Note: Descriptions are shown in the official language in which they were submitted.
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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.
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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
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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;
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- 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
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(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.
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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.
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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
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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)
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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
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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.
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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
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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).
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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.
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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.
