CN110684720A - Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium - Google Patents
Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium Download PDFInfo
- Publication number
- CN110684720A CN110684720A CN201910973104.3A CN201910973104A CN110684720A CN 110684720 A CN110684720 A CN 110684720A CN 201910973104 A CN201910973104 A CN 201910973104A CN 110684720 A CN110684720 A CN 110684720A
- Authority
- CN
- China
- Prior art keywords
- animal
- oocytes
- oocyte
- animals
- obtaining
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2517/00—Cells related to new breeds of animals
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Biotechnology (AREA)
- Reproductive Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of embryo engineering, in particular to a method for obtaining a male pronucleus by taking an oocyte among heterogeneous animals as a medium, which comprises the following steps: step 1, obtaining mature oocytes of an animal A with a first polar body and placing the mature oocytes into in-vitro semen receiving microdroplets of an animal B; step 2, injecting the sperms of the animal B into the in-vitro semen receiving microdroplets containing the oocytes in the step 1 to obtain mixed sperms and ova microdroplets; step 3, putting the sperm-egg mixed micro-droplet obtained in the step 2 into CO2Incubating in an incubator until sperm of the animal B enters cytoplasm of the oocyte to form a male pronucleus; wherein the animal A and the animal B are xenogeneic animals. In the method, the fertilization biological principle of the reproductive isolation of non-cross fertilization of the xenogeneic animals is utilized, the in vitro fertilization method is used for obtaining the male pronucleus of another animal by taking the oocyte of the xenogeneic animal as a medium, and the formation rate of the male pronucleus is up to more than 17.8 percent.
Description
Technical Field
The invention relates to the technical field of embryo engineering, in particular to a method for obtaining a male pronucleus by taking oocytes among dissimilar animals as media.
Background art:
in vitro fertilization refers to a technique in which mammalian sperm and eggs are subjected to a fertilization process in an in vitro artificially controlled environment. The technology is successful in the 50 th of the 20 th century, develops rapidly in the last 20 years, matures day by day, becomes an important and conventional animal breeding biotechnology, is not only applied to animal husbandry production, but also becomes an important auxiliary means for researching other embryo biotechnology, such as cloning, transgene, embryonic stem cell separation culture, sex control and the like, can provide sufficient embryo sources for the introduction of exogenous genes through in vitro fertilization, provides a culture system of mature oocytes and cloned embryos for cloning technology, can carry out sex control on mammals by using separated X and Y sperms and ova in vitro fertilization, and also needs the in vitro fertilization technology to provide embryos and the culture system for the separation of embryonic stem cells, so that the comprehensive development of the biotechnology has great influence on human life. Most importantly, for the time being, the problem of genetic and physiological defects in vitro fertilized animals has never been found.
At present, xenogenic nuclear transplantation may become an effective method for protecting highly endangered animals, establishing a therapeutic cloned non-human primate animal model and a nuclear-cytoplasmic interaction relation research animal model, and can avoid the ethical, legal and clinical experimental limitations in human therapeutic cloning research. The research on the xenosomatic cell nuclear transfer not only can support basic researches such as human therapeutic cloning, the nucleoplasm interaction, the nuclear reprogramming, the epigenetics and the like in the early embryo development and the nuclear transfer process of primates, reveal the characteristics and the rules of the nucleoplasm interaction among different animals, but also can enable the research on the xenosomatic cell nuclear transfer of rare or endangered animals to be feasible in practice. However, the success rate of cloning techniques at present is low, thus greatly hindering the development of cloning techniques, and in addition, many cloned animals show genetic and physiological defects, such as over-size, abnormal obesity, developmental difficulties, organ defects, immune disorders, and the like.
Therefore, it is urgent to establish a more effective technique capable of replacing heterogeneous nuclear transplantation.
Disclosure of Invention
The invention aims to provide a method for obtaining a male pronucleus by taking an oocyte among heterogeneous animals as a medium.
The scheme adopted by the invention for realizing one of the purposes is as follows: a method for obtaining male pronuclei by taking oocytes between xenogeneic animals as media comprises the following steps:
step 1, obtaining mature oocytes of an animal A with a first polar body and placing the mature oocytes into in-vitro semen receiving microdroplets of an animal B;
step 2, injecting the sperms of the animal B into the in-vitro semen receiving microdroplets containing the oocytes in the step 1 to obtain mixed sperms and ova microdroplets;
step 3, putting the sperm-egg mixed micro-droplet obtained in the step 2 into CO2Incubating in an incubator until sperm of the animal B enters cytoplasm of the oocyte to form a male pronucleus;
wherein the animal A and the animal B are xenogeneic animals.
Preferably, in step 1, animal a is a kunming mouse; animal B is high-yield cow, macaque or panda.
The animal A is the source animal of the oocyte, generally adopts the animal which has lower cost, strong reproductive capacity, easy acquisition and no influence by external environment, four-season change and the like; animal B is the animal from which sperm is derived, and is a valuable animal which is not easily obtained by adopting oocyte. And the animal A and the animal B are heterogeneous animals, and the species are far from each other.
Preferably, the step 1 further comprises the following operations: the oocytes were placed in the in vitro fertilization fluid microdroplets after zona pellucida removal treatment.
Preferably, the oocyte is subjected to zona pellucida removal treatment using 0.25% pronase.
Preferably, in the step 2, the high-quality sperm of the animal B with stronger vitality obtained by incubation by the floating method is injected into the in-vitro semen receiving microdroplet containing the oocyte in the step 1.
Preferably, in the step 2, the sperm concentration of the animal B is 3X 105one/mL.
Preferably, in step 3, the animal B is incubated under in vitro fertilization conditions and fertilization environment, and when the animal B is selected from high-producing cows, the incubation conditions are 39 ℃ and CO2The volume percentage is 5 percent, the saturation humidity is high, and the time is 6-8 h.
The invention has the following advantages and beneficial effects: in the method, the fertilization biological principle of the reproductive isolation of non-cross fertilization of the xenogeneic animals is utilized, the male pronucleus of another animal is obtained by taking the oocyte among the xenogeneic animals as a medium by using the in vitro fertilization method, and the formation rate of the male pronucleus is up to more than 17.8 percent.
The method adopts the oocytes which have strong fertility and low cost and are easy to obtain to cultivate the male pronucleus of the target animal, and then transplants the male pronucleus into the rare oocytes which are difficult to obtain, so that the in vitro fertilization rate is improved, the utilization rate of the rare oocytes which are difficult to obtain is further improved, and the method can effectively replace the nuclear transplantation method.
Drawings
FIG. 1 is a microscopic image of a high-yielding cow male pronucleus obtained in example 1 of the present invention.
Detailed Description
The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.
In the embodiment of the invention:
the BO liquid comprises the following components in percentage by weight: 6.55mg/mL sodium chloride, 0.3mg/mL potassium chloride, 0.249mg/mL calcium chloride, 0.0497mg/mL magnesium chloride, 0.0869mg/mL sodium dihydrogen phosphate, 3.104mg/mL sodium bicarbonate, 2.5mg/mL glucose, 0.055mg/mL sodium pyruvate, 0.065mg/mL penicillin, 0.05mg/mL streptomycin sulfate;
the BO-A liquid comprises the following components in percentage by weight: BO liquid, 5mM caffeine, 10mg/mL heparin sodium and 3mg/mL BSA;
the culture solution CRlaa formula: 114.7mM NaCl, 3.1mM KCl, 26.2mM NaHCO30.4mM sodium pyruvate, 5.0mM calcium hemilactate, 3mg/mL BSA, 1.0mM L-glutamine;
culture medium M2, HTF, purchased from sigma;
the super excretion hormones PMSG, HCG were purchased from Ningbo second hormone plant.
[ example 1 ]
Obtaining mature oocytes of Kunming mice; the Kunming mice used in the embodiment are purchased from a disease control center in Wuhan city, and after 6-10 weeks old Kunming white female mice are bred for 1-2 weeks in light control (14h of light and 10h of dark), 10 IU/branch PMSG46-48h is injected into the abdominal cavity, and 10 IU/branch hCG is injected into the abdominal cavity for super-excretion. And (3) after superovulation for 20-22h, killing superovulation mice by adopting a cervical dislocation method, dissecting to obtain a ampulla dilatant part of the superovulation mice to obtain superovulation mother cells, and observing under an inverted microscope to obtain mature oocytes with a first polar body.
Obtaining high-quality sperms of high-yield cows: the semen used in the embodiment is simon tare frozen semen of the dairy cattle, and is purchased from a dairy cattle improvement station in Wuhan city. The method adopts the floating hair to obtain high-quality sperms, and comprises the following specific operation methods: taking frozen semen from a liquid nitrogen tank, putting the frozen semen into a water bath at 37 ℃ for 30-40s for fast thawing, disinfecting with 75% alcohol, cutting off the thin tube, putting the semen into a centrifuge tube containing 1mL of in-vitro receiving semen, blowing and beating the semen lightly with a pipette, turning the semen upside down for several times, putting the semen into a carbon dioxide incubator, standing for 20-30min to enable the semen to float, centrifuging the floated semen for 1500r/min for 5min, centrifuging the in-vitro receiving semen for two times again, 1500r/min for 5 min; discarding the supernatant to obtain the required high-quality sperm.
Placing mature oocyte of Kunming mouse with first polar body in vitro semen BO-A of high-yield cow, balancing for 30min, treating semen of high-yield cow with the in vitro semen BO of high-yield cow, adopting BO-A as in vitro fertilization microdroplet, and controlling the final concentration of semen to be 3 × 105Per mL, then in CO2Incubating for 6h in the incubator, and performing sperm-egg incubation under the in vitro fertilization condition by adopting the in vitro fertilization condition of the high-yield cows: 39 ℃ and 5% CO2And saturation humidity.
After the in vitro fertilization is carried out for 6h, the oocyte is lightly blown by a pipettor, the sperms of the high-yield cows floating on the surface of the oocyte are removed, and the formation condition of the male pronuclei is observed under an inverted microscope. FIG. 1 is a microscopic image of a high-yielding cow male pronucleus obtained in example 1 of the present invention.
[ example 2 ]
This example differs from example 1 in that: placing mature oocytes of Kunming mice in a mouse in-vitro fertilization culture solution HTF for balancing for 30min, treating high-yield cow semen by using the mouse in-vitro fertilization semen HTF, adopting HTF as in-vitro fertilization microdroplets, and adopting the mouse in-vitro fertilization conditions for sperm-egg incubation: 37 ℃ and 5% CO2And saturation humidity.
[ example 3 ]
The Kunming mouse oocyte with the high-yield cow male pronuclei obtained in the example 1 is washed three times by the cow embryo culture solution CRlaa and then placed in the cow embryo culture solution CRlaa, and the culture is carried out by adopting the cow embryo culture conditions: 39 ℃ and 5% CO2And saturation humidity.
[ example 4 ]
This example differs from example 4 in that: the mouse embryo culture solution M for Kunming mouse oocyte with high-yield cow male pronuclei obtained in example 12Washing three times, and placing in mouse embryo culture solution M2In the method, the culture is carried out by adopting the culture conditions of mouse embryos: 37 ℃ and 5% CO2And saturation humidity.
[ example 5 ]
This example differs from example 1 in that: the method comprises the following steps of carrying out zona pellucida removal treatment on the Kunming mouse oocyte by using 0.25% pronase, and then carrying out subsequent operation, and comparing the influence of the zona pellucida on the formation of male pronuclei.
And (4) counting results:
table A record of the formation of the male pronuclei of example 1 and example 2
| Examples | Number of cells processed | Number of formation of Primary nucleus (%) |
| Example 1 | 197 | 23(11.7) |
| Example 2 | 145 | 3(2) |
From the table one data, it can be seen that: adopting the in-vitro fertilization conditions of the high-yield cows: the method comprises a semen receiving environment and a fertilization environment, and is more beneficial to the generation of male pronuclei of high-yield dairy cows, namely the condition of obtaining the male pronuclei by taking the oocyte of a xenogeneic animal as a medium is the in vitro fertilization condition of an animal from sperms.
TABLE two example 4 and example 5 in vitro fertilization embryo development
| Examples | Number of cells processed | Number of cleavage (%) | Number of blastocysts (%) |
| Example 4 | 32 | 8(25) | 0 |
| Example 5 | 28 | 6(21.4) | 0 |
As can be seen from the data in Table two: two animals with species far from each other are fertilized in vitro, male pronuclei of the target animal can be obtained, but a developing embryo can not be obtained, and the ethical problem of animal hybridization does not exist.
TABLE III record of the formation of male pronuclei by zona pellucida of examples 1 and 5
| Examples | Number of cells processed | Number of formation of Primary nucleus (%) |
| Example 1 (with transparent band) | 197 | 23(11.7) |
| Example 6 (without transparent band) | 213 | 38(17.8) |
The data in table three show that: after the zona pellucida of the animal oocyte is removed, the male pronucleus of the target animal can be obtained more conveniently.
While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (7)
1. A method for obtaining male pronuclei by taking oocytes between xenogeneic animals as media is characterized by comprising the following steps:
step 1, obtaining mature oocytes of an animal A with a first polar body and placing the mature oocytes into in-vitro semen receiving microdroplets of an animal B;
step 2, injecting the sperms of the animal B into the in-vitro semen receiving microdroplets containing the oocytes in the step 1 to obtain mixed sperms and ova microdroplets;
step 3, putting the sperm-egg mixed micro-droplet obtained in the step 2 into CO2Incubating in an incubator until sperm of the animal B enters cytoplasm of the oocyte to form a male pronucleus;
wherein the animal A and the animal B are xenogeneic animals.
2. The method of claim 1 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: in the step 1, the animal A is Kunming mouse; animal B is high-yield cow, macaque or panda.
3. The method of claim 1 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: in the step 1, the following operations are further included: the oocytes were placed in the in vitro fertilization fluid microdroplets after zona pellucida removal treatment.
4. The method of claim 3 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: and (3) carrying out zona pellucida removal treatment on the oocyte by adopting 0.25% pronase.
5. The method of claim 1 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: in the step 2, a high-quality sperm of an animal B with stronger activity is obtained by incubation through a floating method and is injected into the in-vitro semen receiving microdroplet containing the oocyte in the step 1.
6. The method of claim 1 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: in the step 2, the sperm concentration of the animal B is 3 multiplied by 105one/mL.
7. The method of claim 1 for obtaining male pronuclei using oocytes from between xenogeneic animals as mediators, wherein: in step 3, in vitro fertilization conditions and fertilization environments of animal B are adopted for incubation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910973104.3A CN110684720B (en) | 2019-10-14 | 2019-10-14 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910973104.3A CN110684720B (en) | 2019-10-14 | 2019-10-14 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110684720A true CN110684720A (en) | 2020-01-14 |
| CN110684720B CN110684720B (en) | 2021-07-20 |
Family
ID=69112440
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910973104.3A Active CN110684720B (en) | 2019-10-14 | 2019-10-14 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110684720B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000012742A1 (en) * | 1998-08-28 | 2000-03-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for remodelling an animal genome by zygotic transfer of a site-specific recombinase |
| CN107365738A (en) * | 2017-08-07 | 2017-11-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of milk cow and the preparation method of ox xenogenesis IVF Embryos |
-
2019
- 2019-10-14 CN CN201910973104.3A patent/CN110684720B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000012742A1 (en) * | 1998-08-28 | 2000-03-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for remodelling an animal genome by zygotic transfer of a site-specific recombinase |
| CN107365738A (en) * | 2017-08-07 | 2017-11-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of milk cow and the preparation method of ox xenogenesis IVF Embryos |
Non-Patent Citations (2)
| Title |
|---|
| SESSIONS-BRESNAHAN, D. R. ET AL.: "Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections", 《THERIOGENOLOGY》 * |
| 全守能: "水牛—猪异种显微受精的初步研究", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
| CN114107180B (en) * | 2021-10-21 | 2024-03-08 | 湖北省农业科学院畜牧兽医研究所 | Zona pellucida-free somatic clone 1-cell stage embryo polymerization and in vitro culture method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110684720B (en) | 2021-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Paramio et al. | Recent advances in in vitro embryo production in small ruminants | |
| Amoah et al. | Biotechnological advances in goat reproduction | |
| CN106520838A (en) | New method for gene injection for somatic cell nuclear transfer reconstructed embryo | |
| CN107365738B (en) | Method for preparing cow and cattle xenogenesis in-vitro fertilization embryo | |
| CN100334205C (en) | Method for producing sex controllable in vitro embryo of buffalo | |
| Chao et al. | Epigenetic reprogramming of embryos derived from sperm frozen at− 20° C | |
| CN110684720B (en) | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium | |
| Saha et al. | Effect of fresh and frozen semen on in vitro fertilization and subsequent development of goat embryos. | |
| CN101492669A (en) | Method for cloning embryo with cattle spermatozoon | |
| BG61041B1 (en) | Method for producing non human transgenetic animals by the introduction of exogenous dna in somatic embryonic animal cells | |
| Blakewood et al. | Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian embryos | |
| RU2410063C1 (en) | Method for extracorporeal cultivating bovine oocytes | |
| Wang et al. | Actin filament distribution in blocked and developing pig embryos | |
| Tao et al. | Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda (Ailurus fulgens) | |
| Goel et al. | Status and Prospects of Reproductive Biotechnologies of Small Ruminants in India: An Overview | |
| KR102552211B1 (en) | Method for producing in vitro rat 0vary | |
| CN100526456C (en) | Method for transplanting consubstantial cell nucleus | |
| RU2823596C1 (en) | Method for producing in vitro cattle embryos | |
| Kumar et al. | Advancement in Reproductive Biotechnologies in Livestock | |
| KR101934969B1 (en) | Composition for low or room temperature preservating of bovine embryos | |
| CZ297528B6 (en) | Use of MAP kinase JNK inhibitor for preparing culture medium for prolongation of life of mammalian oocytes in vitro | |
| Bavister et al. | In vitro fertilization and embryo transfer technology as an aid to the conservation of endangered primates | |
| Galli et al. | 40 years of AETE: the contribution of scientists and practitioners to the progress of reproductive biotechnologies in Europe | |
| Bekseitov et al. | Quality of biological material in embryo transplantation | |
| Babenkov et al. | Microsurgical separation of deconserved embryos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230202 Address after: 430070 Room 16, 28/F, Unit A, Commercial Building, No. 5, West District, Wuchangfu Phase II, No. 32, Wenzhi Street, Beigang Village, Hongshan District, Wuhan City, Hubei Province Patentee after: Wuhan newlite Biotechnology Co.,Ltd. Address before: 430064, No. 1, Nanhu Yao yuan, Hongshan District, Hubei, Wuhan Patentee before: INSTITUTE OF ANIMAL SCIENCE AND VETERINARY, HUBEI ACADEMY OF AGRICULTURAL SCIENCES |