EP1161144A1 - Cryopreservation of oocytes and embryos and methods for producing animals involving the same - Google Patents
Cryopreservation of oocytes and embryos and methods for producing animals involving the sameInfo
- Publication number
- EP1161144A1 EP1161144A1 EP99957762A EP99957762A EP1161144A1 EP 1161144 A1 EP1161144 A1 EP 1161144A1 EP 99957762 A EP99957762 A EP 99957762A EP 99957762 A EP99957762 A EP 99957762A EP 1161144 A1 EP1161144 A1 EP 1161144A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- embryos
- oocytes
- lipid
- embryo
- freezing
- 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.)
- Withdrawn
Links
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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Definitions
- the present invention relates to methods for the cryopreservation of oocytes and embryos, use of oocytes and embryos, and methods for producing live animals from such embryos.
- cryopreservation of animal oocytes and embryos remains largely illusionary.
- cryopreservation techniques used for cryopreservation of embryos from other species are generally not successful.
- lipid was forced from the embryonic cells, resulting in a layer of lipid between the blastomeres and the zona pellucida. This lipid was moved by aspirating a lipid from the embryo using micro manipulation techniques. Removing lipids from embryos requires considerable technical skill, as well as much complexity.
- the present invention seeks to overcome the problems associated with cryopreservation of oocytes and embryos, and seeks to provide simple, convenient and easily performed methods for the cryopreservation of oocytes and embryos, such as zona intact porcine embryos, and for producing live animals therefrom.
- a method for the cryopreservation of oocytes or embryos which comprises centrifugation of oocytes or embryos to polarise cytoplasmic lipid outside the oocyte or embryonic cells, subjecting the oocytes or embryos to low temperature conditions in the presence of a cryoprotectant which results in freezing of the oocytes or embryos prior to lipid depolarisation, followed by low temperature storage of the frozen lipid polarised oocytes or embryos.
- the oocytes or embryos are vitrified by freezing in liquid nitrogen or other very cold fluid or gas which allows rapid temperature reduction.
- a method for producing animals from embryos which comprises thawing a cryopreserved lipid polarised embryo and thereafter transferring the embryo to a synchronised recipient female, and allowing the embryo to develop to term to give rise to live animals.
- the present invention provides for the cryopreservation of animal embryos, for example zona intact porcine embryos, which hitherto have not been amenable to cryopreservation, and more particularly the successful production of animals from the cryopreseved embryos.
- Cryopreserved oocytes can, on thawing, be fertilised, or genetically manipulated and fertilised, and then transferred into a pregnancy competent female to give live animals.
- the inventors have surprisingly found that centrifugation of oocytes and embryos, such as zona intact porcine embryos, which polarises cytoplasmic lipid outside the oocyte and embryonic cells, followed by exposure to low temperature conditions, preferably vitrification, in the presence of cryoprotectant, enables successful cryopreservation of the polarised oocytes and embryos which maintains their viability such that on implantation into the uterus of a pregnancy competent female animals, progeny animals can develop.
- oocytes and embryos such as zona intact porcine embryos, which polarises cytoplasmic lipid outside the oocyte and embryonic cells
- a method for the cryopreservation of animal oocytes embryonic cells comprising centrifugation of oocytes and embryos to polarise cytoplasmic lipid outside the oocyte or embryonic cells, subjecting the oocytes or embryos to low temperature conditions in the presence of a cryoprotectant which results in freezing of the embryo prior to lipid depolarisation, followed by low temperature storage of the frozen lipid polarised oocyte or embryo cells.
- Embryos which may be subject to the methods of the present invention include zona intact embryos (blastomeres) from the oocyte stage, through to late blastocysts, including morulae to mid-blastocysts stage, and hatched (non-zona intact) blastocysts.
- Oocytes and embryos may be from any animal, that is any mammal, including companion animals (for example dogs and cats), domestic/livestock animals (for example horses, cows, sheep, goats, pigs, llamas, and alpacas), laboratory animals (for example mice, rats, and monkeys), and humans.
- companion animals for example dogs and cats
- domestic/livestock animals for example horses, cows, sheep, goats, pigs, llamas, and alpacas
- laboratory animals for example mice, rats, and monkeys
- the invention relates to pigs, that is pig oocytes and embryos.
- Oocytes and embryos may be recovered from donor animals by surgical or non-surgical methods.
- Non surgical methods can be used to recover oocytes and embryos from live cattle, but surgical methods are used for recovery from some other live animals, including pigs.
- embryos may be surgically recovered from pigs within one to six days following mating.
- oocytes and embryos may be flushed from reproductive tracts of slaughtered female animals.
- a second alternative is to obtain immature oocytes from the ovaries of slaughtered animals, and mature and fertilise them in vitro.
- the embryos obtained by any of these procedures may be briefly cultured in a medium standardly used for oocyte and/or embryo culture to an appropriate stage. Whilst in no way essential, it is generally desirable to briefly culture embryos prior to the methods of this invention.
- the oocytes and embryos are cultured in a cryoprotectant-containing solution prior to vitrification.
- the oocyte and embryos need only be incubated in the cryoprotectant solution for a short period of time, for example from two minutes to one hour, more preferably from two minutes to 30 minutes, still more preferably from 3 minutes to 20 minutes.
- Oocytes and embryos may be incubated in a cryoprotectant-containing solution either prior to, during or after centrifugation, or both.
- the cryoprotectant-containing solution in which oocytes and embryos are incubated either prior to, during centrifugation, or after centrifugation may contain any standard cryoprotectant established for use in the freezing of animal oocytes and embryos, including glycerol, ethylene glycol, dimethylsulfoxide, propylene glycol and poly vinyl pyrrolidine, sucrose, trehalose, Ficoll, acetamide, egg yolk and the like.
- the concentration of cryoprotectant is used in an amount sufficient to replace to at least some extent water within the oocyte or embryo, such that on rapid freezing ice crystal formation is prevented.
- cryopreservatives may be present in an amount from 0.5M to 8M.
- One or more cryoprotectants may be used.
- the time in which oocytes and embryos may be incubated in a cryoprotectant solution following centrifugation is insufficient to allow lipid repolarisation into the tissues of the oocyte or embryo.
- Oocytes and embryos are centrifuged for a time sufficient to polarise cytoplasmic lipid from the oocytes and embryonic cells to the outside of the cells, for example 1 to 15 minutes at 10,000 to 20,000g.
- the time period of centrifugation will depend upon the centrifugal force applied during centrifugation. At a centrifugal force of about 13000g polarisation takes place after about 8 minutes of centrifugation. It may be more convenient to centrifuge the oocytes and embryos in the presence of embryo culture medium, rather than in the presence of more viscous cryoprotective-containing solutions.
- Culture medium and cryoprotectant-containing solution for culture either before or after centrifugation may contain inhibitors of actin polymerisation such as Cytochalasin B which relaxes cytoskeletal elements.
- lipid polarised oocytes and embryos are subject to low temperature conditions in the presence of a cryoprotectant which results in freezing of the oocyte or embryo prior to lipid depolarisation.
- lipid depolarisation is meant the return of lipid to the cells which lipid was polarised outside the cells by centrifugation. It is to be noted that on polarisation cytoplasmic lipid may be attached to cells but displaced outside the cells.
- Low temperature conditions may be provided by slow cooling, rapid freezing and vitrification.
- the oocyte or embryo is frozen before the lipid returns to the cells. Vitrification may take place by placing the oocytes or embryos in a vessel, and plunging the vessel into an extremely cold environment, such as liquid nitrogen or other liquefied and/or gaseous extremely cold substance.
- a vessel containing oocytes or embryos may be rapidly frozen in an ultra-cold freezer, for example at temperatures below about -30°C. Any other apparatus or methods that enable rapid freezing may be used.
- oocytes or embryos may be loaded into a straw which is heat sealed, and then plunged into liquid nitrogen.
- embryos may be pulled by capillary action into a open pulled straw, which is then plunged into liquid nitrogen and subsequently stored (Vajta et al (1997) Cryoletters 18 191-5).
- Oocytes and embryos may be stored in a conventional freezer facility, at temperatures, for example, from -10°C to -70°C or more.
- Frozen lipid polarised oocytes or embryos may be thawed according to conventional oocyte and embryo thawing techniques, such as incubation of a frozen straw at a temperature of 35°C to 39°C in a suitable culture medium. Thawed embryos may be washed in culture medium, further cultured briefly, and then transferred to a synchronised recipient female, such as to the uterus of a pregnancy competent female animal. At the conclusion of pregnancy term, that is embryo development to term, the introduced embryos have developed to live animals.
- the present invention provides a simple and straight forward procedure for cryopreservation of oocytes and embryos, particularly zona intact frozen embryos. On thawing and implantation of the embryos into the uterus of a pregnancy competent animal, animals may be produced in a manner which has not been achievable by the prior art.
- an animal when produced from an oocyte (subsequently fertilised) or embryo which has been cryopreserved in accordance with the invention hereinbefore described.
- Oocytes or embryos may be subject to genetic manipulation prior to the process of this invention.
- one or more genes of interest may be inserted into an oocyte or embryo by established techniques, such as using pronuclear microinjection, homologous recombination using embryonic stem cell technologies and other established techniques for introducing genes into oocytes and embryos (Nottle et al (1997), Reprod Fertil Suppl 52, 237-244.
- This invention allows banks or libraries of embryos or oocytes to be prepared. These banks or libraries may be provided in frozen form and presented in a convenient manner. Examples include a straw or tube, and a plurality of straws or tubes with multiple oocytes or embryos.
- the bank or library may contain oocytes or embryos from different animals and may find use in artificial insemination and breeding programs.
- Example 1 Porcine embryos were collected and washed thoroughly in culture medium (mPBl - Quinn et al (1982) J. Reprod. Fert. 66:161-168) of 39°C three times. The embryos, in the early blastocyst stage were cultured for 40 minutes in the standard embryo culture medium NCSU-23 (Peters & Reed (1991), Theriogenology 35, 253) with 10%) foetal bovine serum (FBS) containing 7.5 ⁇ g/ml Cytochalasin B at 39°C in a humidified environment of 5%> CO and air.
- NCSU-23 the standard embryo culture medium
- FBS foetal bovine serum
- the embryos were cultured for 5 minutes in 6%> BSA in BECM-3h, and then washed in 25% VS3a for approximately three minutes (VS3a containing 6.5 m glycerol, 6% bovine serum albumin in BECM-3h (Dobrinsky et al (1996) Biol. Reprod. 55: 1069-1074).
- the embryos were centrifuged in a 1.5 Eppendorf tube (in the same media) at 13000g for about 12 minutes, recovered back into 25%> VS3a, and then left in that media for a further five minutes.
- the embryos were then washed for 30 seconds in 65%> VS3a, followed by a wash in 1ml VS3a before being loaded into a straw, heat sealing the straw, and plunging the embryos in the straw into liquid nitrogen.
- the embryos following a wash in the vitrification solution are placed into a small drop of vitrification solution and drawn by capillary action into a narrowed hand pulled 0.25ml freezing straw (unsealed pulled straw, UPS). The straw is then plunged directly into liquid nitrogen.
- UPS unsealed pulled straw
- Porcine Embryos frozen at various stages, thawed and then cultured for 48h.
- the morphological stages examined were Mor, morulae, MBI, early to mid blastocysts; Peri, peri-hatching blastocysts.
- the treatment used were BEVS, Beltsville embryo vitrification system (Dobrinsky et al (1997) Theriogenology 47:, 343); Sp, centrifuged, UPS, unsealed pulled straw.
- Embryos were collected, washed thoroughly in mPBl and then cultured for 35 minutes in NCSU-23 + 10% FBS with 7.5 ⁇ g/ml Cytochaiasin B at 39°C in an atmosphere of 5% CO 2 in air and 100% humidity. Morulae to middle blastocyst stages were centrifuged at 13000g for the last 10 minutes of this incubation in the culture medium containing the Cytochaiasin B. The embryos are then transferred to 2M ethylene glycol in mPBl at 25°C for five minutes before being washed thoroughly in 8M ethylene glycol and 7% PVP in PBS and then placed into a small droplet of the vitrification media and loaded into an unsealed pulled glass by capillary action. The straw is then plunged into liquid nitrogen and stored. 5
- Thawing is by placing the end of the straw containing the embryos into 1.2mls of 1M sucrose in PBS at 39°C. By blocking the open end with a finger, the fluid containing the embryos is forced out once, thawed, by the warming of the straw. Once collected, the embryos are placed into 1M ethylene glycol in mPBI for two minutes followed by 0.5M 10 ethylene glycol in mPBI for a further 2 minutes, both at 25°C. Five minutes in mPBl at 39°C completes the rehydration procedure. The embryos can then be prepared for culture or transfer.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dentistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPP7299A AUPP729998A0 (en) | 1998-11-24 | 1998-11-24 | Cryopreservation of porcine embryos and methods for producing piglets involving the same |
AUPP729998 | 1998-11-24 | ||
PCT/AU1999/001048 WO2000030441A1 (en) | 1998-11-24 | 1999-11-24 | Cryopreservation of oocytes and embryos and methods for producing animals involving the same |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1161144A1 true EP1161144A1 (en) | 2001-12-12 |
EP1161144A4 EP1161144A4 (en) | 2002-04-17 |
Family
ID=3811507
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99957762A Withdrawn EP1161144A4 (en) | 1998-11-24 | 1999-11-24 | Cryopreservation of oocytes and embryos and methods for producing animals involving the same |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1161144A4 (en) |
AU (1) | AUPP729998A0 (en) |
WO (1) | WO2000030441A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPR386101A0 (en) * | 2001-03-21 | 2001-04-12 | Monash University | Culture system for embryos |
KR100427850B1 (en) * | 2001-05-04 | 2004-04-28 | 충남대학교산학협력단 | Freezing method of semen or embryo derived from small-species dogs |
WO2007028396A2 (en) * | 2005-09-08 | 2007-03-15 | Aarhus Universitet | Cell nuclear transfer |
WO2013096659A1 (en) * | 2011-12-20 | 2013-06-27 | Cook General Biotechnology Llc | Methods and compositions for storage of animal cells |
CN104206376A (en) * | 2014-09-17 | 2014-12-17 | 南宁培元基因科技有限公司 | Lowline embryo freezing liquid, freezing method and unfreezing method |
CN114214269A (en) * | 2021-12-17 | 2022-03-22 | 浙江大学 | Porcine embryo culture solution and application |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995005075A1 (en) * | 1993-08-13 | 1995-02-23 | Bresatec Limited | Cryopreservation of porcine embryos |
-
1998
- 1998-11-24 AU AUPP7299A patent/AUPP729998A0/en not_active Abandoned
-
1999
- 1999-11-24 EP EP99957762A patent/EP1161144A4/en not_active Withdrawn
- 1999-11-24 WO PCT/AU1999/001048 patent/WO2000030441A1/en not_active Application Discontinuation
Non-Patent Citations (4)
Title |
---|
NAGASHIMA H ET AL: "CRYOPRESERVATION OF PORCINE EMBRYOS" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 374, 30 March 1995 (1995-03-30), page 416 XP002947390 ISSN: 0028-0836 * |
NAGASHIMA H ET AL: "NUCLEAR TRANSFER OF PROCINE EMBRYOS USING CRYOPRESERVED DELIPATED BLASTOMERES AS DONOR NUCLEI" MOLECULAR REPRODUCTION AND DEVELOPMENT, LISSS, NEW YORK, NY, US, vol. 48, no. 3, November 1997 (1997-11), pages 339-343, XP000884362 ISSN: 1040-452X * |
NAGASHIMA H ET AL: "Vitrification of porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid droplets." THERIOGENOLOGY, vol. 45, no. 1, 1996, page 180 XP008000289 Annual Conference of the International Embryo Transfer Society;Salt Lake City, Utah, USA; January 7-10, 1996 ISSN: 0093-691X * |
See also references of WO0030441A1 * |
Also Published As
Publication number | Publication date |
---|---|
AUPP729998A0 (en) | 1998-12-17 |
WO2000030441A1 (en) | 2000-06-02 |
EP1161144A4 (en) | 2002-04-17 |
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