and pregnancy following the use of frozenlthawed embryos by bio-spectrum irradiation in the process of cryopreservation of '10 embryos;
- the efficiency of micro-manipulation and repair of embryos' injuries by bio-spectrum irradiation in the process of micro-manipulation;
- the rate of embryos survival, ovulation, and fertilization as well as development of the uterus by bio-spectrum irradiation to live female animals; and - the sperm quality by bio-spectrum irradiation to live male animals.
The simulated bio-spectrum mentioned above is described in Chinese patent application No. 91109014.2. This bio-spectrum has a wide band synthesized physical field. lts wavelength ranges from 0.2 Nm to 10 cm. The irradiation signal 20 is very weak and has a wavelength ranging from 30 pm to 10 cm. Same parts of the physical field can produce certain effects.
The objects of the present irEVention were reached by the following technical solutions, Methods of applying the bio-spectrum to in vitro production of animal embryos 1. Collection of coccyges.
2. In vitro maturation of oocytes: oocytes deposited in a standard or defined medium were irradiated with the bio-spectrum generator for 3 to 20 minutes.
During this time period, the average temperature was kept at no higher than 40°C.
3. Capacitation of spermatozoa: semen diluted in a standard or defined medium was irradiated with the bio-spectrum generator for 3 to 20 minutes.
During this time period, the average temperature was kept at no higher than 40°C.
4. In vitro fertilization of oocytes: both matured oocytes and capacitated sperms were co-cultured in one test tube containing a standard or defined medium and irradiated with the bio-spectrum generator from once to 3 times, for 3 to 25 minutes each time. During irradiation, the average temperature was not higher than 40°C.
In vifro culture of embryos: zygotes transferred to a standard or defined medium were irradiated with the bio-spectrum generator for 3 to 30 minutes. During irradiation, the average temperature was not higher than 40°C.
Methods of applying the bio-spectrum to cryopreservation of animal embryos Thawed embryos deposited in a culture medium were irradiated with the bio-spectrum generator for 3 to 20 minutes. During irradiation, the average temperature was not higher than 40°C.
Methods of applying the bio-spectrum to micro-manipulation of animal embryos After micro-manipulation, embryos were deposited in a culture medium and were irradiated with the bio-spectrum generator for 3 to 25 minutes. In order to get stronger effects, embryos were irradiated with the bio-spectrum generator for 3 to 25 minutes before micro-manipulation. During irradiation, the average temperature was not higher than 40°C.
Methods of applying bio-spectrum to animal reproduction, development and growth Animals were irradiated with the bio-spectrum generator once or twice daily, for 30 to 60 minutes each time. During irradiation, the surface temperature of the animals was kept at no higher than 45°C. The results will be even better when conventional techniques of anima( reproduction, development and growth, will be used together with the irradiation. For example, all kinds of gonadotrophin can be envisioned as potential partners.
Methods of applying the bio-spectrum to prevent and cure animal diseases Parts of or whole animal bodies were irradiated with the bio-spectrum generator from 1 to 3 times daily, for 6 to 60 minutes each time. During irradiation, the temperature of the irradiated part was not higher than 45°C.
By using irradiation with the bio-spectrum in in vitro production of embryos, in vitro fertilization increased by at least 18% and the development rate of in vitro fertilized embryos increased by at least 19%.
By using irradiation with the bio-spectrum in cryopreservation of embryos, the survival rate of embryos, after they were frozen/thawed, increased by at least 16% and the rate of pregnancy induced by transferred embryos was also significantly improved.
By using irradiation with the bio-spectrum in micro-manipulation of embryos, the in vifro development rate of demi-morulae and demi-blastocysts and the rate of embryos sustained success was significantly improved.
By using irradiation with the bio-spectrum in animal reproduction, 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 was improved.
By using irradiation with the bio-spectrum in animal disease control, the diseases were alleviated and cured.
DESCRIPTION OF PREFERRED EMBODIMENTS
Embodiment 1 The oocytes were collected from donor cows by conventional methods, and then put in regular or special medium for maturation. During maturation, oocytes were irradiated with the bio-spectrum device (model WS-101 D) for 15 minutes (at weak level). The temperature was kept at 38-40°C by adjusting the distance between the embryo container and the irradiation device. The special medium was made according to the procedure of Brackett et al. (1978, Fertility and Sterility, 29(5):571-582). The recipe for the special medium was as follows:
Components g/L
NaCI 6.55 KCI 0.30 CaCl2 ~ 2H20 0.33 NaH2P04 ~ H20 0.11 MgCl2 ~ 6H20 0.11 NaHCl3 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 was sterilized by filtration after the components had completely dissolved in 1000 ml water. The sterile medium was then equilibrated in an incubator, at 38°C in 59% C02 in the air. The osmolality of the medium was about 300 mOsm/kg H20. The special medium can also be made by adding 84 mg NaCH03 to 100 ml of medium. The m-HIS (modified high ionic strength solution) was made by adding 34 mg NaCI to 10 ml of the above medium.
Sperm capacitation First, the fresh semen was treated according to the procedure of Brackett et al.
(1978, Fertility and Sterility, 29(5):571-582). Semen was pre-diluted with the special medium. After a centrifugation at 350 g, the sperm pellet was resuspended with m-HIS medium and put in a water bath at 38°C for 15 minutes. After another centrifugation, the sperm pellet was resuspended with the special medium in a tube and irradiated with the bio-spectrum device at a weak irradiation level for 10 minutes. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the tube and the irradiation device.
In vitro fertilization of oocytes The in vitro matured oocytes and in vitro capacitated sperm were put in a tube containing the special medium, and irradiated with the bio-spectrum device for 20 minutes. The tube was put in a C02 incubator for a one-hour culture at 38°C.
The tube was then again irradiated with the bio-spectrum for 18 minutes and put in the incubator for a 5-hour culture. A weak level of irradiation was used and the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the tube and the irradiation device.
In vitro culture of embr)ros Before incubation, the in vitro fertilized eggs were put in the special medium for a 25-minute irradiation with the bio-spectrum. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C
by adjusting the distance k~etween the medium container and the irradiation device.
Embodirrtent 2 Bovine embryos were put in the special medium and irradiated with the bio-spectrum for 15 minutes. A weak irradiation level was used. During irradiation, the temperature of rr~edium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device. The cryoprotectant was added according to the procedure set forth in the Japanese Journal of 10 Animal Reproduction 28: 7 50-153, 1982. llAore specifically, the embryos were transferred in a stepwise fashion to PBS plus 2p% CS and 0.18, 0.33, 0.~5, 0.88, 1.0M Glycerol, respectively, each step for 5 minutes. The embryos were put in freezing medium (PBS plus 20°/o CS and ~.OM Glycerol) for 30 minutes were then placed in a 0.5 rnt straw. The straws were ct~oled in a freezer to -7°C
at a rate of t°ClmirZ., then artificially seeded, slowly cooled at -0.3°Clmin. to -35~C and cooled at -0.1 °Clmin. to -36°C, then plunged into liquid nitrogen (-19~6°C) and stored.
Embryo thawing: the straws were removed from the liquid nitrogen, inserted into a 21 °C water bath, and slightly shaken until the 'tce melted. The rate of thawing was 360°Clmin. After thawing, the cryoprotectant was removed by holding embryos each step in reverse of adding eryoprotectant. Embryos were then suspended in the special medium before their irradiation with the bio-spectrum for 20 minutes. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
Embodiment 3 Cryapreservation of embryos was preferably achieved by vitrification according ko the method disclosed by Kasai et al. in J. Reproduction & Fert. 89:91-97.
More specifically, the bovine embryos were suspended in the special medium before their irradiation with the bio-spectrum for 15 minutes. A weak level of irradiation was used. During irradiation, the temperature of the medium was kept at 38 to 40°C, by adjusting the distance between the medium container and the irradiation device. A 0.25 ml straw was used to take in 100 pl S-PBS medium (Brackett ef al., 1978, Fertility and Sterility, 29(5):571-582), 20 NI air, 6 pl EFS
medium (see Kasai et al., J. Reproduction & Fert. 89:91-97), 6 NI air, 40 NI
EFS
medium with embryos after a 3-minute equilibration in EFS medium at room temperature, 6 pl air, 6 NI EFS medium, 15 pl air and 20 pl S-PBS medium.
Finally, the end of the straw was sealed using hot forceps.
Thawing:
The straws were taken out of the nitrogen, inserted into a 20°C water bath, and slightly shaken until the ice melted. The embryos were then quickly flushed out from the straws with 0.5 ml S-PBS medium (see following recipe), transferred to S-PBS medium for 5 minutes and transferred to m-PBS medium (ie. an S-PBS
medium minus glycerol, see R. Dulbeco & M. Vogt, 1954, J. Exp. Med., 99,167).
After three washes in the special medium, the embryos were irradiated with the bio-spectrum for 10 minutes. A weak irradiation level was used. The temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
The recipe for the S-PBS standard medium was as follows:
Components gIL
NaCI 8 KCI 0.2 NaH2P04 1.15 KH2P04 0.2 CaCl2 0.1 MgCI ~ 6H20 0.1 Napyruvate 0.036 Glucose 1 Penicillin 100 IV/ml Streptomycin 0.05 g/L
Glycerol 0.5 M
The EF medium was a 0.5 M sucrose solution (EF medium) which contained 30% polysucrose. The EFS medium was made by mixing ethylene (40%) with the EF medium (60%).
Embodiment 4 Embryo splitting: Rat, goat and bovine embryos were split with a metal knife according to the procedure of Matsu Moto Katsu Ya et al. (Japanese Journal of Animal Reproduction 33, 1-5, 1987). The micro-surgical razor blade was fixed within a micro-manipulator. Before splitting, the embryos were suspended in the culture medium before the irradiation with the bio-spectrum for 15 minutes.
After irradiation, the embryos were kept within a droplet of 0.5 ml PBS (see R.
Dulbeco & M. Vogt, 1954, J. Exp.Med, 99, 167) plus 20% FCS (see Matsu Moto Katsu Ya et al., Japanese Journal of Animal Reproduction, 33, 1-5, 1987) at the center of a plastic dish (diameter 8 cm, height 1 cm). The micro-surgical bisection was performed using a micro-manipulation unit consisting of an inverted microscope.
After splitting and treatment, demi-embryos were suspended in the culture medium before their irradiation with the bio-spectrum for 30 minutes. A weak irradiation level was used. During irradiation, the temperature of the medium was kept at 38 to 40°C by adjusting the distance between the medium container and the irradiation device.
Embodiment 5 Sex identification: The sex of an embryo can be accurately determined through sampling several cells therefrom. After sampling, the embryos were partly damaged, and their viability decreased. The viability of the embryos was increased by treating them with the bio-spectrum for 20 minutes (Model WS-101 D).
Embodiment 6 Reproduction, Development and Growth of the Animals Mice were used as experimental animals. Female mice were randomly divided into 2 groups, A and B with 20 mice in each group. Group A mice were irradiated with the bio-spectrum; group B mice were not irradiated and were thus used as controls. Both group A and group B mice were under the same experimental conditions. Each treatment had 2 replicates, with 10 mice per replicate. The mice in group A were irradiated with the bio-spectrum for 20 minutes once a day at a set time. The WS-101 bio-spectrum model, made by Zhoulin Bio-Spectrum Company, was used. A high level of irradiation was used. The temperature over the mouse back was kept under 38°C. The mice were irradiated 10 times in 10 days. On the 4th day, every mouse in group A and group B were injected with PMSG101U (a hormone produced by Tianjin Experiment Animal Center, Tianjin, China). On the 6th day, they were further injected with HCG101U (a hormone produced by Shanghai Biochemistry Pharmaceutical Factory, Shanghai, China).
After treatment, one male mouse was used for mating in each cage. On the 7th day, fertilized eggs were 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 day 10, blastocysts were collected from the remaining 10 mice in each group. The results showed that bio-spectrum improved the ovulation and fertilization ability of eggs, and significantly improved the development of blastocysts. The comparison between group A and group B
also revealed that bio-spectrum irradiation strongly stimulated uterine development in female mice. This experiment showed that bio-spectrum irradiation improved the reproduction, development and growth of the animals.
Embodiment 7 The AES-101 Bio-Spectrum Health Care Device (the bio-spectrum device) made by Beijing Zhoulin-Bio-Spectrum Company was used for irradiating the abdomen of Iambs afflicted with diarrhea. These Iambs were treated with a high level of irradiation, once a day for 20 minutes each time.
The surface temperature of the area being irradiated was maintained at no more than 45°C. The total treatment lasted for 2 to 4 days. Diarrhea was clearly controlled.
In view of the above, it will be understood that the bio-spectrum can have many applications in bio-engineering in addition to the above examples. The use of the bio-spectrum can understandably solve many problems in areas related to both embryonic engineering and bio-engineering, by simplifying the complex and difficult techniques in such areas, for example.
While the invention has been described in conjunction with example embodiments, it will be understood that it is not intended to limit the scope of the invention to such embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents which may be included as defined by the appended claims.
Claims (4)
1. A method of applying a bio-spectrum to animal embryonic engineering, said method including the steps of:
(1) in vitro maturation of oocytes to obtain matured oocytes;
(2) capacitation of spermatozoa to obtain in vitro capacitated spermatozoa;
(3) in vitro fertilization of oocytes to obtain fertilized oocytes; and (4) in vitro culture of embryos;
said method being characterized in that:
- the bio-spectrum, is used at each of the steps (1) to (4) and has a wavelength spectrum ranging from 0.2 µm to 10 cm;
- in step (1), the oocytes are cultured in a standard or special medium, irradiated with a bio-spectrum generator for 3 to 20 minutes, while keeping temperature of the medium at no higher than 40°C;
- in the step (2), semen diluted with the standard or special medium is irradiated with the bio-spectrum generator for 3 to 20 minutes, while the temperature of the medium is kept at no higher than 40°C;
- in step (3), the matured oocytes and in vitro capacitated spermatozoa are co-cultured in a test tube containing the standard or special medium, and irradiated with the bio-spectrum from 1 to 3 times, for 3 to 25 minutes each time, while keeping the temperature of the medium at no higher than 40°C; and - in step (4), the fertilized oocytes are transferred to the standard or special medium and irradiated with the bio-spectrum generator for 3 to 20 minutes, while keeping the temperature of the medium at no higher than 40°C.
(1) in vitro maturation of oocytes to obtain matured oocytes;
(2) capacitation of spermatozoa to obtain in vitro capacitated spermatozoa;
(3) in vitro fertilization of oocytes to obtain fertilized oocytes; and (4) in vitro culture of embryos;
said method being characterized in that:
- the bio-spectrum, is used at each of the steps (1) to (4) and has a wavelength spectrum ranging from 0.2 µm to 10 cm;
- in step (1), the oocytes are cultured in a standard or special medium, irradiated with a bio-spectrum generator for 3 to 20 minutes, while keeping temperature of the medium at no higher than 40°C;
- in the step (2), semen diluted with the standard or special medium is irradiated with the bio-spectrum generator for 3 to 20 minutes, while the temperature of the medium is kept at no higher than 40°C;
- in step (3), the matured oocytes and in vitro capacitated spermatozoa are co-cultured in a test tube containing the standard or special medium, and irradiated with the bio-spectrum from 1 to 3 times, for 3 to 25 minutes each time, while keeping the temperature of the medium at no higher than 40°C; and - in step (4), the fertilized oocytes are transferred to the standard or special medium and irradiated with the bio-spectrum generator for 3 to 20 minutes, while keeping the temperature of the medium at no higher than 40°C.
2. The method according to claim 1, characterized in that components of the special medium are: NaCl 6,55 g/l, KCl 0.30 g/l, CaCl2.cndot.2H2O 0.33 g/l, NaH2PO4-H2O 0.11 g/l, MgCl2-6H2O 0.11 g/l, NaHCO3 3.10 g/l, glucose 2.50 g/l, Na pyruvate 0.14 g/l (or Pyruvate 0.11 g/l), bovine serum albumin 3.00 g/l, and sodium salt penicillin 0.031 g/l; the components being completely dissolved in 100 ml water to form a solution, and 84 mg NaHCO3 being added to 100 ml of said solution to form the special medium.
3. A method of applying bio-spectrum to cryopreservation of animal embryos, said method comprising the steps of:
(1) putting the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio-spectrum generator for 3 to 35 minutes before cryopreservation, after cryopreservation or after thawing;
said method being characterized in that:
- the bio-spectrum has a wavelength spectrum ranging from 0.2 µm to cm; and - during step (2), temperature of the medium is kept at no higher than 40°C.
(1) putting the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio-spectrum generator for 3 to 35 minutes before cryopreservation, after cryopreservation or after thawing;
said method being characterized in that:
- the bio-spectrum has a wavelength spectrum ranging from 0.2 µm to cm; and - during step (2), temperature of the medium is kept at no higher than 40°C.
4. A method of applying bio-spectrum to micro-manipulation of embryos, said method comprising the steps of:
(1) placing the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio-spectrum generator for 3 to 35 minutes before or after micro-manipulation, while temperature of the medium is kept at no higher than 40C.
(1) placing the embryos in a culture medium; and (2) irradiating the embryos of step (1) with a bio-spectrum generator for 3 to 35 minutes before or after micro-manipulation, while temperature of the medium is kept at no higher than 40C.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94118306 CN1068195C (en) | 1994-11-24 | 1994-11-24 | Application of biological spectrum in animal embryo engineering and animal |
| CN94118306.8 | 1994-11-24 | ||
| PCT/CN1995/000087 WO1996015732A1 (en) | 1994-11-24 | 1995-11-02 | Use of the bio-frequency spectrum in animal embryo-engineering |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2207013A1 CA2207013A1 (en) | 1996-05-30 |
| CA2207013C true CA2207013C (en) | 2006-07-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002207013A Expired - Lifetime CA2207013C (en) | 1994-11-24 | 1995-11-02 | Application of bio-spectrum in animal embryonic engineering |
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| Country | Link |
|---|---|
| EP (1) | EP0872219B1 (en) |
| CN (1) | CN1068195C (en) |
| AU (1) | AU704690B2 (en) |
| CA (1) | CA2207013C (en) |
| DE (1) | DE69528295T2 (en) |
| WO (1) | WO1996015732A1 (en) |
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| IL137366A0 (en) * | 2000-07-18 | 2001-07-24 | Shladot Metal Works Ltd | A method for increasing the fertilizing capability of sperm cells |
| KR100516881B1 (en) * | 2002-12-13 | 2005-09-27 | (주)아비코아생명공학연구소 | Method for Inactivating Nuclear Chromosomal Materials of Recipient Oocyte for Manufacturing Clone Embryo from Somatic Cell Using X-ray Irradiation |
| CN102250832B (en) * | 2011-05-30 | 2012-08-15 | 中国农业大学 | Culture liquid for promoting ectogenesis of frozen embryo after thawing |
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| SU731976A1 (en) * | 1975-04-18 | 1980-05-05 | Ленинградский Научно-Исследовательский Институт Радиационной Гигиены | Method for improving fertility of small rodents |
| SU1152583A1 (en) * | 1982-03-01 | 1985-04-30 | Кубанский Ордена Трудового Красного Знамени Сельскохозяйственный Институт | Method of insemination of sow |
| EP0263192A1 (en) * | 1986-10-03 | 1988-04-13 | HENKEL CORPORATION (a Delaware corp.) | Dicyanoethenyl fatty compounds and derivatives thereof |
| SU1402343A1 (en) * | 1985-06-04 | 1988-06-15 | Кубанский сельскохозяйственный институт | Method and apparatus for treatment of sperm of animals |
| SU1724204A1 (en) * | 1989-10-09 | 1992-04-07 | Гродненский государственный медицинский институт | Method of boar sperm treatment |
| SU1757676A1 (en) * | 1989-11-27 | 1992-08-30 | Горский Сельскохозяйственный Институт | Method for stimulation of specific immunity induction in hens to newcastle disease |
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1994
- 1994-11-24 CN CN 94118306 patent/CN1068195C/en not_active Expired - Lifetime
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- 1995-11-02 WO PCT/CN1995/000087 patent/WO1996015732A1/en active IP Right Grant
- 1995-11-02 CA CA002207013A patent/CA2207013C/en not_active Expired - Lifetime
- 1995-11-02 EP EP95936416A patent/EP0872219B1/en not_active Expired - Lifetime
- 1995-11-02 AU AU38386/95A patent/AU704690B2/en not_active Expired
- 1995-11-02 DE DE69528295T patent/DE69528295T2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU3838695A (en) | 1996-06-17 |
| CN1068195C (en) | 2001-07-11 |
| DE69528295D1 (en) | 2002-10-24 |
| AU704690B2 (en) | 1999-04-29 |
| EP0872219A4 (en) | 1999-02-03 |
| DE69528295T2 (en) | 2003-05-08 |
| WO1996015732A1 (en) | 1996-05-30 |
| EP0872219A1 (en) | 1998-10-21 |
| CN1123131A (en) | 1996-05-29 |
| CA2207013A1 (en) | 1996-05-30 |
| EP0872219B1 (en) | 2002-09-18 |
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