WO1996015732A1 - Utilisation du spectre de frequences biologiques dans l'embryogenese animale - Google Patents

Utilisation du spectre de frequences biologiques dans l'embryogenese animale Download PDF

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Publication number
WO1996015732A1
WO1996015732A1 PCT/CN1995/000087 CN9500087W WO9615732A1 WO 1996015732 A1 WO1996015732 A1 WO 1996015732A1 CN 9500087 W CN9500087 W CN 9500087W WO 9615732 A1 WO9615732 A1 WO 9615732A1
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animal
irradiation
embryo
minutes
culture medium
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PCT/CN1995/000087
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English (en)
French (fr)
Inventor
Lin Zhou
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Lin Zhou
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Application filed by Lin Zhou filed Critical Lin Zhou
Priority to AU38386/95A priority Critical patent/AU704690B2/en
Priority to CA002207013A priority patent/CA2207013C/en
Priority to DE69528295T priority patent/DE69528295T2/de
Priority to EP95936416A priority patent/EP0872219B1/en
Publication of WO1996015732A1 publication Critical patent/WO1996015732A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation
    • A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation

Definitions

  • the invention belongs to the field of biological engineering, and particularly relates to the application of biological frequencies in animal embryo engineering.
  • Animal embryo engineering mainly includes technologies such as embryo production, fetal cryopreservation, and embryo micromanipulation.
  • in vitro embryo production technology can be divided into using the genetic resources of good animals to accelerate genetic improvement; overcoming the infertility of certain rare animals, saving rare or endangered animal sources; providing animals for the production of genetically modified animals and animal sex identification Embryo resources.
  • the current in vitro embryo production technology is very inefficient, and in addition, the quality of embryos produced outside the embryo is worse than that of the embryo, and the pregnancy rate of embryo transfer is also low.
  • Animal embryo cold storage technology is the key technology for whether animal embryo transfer technology can be applied to production.
  • the application of this technology can establish an embryo germplasm bank and facilitate the transportation and exchange of animal breed resources internationally.
  • the current methods commonly used in the country include conventional steam freezing (slow cooling control) and vitrification (quick cooling).
  • steam freezing slow cooling control
  • vitrification quick cooling
  • the embryos produced outside the field are thawed after freezing, and the survival rate is reduced by about 65.
  • the pregnancy rate after transplantation is at least 20% lower than that of normal fresh embryos.
  • Animal embryo micromanipulation techniques include: gender identification to obtain animal offspring of the desired sex; animal embryo immunization, including embryo cutting and nuclear transfer techniques' to produce a large number of identical offspring from the same it show animal (clone animal) Greatly improve the reproduction rate and genetic improvement of animals.
  • Transgenic technology mainly refers to the technique of marked injection and sperm loading. The purpose is to promote animal growth, enhance animal resistance, improve the quality of animal products, or provide humans with a large amount of Precious medicines (depending on the gene transferred). Because animal embryo micromanipulation techniques will inevitably damage animal embryos, their success rate is very low.
  • the present invention is based on the following recognitions: For various reasons, humans rarely study the use of physical methods to regulate and influence the reproductive growth of animals, and rarely apply physical methods to embryo engineering to solve technical problems.
  • the inventor believes that all living matter has both chemical and physical properties. Living matter itself has characteristic physical properties (such as the charge of a cell, the biological field of the body, etc.). When the external electric field is similar to some of the main physical characteristics of living matter, the charged matter in the living matter will interact with the external electromagnetic field, which may affect the molecules, atoms, and electrons in the cell at the same time, resulting in obvious Biological effects. Taking isolated tissues and cells as an example, tissues and cells are supported by the chemical and physical environments in the body, and their growth and development are normal.
  • the purpose of the present invention is to use simulated biological frequencies to act on animal embryo engineering technology and animals, and cause radiation biological effects of animal embryos and animal machinery, specifically including: using simulated biological spectrum irradiation in the production of embryos to improve Mother cell maturation rate, in vitro fertility rate, embryo maturation rate. Improve embryo quality;
  • simulated biological frequency f irradiation is used to improve embryo survival and pregnancy development:
  • simulated biological frequency irradiation is used to repair damaged embryos and increase their success rate.
  • Simulated biological frequency irradiation was used in animal reproduction and disease treatment to improve the survival rate of fertilization marks: to promote the animal's uterine night twilight; to improve the printing rate, fertilization rate of printing, and the development rate of fertilization.
  • For male animals can improve sperm quality, etc .:
  • the simulated biological frequency described in Chinese Patent Application No. 91 10901 4 ⁇ The disclosed broad-band integrated physical field has a wavelength from micrometer (0.2 ⁇ ) to centimeter (10cm): among 5 Gum-1 10cm ⁇
  • the radio signal is weak. Use the object 3 ⁇ 4 part of the field
  • Correction page ISA / CN The field can also get a certain amount of fruit.
  • the mature Indoblast and Pseudosaurus sylvestris are placed in a test tube containing a common culture medium or a specific culture solution, and irradiated with a frequency chirp generator 1 to 3 times for 3 to 25 minutes each time, The temperature of the culture medium must not be higher than 40 ° C.
  • Embryo culture in vitro. The fertilized India mother is transferred into the ordinary culture medium or specific culture medium, and the irradiation takes place for 3 to 30 minutes. During the irradiation, the temperature of the culture medium must not be higher than 40 ° C.
  • Method for applying biofrequency in cryopreservation of animal embryos Place animal embryos in culture medium before or after thawing, and irradiate with spectrum for 3 to 30 minutes. To enhance the effect, animals can also be thawed before and after thawing The embryos are placed in the culture medium and irradiated with frequent monks for 3 to 20 minutes. During the irradiation, the temperature of the culture medium must not be higher than 40 ° C.
  • Method for applying biological frequency in animal embryo microsurgery After the micromanipulation, the animal embryo is placed in the culture medium and irradiated with the frequency for 3 to 35 minutes. In order to enhance the effect, it can also be used in Xianzheng operation. Before the animal embryos were placed in culture, they were irradiated with a spectrum generator for 3 to 35 minutes. During the irradiation, the temperature of the culture medium should not be higher than 40 °. Irradiate the animal 1-2 times a day for 3-60 minutes each time. During the irradiation, the surface temperature of the animal should not be higher than 45 ° C. When using this method, supplemented with conventional reproductive development techniques C (such as injection of various gonadotrophins, etc.) is more effective.
  • reproductive development techniques C such as injection of various gonadotrophins, etc.
  • Application method of biological frequency in animal disease prevention and treatment Use frequent words to irradiate the local or whole body of the animal, once a day, 3 times a day, 6-60 minutes each time, during the irradiation process, the temperature of the surface where the object is irradiated must not Above 45 ° C.
  • the use of simulated biological spectrum irradiation can improve the fetal survival rate by at least 16%, and also significantly improve the pregnancy development rate of embryos.
  • simulated biological frequency irradiation in animal reproduction and development can promote the development of female animals' uterus and increase the rate of typesetting, the rate of printed fertilization, and the rate of survival and development of sentimental printing. Improve the sperm quality of male animals.
  • simulated biological frequency radiation in the treatment of diseases in animals can alleviate and control the disease and achieve a therapeutic and health effect.
  • Indo-mother cells were collected from donor cows by conventional methods, and the collected indo-mother cells were placed in a container containing a normal culture medium or a specific culture solution for in-mature maturation of the indo-mother cells.
  • the WS-101D frequency treatment device is irradiated for 15 minutes.
  • the dose switch of the treatment device is selected to be weak.
  • the temperature of the culture medium is controlled between 38 ° C and 40 ° C. The temperature is controlled by adjusting the irradiation distance. .
  • the specific culture solution was prepared according to the method of Bracket! With aCl 6. 55 g, liter, C1 0. 30 z liter, QiCl :.
  • Correction page ISA / CN Add the above-mentioned special culture solution to the semen, centrifuge at 350 g for 5 minutes, remove the supernatant, add m-HIS solution to the precipitated dragon, and incubate in a water bath at 38 ° C for 15 minutes. Then centrifuge and remove the supernatant.
  • Into a test tube containing a specific culture solution irradiate the WS-101D frequency therapy device produced by Zhou Linpin Zhu Corporation for 10 minutes. The dose switch of the treatment device is selected as a weak range.
  • the temperature of the culture solution is controlled at 38. Between ° C and 40 ° C, the temperature is controlled by adjusting the irradiation distance.
  • the fertilized imprinted mother cells were transferred into a specific culture solution, and irradiated with the WS-101D spectrum therapy device for 25 minutes.
  • the dose switch of the therapy device was selected to be weak, and the temperature of the culture solution was controlled at 38 ° C to 40 ° C. Between C, the temperature is controlled by adjusting the irradiation distance.
  • the bovine embryos were placed in a specific culture solution, and treated with WS-101D collar maggots for 15 minutes, and the dose switch of the treatment device was selected to be a weak range.
  • the temperature of the culture solution was controlled at 38 ° C to 10 °. Between C, the temperature is controlled by adjusting the irradiation distance.
  • Cryoprotective agents were added using methods such as Tadashi Inoue (Fujian Naoto 28: 150-153, 1982). That is, PBS containing 20% CS was prepared in advance, and glycerin was used to prepare 0.18, 0.33, 0.775, 0.88, and 1.0M solutions, and respectively filled into small cultures for use.
  • the frozen bovine embryos were shifted at intervals of 5 minutes, placed in a 1.0 M solution for 30 minutes, and then loaded into 0.5 ml thin tubes, and then transferred to a freezer to start cooling. Take 1. The speed of C./min is reduced to a temperature of 7 ° C. At this temperature, crystallization is induced. After forced ice planting, the temperature is lowered to -35X at a rate of 0.3 ° C / min, from 35 ° C to _36 ° C. After cooling at a rate of 0.1 ° C / min, liquid gas (-196 ° C) was directly injected.
  • Embryo solution Remove the thin tube containing the embryo from the liquid gas, immediately put it into warm water at 21 ° C, and gently shake it. The seed speed is about 360 ° C / min. Embryo solution) a in time
  • correction page ISA / CN The method of slow dilution removes the cryoprotectant slowly, that is, at room temperature, the dilution is performed in the reverse order of adding the cryoprotectant. Then, the blasted embryos were placed in a specific culture solution, and irradiated with a WS-101D spectrum therapy device for 20 minutes.
  • the dose switch for treatment was selected as a weak range, and the temperature of the culture solution was controlled between 38 ° C and 40 ° C. The temperature is controlled by adjusting the irradiation distance.
  • the embryos were equilibrated with the EFS solution at room temperature for 3 minutes, they were quickly sucked into the Niechang EFS 3 ⁇ 4 ( ⁇ .40LI1) section of the thin tube, and then the air ( ⁇ 6 1), £! 3 ⁇ 4 solution (: ⁇ 6 ⁇ 1) were sequentially sucked in. ), Air ( ⁇ I 5ul) and S-PBS solution (; ⁇ 20 ⁇ 1), and finally sealed with pre-tweezers.
  • the therapy device is irradiated for 10 minutes, the dose switch is weak, the culture temperature is controlled between 38 ° C and 40 ° C, and the temperature is controlled by the irradiation distance of the joint.
  • EFS solution preparation method First prepare 0.5M sugar solution containing 30% polysaccharin solution: 3 ⁇ 4 (EF solution), and then mix with 40% diol and 60% EF 3 ⁇ 4 to form EFS solution.
  • Embryo segmentation was performed using Matsumoto Kazuya et al. ("S Makes", “,” and “C Embryo Cells, Cells, Embryo Cuts, and Cuts”. ⁇ Animal 5? Metal word cutting method used. First, use the tip of a razor with tweezers Open it and fix it on the capillary glass tube, and then install the capillary glass tube on the microscope to make the operation.
  • the half embryos were placed in the culture medium and irradiated with the WS-101D spectrum therapy device for 30 minutes.
  • the dose switch of the therapy device was selected to be a weak range.
  • the temperature of the culture solution was controlled between 38 ° C and 40 ° C. Adjust the irradiation distance to control the temperature.
  • Gender identification Taking a small number of cells from an embryo allows accurate sex identification. However, it is easy to damage the embryo and affect its vitality. After the cells are taken, the embryos are irradiated with the WS-101D frequency universal therapy instrument for 20 minutes, which can improve the vitality of the embryos.
  • mice were used as experimental animals. The mice were randomly divided into two groups of 20 animals each. Group A was a bio-frequency irradiation group, group B was a control group, and the control group was not subjected to irradiation treatment. The other experimental conditions of the two groups were the same. Each group of animals was divided into 2 batches, and 10 animals in each batch were tested.
  • the WS-101 spectrum therapy device made by Zhou Linbin Qiong Headquarters was used to irradiate Group A once a day at a regular time, the exposure time was 20 minutes, and the Yiyi dose switch was selected as a strong file.
  • the rat back temperature is not higher than 38 ° C, and the total irradiation time is 10 days / 10 times.
  • the rats in the A and B groups were printed with 10 P rats and mouse infusion tubes. The comparison of the two groups showed that irradiation with the frequent therapeutic apparatus had a significant protective effect on the survival of the fertilized marks.
  • the oviducts of each of the two groups A and B were flushed with oviducts to collect eggs, and the number of blastocysts was collected.
  • the comparison between the two groups showed that the treatment with frequent irradiation had the effect of promoting fertilization and printing. Blastocyst development is obvious.
  • Comparison of the two groups A and B also showed that the irradiation of the frequency-frequency therapy device significantly increased the congestion and development of the mouse uterus. This embodiment illustrates that treating animals with irradiance by using frequent language can promote the animal's reproductive development and growth.
  • the lambs were treated with WS-1 01 frequency treatment apparatus made by Zhoulin Spectrum Corporation. The treatment was written once a day. Each time 20 minutes, the switch was chosen to block the lamb's abdomen.
  • the biofrequency will be applicable to a variety of biological engineering objects.
  • it can also make complicated and difficult processes in embryo engineering and biological engineering easier. Therefore, there may be other different implementation schemes in the field of bioengineering, and these schemes should all belong to the protection scope of the present invention.

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  • Veterinary Medicine (AREA)
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  • Engineering & Computer Science (AREA)
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Description

生物频 i蒈在动物胚胎工程中的应用
技术领域
本发明属于生物工程领域， 具体涉及生物頻诸在动物胚胎工 程中的应用。
背景技术
近年来， 有关动物胚胎工程的基础研究和技术开发取得了惊 人的进展， 胚胎工程研究不仅停留在研究室阶段， 而以极快的速 度向实用化、 产业化方面迈进和普及。 动物胚胎工程主要包括胚 胎休外生产、胚胎冷 保存和胚胎显微操作等技术。
胚胎体外生产技术的发展可以 分利用 i 良动物的遗传 源， 加快遗传改良； 克服某些珍稀动物的不孕 ώ , 保存稀有动物或濒 危动物 i 源； 为生产转基因动物及进行动物性别鉴定提供动物胚 胎資源。 然而， 目前胚胎体外生产技术的效率很低， 另外， 休外生 产的胚胎质量比休内的差， 胚胎移植 娠率也 低。
动物胚胎冷 保存技术是动物胚胎移植技术能否应用于生产 的关键技术， 该技术的应用可以建立胚胎种质库， 便于国际间动 物品种資源的运输和交流。 目前国 ί示上通用的方法有常規汽冻（慢 速控制降温） 和玻璃化冷 （快速降温）两种方法。 但是， 休外生产 的胚胎经冷冻后解冻，存活率降低， 约 65 , 移植后妊娠率比正常 鲜胚胎移植降低至少 20%。
动物胚胎显微橾作技术包括： 性别鉴定， 以荻得期望性別的 动物后代； 动物胚胎免隆， 包括胚胎切割及核移植技术' 以生产来 自同一 it秀动物的大量相同的后代（克隆动物）， 大大提高繁殖率、 速动物遗传改良； 转基因技术， 主要指显徵注射技术及精子载 ί本技术， 目的在于促进动物生长， 增强动物抗^性， 提高动物产品 质量， 或为人类提供大量的珍贵药品（依转移的基因不同而异）。由 于动物胚胎显微操作技术必然会损伤动物胚胎， 因此， 其成劝率 都很低。
更 正 百 ISA/CN 发明内容
本发明基于如下认识： 由于种种原因， 人 ί门很少研究应用物 理方法调节影响动物的繁殖生长劝能， 也很少 物理方法应用到 胚胎工程中解决技术难题。发明人认为： 一切生命物质都同时具有 化学特性和物理特性 生命物质本身就具有有特征的物理特性（如 细胞的电荷，机体的生物场， ）。 当外界的电 ¾场与生 命物质中的某些主要物理特性相似时， 生命物质中的带电荷物质 就会与外界电磁场相互作用， 可能使细胞内分子、原子、电子同 时受到影响而产生明显的生物效应。以离体组织和细胞为例， 组织 和细胞在体内受到化学环境和物理环境的支持，生长发育正常。当 组织或细胞离休后， 尽管竭力采用各种化学性质的保护剂和營养 成份来改善培养环境， 但是， 离体組织或细胞的生长发育仍然明 显比休内生长发育时差。发明人进一步认为： 在离体組织和细胞培 养中， 施加一个模拟生物频 的弱电磁场将改善离体组织和细胞 的生长状况。 因此， 模拟生物頻诸方法应用到胚胎工程中具有意 义。 同理， 模拟生物頻诸 对动物的繁殖、发育生长和防治疾病 具有明显的促进作用
本发明目的是利用模拟的生物頻语作用于动物胚胎工程技术 和动物中， 引起动物胚胎和动物机休的辐射生物效应， 具体包括： 在胚胎休外生产中利用模拟的生物频谱照射， 提高印母细胞 成熟率， 体外受锖率 胚胎成熟率.提高胚胎质量；
在胚胎冷冻保存过程中利用模拟的生物頻 f照射， 提高胚胎 存活率和妊娠发育率：
在胚胎显微搡作技术中利用模拟的生物頻语照射， 修复损伤 胚胎， 提高其成劝率。
在动物繁 发育和 ^ίτ治疾病中采用模拟生物频 ¾照射， 以提 高受精印的存活率： 促进动物子宫发宵； 提高排印率、印的受精率 和受蜻印的发育率。 对雄性动物能改善精子的品质等：
这里所述的模拟生物频诸栺中国专利申请 91 10901 4 · 所公 开的宽频带综 ^物理场， 其波长从微米（0. 2μηι )到厘米（10cm ) : 其 中在 5 Gum一 1 0cm之^， 射信号很弱。 用该物 ¾场中的某一部份
更 正 页 ISA/CN 场也可以获得一定的敖果。
实现本发明目的的技术方案是：
生物频诸在动物胚胎体外生产中应用的方法：
( 1 )印母细胞收集。（2 )印母细胞体外成熟。 收集的印母细胞 放置于普通培养液或特定培养液中， 用频醤发生 照射 3至 20分 钟， 照射过程中， 培养液的温度不得高于 40°C。 ( 3 ) 精子荻能。 采集的锖液 用普通培养液或特定培养液稀释后用频谱发生^照射 3至 20分钟，照射过程中，培养液的温度不得高于 40°C。 （4 ) 印母 细胞休外受精。 成熟的印母细胞和荻能蜻子放置于装有普通培养 液或特定培养液的试管中， 用频镨发生器照射 1到 3次，每次照射 3到 25分中，每次照射过程中，培养液温度不得高于 40°C。 （5 )胚 胎体外培养。 ^受精印母移入普通培养液或特定培养液中， 用频镨 发生^照射 3至 30分钟. 照射过程中， 培养液温度不得高于 40° C。
生物频诸在动物胚胎冷冻保存中应用的方法： 解冻前或后将 动物胚胎置于培养液中， 用频谱发生 ^照射 3至 30分钟， 为了增 强作用， 也可以在解冻前和解冻后将动物胚胎置于培养液中， 先 用频僧发生^照射 3至 20分钟， 照射过程中， 培养液温度不得高 于 40°C。
生物頻镨在动物胚胎显微橾作中应用的方法： 显微操作后将 动物胚胎置于培养液中， 用頻偖发生^照射 3至 35分钟， 为了增 强作用， 也可以在显徵橾作前将动物胚胎置于培养 ¾中先用频谱 发生器照射 3至 35分钟， 照射过程中， 培养液温度不得高于 40° 生物頻 在动物繁殖发育生长中应用的方法： 用频 i#发生^ 照射动物，每天 1—2次，每次 3— 60分钟，照射过程中，动物表面 温度不高于 45°C。 使用本方法时， 辅以常規的繁殖发育生长技术 C如注射各种促性腺激素等）效果更佳。
生物頻诸在动物疾病防治中的应用方法： 用频语发生^照射 动物的局部或全身， 每天】一3次， 每次 6— 60分钟， 照射过程中， i-f]物被照射处表面温度不得高于 45°C。
更 正 百 ISA/CN 在胚胎体外生产'中利用模拟的生物频 -i 照射， 可以使休外受 情率至少提高百分之十八， 体外受精印的发育率至少提高百分之 十九。
在胚胎冷冻保存过程中利用模拟的生物频谱照射， 可以使胚 胎存活率至少提高百分之十六， 对胚胎的妊娠发育率也有明显的 提高
在胚胎显微操作技术中利用模拟的生物频谱照射， 可以显著 地提高半分桑椹体外发育率和半分囊胚发育率， 提高胚胎染色体 标本制作效率。
在动物繁殖发育生长中利用模拟的生物頻 照射， 可以促进 雌性动物子宫发育， 提高排印率、 印的受精率、受情印的存活率和 发育率。 改善雄性动物的精子品质等。
在动物 f¾治疾病中利用模拟的生物频镨照射， 可以緩解和控 制疾病，达到治疗保健作用。
本发明的最佳实施方式
实施例 1
用常規方法从供体母牛中收集印母细胞， 将所采集的印母细 胞放置于装有普通培养液或特定培养液的容器中进行印母细胞体 外成熟， 用周林频醤总公司生产的 WS— 101D频诸治疗仪照射 15 分忡， 治疗仪的剂量开关迭择为弱档， 照射过程中， 培养液温度控 制在 38°C至 40°C之 间 ， 通过调节照射距离来控制温度。 特定培 养液按照 Bracket!等的方法配制。 用 aCl 6. 55克 ,升, C1 0. 30 z升， QiCl:. · 2H.O 0. 33 /升、 Nal^PC^^O 0. 1 1 克 /升、 MgCl2 · 6H20 0. 1 1 /升、 NaHCO, 3. 1 0 /升、 萄糖 2. 50克/升、 无水 丙酮 ¾钠 0. 14克 /升（或丙 δ ¾ 0. 1 1 /升）、牛血清白蛋白 3. 00 乏 /升、 青零素钠盐 0. 031 ( 50单位 /毫卄）加入蒸馏水 1000 ¾升， 待其 分溶解后过^灭¾。 在舍 59 %CO2空气的 38°C培养箱中平 衡， 滲透压约为 300毫滲量 公斤。 在 100 ¾ 上述配制的溶液中 加入 84毫升 NaHO¾即配制为特定培养液， 在 10毫升上述配制的 溶液中加入 34 ¾克 aCl即配制 m-HlS液。
锖子荻能。 ^采集的新鲜情¾先用 Bmd tt等的万法处理. 即
更 正 页 ISA/CN 于精液中加入上述特 '定培养液， 350克离心 5分钟， 去上清， 于沉 淀的蜻子中加入 m— HIS液， 在 38°C水浴培养 15分钟， 随后离 心， 去上清， 置入装有特定培养液的试管中用周林频诸总公司生 产的 WS— 101D频语治疗仪照射 10分钟， 治疗仪的剂量开关逸择 为弱档， 照射过程中. 培养液温度控制在 38°C至 40°C之间， 通过 调节照射距离来控制温度。
印母细胞体外受情。^体外成熟的印母细胞和休外获能精子放 置于装有特定培养液的试管中， 用周林频诸总公司生产的 WS - 101D频诸治疗仪照射 20分钟， 然后将试管置于 38°C CO,培养箱 中培养， 一个小时后^试管取出， 再用频镨治疗仪照射 18分钟后 置于培养箱中培养 5小时。照射过程中，频诸治疗&的剂量开关逸 择为弱档， 培养液温度控制在 38°C至 40°C之间， 通过调节照射距 离来控制温度。
胚胎休外培养。 ^受精印母细胞移入特定培养液中， 用 WS— 101D頻谱治疗仪照射 25分钟， 治疗仪的剂量开关逸择为弱档，培 养液温度控制在 38°C至 40。C之间， 通过调节照射距离来控制温 度。
实施例 2
将牛胚胎置于特定培养液中， 用 WS— 101D领镨治疗汉照射 15分忡， 治疗仪剂量开关逸择为弱档， 培养液温度控制在 38°C至 10。C之间，通过调 节照射距离控制温度。采用井上忠恕等方法（家 畜繁直杂志 28 ： 150- 153 , 1982年）添加冷冻保护剂。即预先准备 含 20%CS的 PBS , 用甘油配制成 0. 18、 0· 33、 0. 75、 0. 88 以及 1. 0M溶液， 并分别装入小培养 中待用。 将冷冻用牛胚胎以 5分钟 间隔过档， 并在 1. 0M溶液中放置 30分钟后装入 0. 5毫升细管中， 再转入冷冻仪中开始降温冷 。 以 1。C./分的速度降温到一 7°C， 在 此温度诱发结晶， 进行强制植冰后， 再以 0. 3°C/分的速度下降到 - 35X , 一 35°C至 _ 36°C以 0. 1°C/分速度降温后， 直接投入液氣 (一 196。C)。
胚胎解 。 从液氣中取出装有胚胎的细管， 立即投入 21°C的 温水中，轻轻搖荡 . 籽 速度约 360°C/分。胚胎解 )a及时以阶段
更 正 页 ISA/CN 性稀释法缓慢地除去 '冷冻保护剂， 即在室温条件下， 按照添加冷 保护剂的相反的顺序进行稀释。 然后， 解 后的胚胎置于特 定培养液中， 用 WS— 101D频谱治疗仪照射 20分钟， 治疗 f义的剂 量开关选择为弱档，培养液温度控制在 38°C至 40°C之间， 通过调 节照射距离控制温度。
实施例 3
采用 Kasai等的玻璃化法 U. Reporod. Pert. . 89： 91— 97 )冷冻。 即在室温下往 0. 25ml细管内依次吸入 S— PBS液（ 〜l 00ul )、空气 (：〜 20W)、EFS液 (：〜 6μ1)、空气 (：〜 6μ1 ) 以及 EFS ¾ ( 〜40ul)待用。 将牛胚胎置于特定培养液中， 用 WS—101D频语治疗仪照射 15分 忡， 剂量开关逸择为弱挡， 培养液温度控制在 38°C至 40°C之间， 通过调节照射距离控制温度。 将胚胎用 EFS液在室温下平衡 3分 钟后， 迅速吸入上述细管的聂长 EFS ¾ ( 〜.40LI1)段中， . 后依次 吸入空气（〜6 1)、£!¾液 (：〜 6μ1 )、空气（〜 I 5ul )和 S— PBS液 (；〜 20μ1) , 最后用预 的镊子封口。
解冻。 从液氮中取出装有胚胎的细管， 立即投入 20°C水中， 径轻摇荡。 迅速用 0. 5mlS— PBS液沖出细管内胚胎， 然后移入 S— PBS液内， 经过 5分钟移到 m— PBS；¾， ；先涤三次后置于特定培养 液中用 WS— 101D頻镨治疗仪照射 1 0分钟， 剂量开关逸择弱挡， 培养温度控制在 38°C至 40°C之间， 通过谰节照射距离控制温度。
用 NaCl 8克. /升、 KC1 0. 2免 /升、 Na2HP04 1. 1 5 /升、 KH2PCX 0. 2 f升、 CaCl, 0. 1 升、 MgCl2 · 6H?0 0. 1 卄、丙 8 ¾纳 0. 036免 /升、 萄糖 1 免/升、青霎素 】00IU /ml、¾ 素 0. 05 /升、 配制 0. 5M蔗糖溶液即为 S— PBS液。
EFS液配制方法： 先配制含 30 %聚蔗餹溶液的 0· 5M ^糖溶 :¾ (EF液）然后以 40 % 二醇和 60 %EF ¾相混 ^即成 EFS液。
实施例 4
胚胎分割。 胚胎分割采用松本和也等（ 『 S 使 ' 卜、 '及 C 胚胎胞紬胞胚 ^切断分 』 . ^畜 5?殖字 使用过的金 属刀切割法。先将剃须刀的刀尖用镊子掰开， 并 ®定在毛细玻璃管 上， 然后把毛细玻璃管安装在显微橾作^ ^手上 操作时， 0.
更 正 百 ISA/CN 05mlPBS+ 20¾CS培养液滴于塑料培养 (直径 8cm、高 lcm )中 央。 待切割胚胎置于培养液中， 用 WS— 101D频语治疗仪照射 15 分钟， 取出胚胎放入培养亚中央， 在装有显微橾作仪的倒置显微 镋下切割胚胎。
切割和处理完毕后 半胚置于培养液中， 用 WS— 101D频谱 治疗仪照射 30分钟， 治疗仪的剂量开关逸择弱档， 培养液温度控 制在 38°C至 40°C之间， 通过调节照射距离控制温度。
实施例 5：
性别鉴定。 从胚胎中采取少量细胞可以准确地进行性別鉴定。 但是容易对胚胎迳成损伤， 影响其活力。 在采取细胞之后用 WS— 101D頻普治疗仪照射胚胎 20分钟， 可以提高胚胎的活力。
实施例 6 :
动物的繁殖发育生长劝能。以小鼠作为实验动物， 将 鼠随机 分成两組， 每組 20只， A組为生物频诸照射组， B组为对照组， 对 照组不作照射处理， Α . Β两組其它实验条件相同。 每组动物分 成 2批， 每批 10只进行实验。 采用周林频镨总公司制達的 WS— 101頻谱治疗仪， 每天定时照射 Α組 1次， 照射时间为 20分钟， ί义 · 剂量开关逸择为强档。鼠背温度不高于 38°C，共照射 10天 /10 次。 其中在照射第 4天， A、B两組每尸、雌鼠注射孕马血促性腺激素 (PMSG10IU ) , 照射第 6天， A、B两組每, σ、雌鼠各注射人绒毛膜促 性腺激素（HCG10IU) , 注射后，每只雌鼠笼内放 1 公鼠交配。 第 7天， Α、Β两组各 10 Ρ、 鼠输印管沖印， 两组比较的结果显示： 用 频诸治疗仪照射对受精印的存活有明显的保护作用。 在第 10天对 Α、Β两组剎余各 10只 鼠输卵管沖卵， 收集囊胚数， Α、Β两組比 较结果显示：用频诸治疗 照射有促进排印和印的受精作用， 并对 囊胚的发育作用明显。 Α、Β两组比较还显示： 频镨治疗仪照射， 明 显使 鼠子宫充血发育。 本实施例说明： 用频语治疗 ί义照射动物， 可促进动物的繁殖发育生长劝能。
实施例 7：
用周林頻谱总公司制達的 WS— 1 01频诸治疗仪照射治疗羔羊 復;'写每天一次. 每次 20分忡， 丫 ^开关逸择强挡， 羔羊腹部被照
— 7 —
更 正 百 ISA/CN 射时的表面温度不得 过 45°C， 共照射 2— 4 天， 可控制腹泻. 荻 得明显的治疗效果。
发明人认为： 除了使用上述实施例外， 根掂本发明的主要构 思，生物频镨将适用于多种生物工程对象。除能使胚胎工程乃至生 物工程中的不少难题得到解决， 还能使胚胎工程和生物工程中的 复杂、高难度工艺变为^单易行。 因此， 在生物工程领域还可以有 其它不同的实施方案， 而这些方案都应属于本发明的保护范围。
- 8 ―
更 正 页 ISA/CN

Claims

权利要求
1. 一种生物频诸在动物胚胎工程中应用的才法， 所述方法包 括：
( 1 ) 卵线细胞收集，
(2) 卵母细胞体外成熟，
(3 )精子获能，
(4) 卵母细胞体外受精，
(5)胚胎体外培养，
其特征在于：
印母细胞体外成熟过程中将卵母细胞放置于普通培养液或特 定培养液中， 用频语发生器照射 3至 20分钟， 照射过程中， 培养 液温度不得高于 40°C ;
情子荻能过程中将精液用普通培养液或特定培养液稀释后用 频镨发生^照射 3至 20分钟， 照射过程中， 培养液温度不得高于 0°C ;
印母细胞体外受精过程中将卵母细胞和体外荻能精子放置于 装有普通培养液或特定培养液的试管中， 用频镨发生! ^照射 1至 3 次，每次照射 3至 25分钟， 照射过程中，培养液温度不得高于 40° C;
胚胎体外培养过程 受椅印母细胞移入普通培养液或物特定 培养液中， 用频谱发生^照射 3到 30分钟， 照射过程中， 培养液 温度不得高于 40°C
2. 如权利要求 1所述生物频镨在动物胚胎工程中应用的方 法， 其特征在于所述特定培养液的成分为在 NaCl 6. 55克 /升、 KC1 0. 30克 /升、 CaCl2 · 2H20 0. 33克 /升、 NaH2PC¾ · H20 0. 11克 /升、 MgCl2 · 6H20 0. 1 1克 /升、 NaHC03 3. 10克 /升、葡萄糖 2. 5克 /升、 无水丙酮駿钠 0. 14克 /升（或丙酮酸 0. 11克 /升）、牛血清白蛋白
3. 00克 /升、 表雷素钠盐 0. 031 (50单位 /亳升）中加入上蒸水到 1000毫升， 然后在 100毫升以上溶液中加入 84毫克 NaHC03。
3. 一种生物頻语在动物胚胎冷冻中应用的方法， 其特征在
- 9 - 更 正 百 ISA/CN 二 ： 冷^前或后 ^动物胚胎置于培养液中 . 发生 照射 3至 35分钟， 可以在解冻前和解 后 ?动 ^胚胎置亍培养液中， 用 ^语发生 照射 3至 35分钟， 照射过程中， 培养液 S度不得高于 -10°CD
4. 一种生物频镨在动物胚胎显徵操作中应用的方法， 其特征 在于显徵操作前或后 动物胚 ½置于培养液中. 用 ^发生 ^照 射 3至 35分钟， 也可以是在显徵橾作前和显微操作后 动物胚胎 置于培养液中， 用频谱发生^照射 3至 35分钟， 照射过程中， 培 养液温度不得高于 40°C。
5. 一种生物频谱在动物繁殖发育生长中应用的方法， 其特征 在于： 用生物頻谱照射动物，每天 1—2次，每次 3— 60分钟， 照射 过程中， 动物表面温度不高于 45°C。
6. 一种生物頻语在动物昉治疾病中应用的方法， 其特征在 于： 用生物頻镨照射动物的局部或者全身，每天 1— 3次，每次 6— 60分钟， 照射过程中， 动物表面温度不高于 45°C。
- 10 - 更 正 百 ISA CN
PCT/CN1995/000087 1994-11-24 1995-11-02 Utilisation du spectre de frequences biologiques dans l'embryogenese animale WO1996015732A1 (fr)

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AU38386/95A AU704690B2 (en) 1994-11-24 1995-11-02 Use of the bio-spectrum in animal embryo-engineering
CA002207013A CA2207013C (en) 1994-11-24 1995-11-02 Application of bio-spectrum in animal embryonic engineering
DE69528295T DE69528295T2 (de) 1994-11-24 1995-11-02 Anwendung des biofrequenzspektrums in der embryotechnologie bei tieren
EP95936416A EP0872219B1 (en) 1994-11-24 1995-11-02 Use of the bio-frequency spectrum in animal embryo-engineering

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CN 94118306 CN1068195C (zh) 1994-11-24 1994-11-24 生物频谱在胚胎工程中的应用
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