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WO1995005075A1 - Conservation cryogenique d'embryons porcins - Google Patents

Conservation cryogenique d'embryons porcins Download PDF

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Publication number
WO1995005075A1
WO1995005075A1 PCT/AU1994/000474 AU9400474W WO9505075A1 WO 1995005075 A1 WO1995005075 A1 WO 1995005075A1 AU 9400474 W AU9400474 W AU 9400474W WO 9505075 A1 WO9505075 A1 WO 9505075A1
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WIPO (PCT)
Prior art keywords
embryo
embryos
lipid
temperature
droplets
Prior art date
Application number
PCT/AU1994/000474
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English (en)
Inventor
Hiroshi Nagashima
Mark Brenton Nottle
Naomi Kashiwazaki
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Bresatec Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Bresatec Limited filed Critical Bresatec Limited
Priority to AU74532/94A priority Critical patent/AU7453294A/en
Publication of WO1995005075A1 publication Critical patent/WO1995005075A1/fr

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Classifications

    • 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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • 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
    • A01N1/10—Preservation of living parts

Definitions

  • This invention relates to the cryopreservation of porcine embryos.
  • cryoprotectants have been used in conjunction with embryos for several species to minimize the impact of ice crystal formation.
  • Two general types of cryoprotectants have been used, permeating substances and non permeating substances.
  • Permeating cryoprotectants enter cells and include substances, such a DMSO, or glycerol and act to effectively lower the freezing point These types of cryoprotectants are used to replace intracellular water reducing the likelihood of lethal intracellular ice crystal formation.
  • Non-permeating cryoprotectants do not enter the cell. The presence of cryoprotectant outside of a cell can cause the cells to dehydrate, removing intracellular water preventing the formation of large ice crystals within the cell, which would otherwise be lethal to cells.
  • membranes that form part of the cellular structure; either as the external membrane that envelope cells to ensure integrity of the cells or internal membrane structures such as those forming the endoplasmic reticulum.
  • These membranes comprise a bilayer of phospholipid molecules with other structural components, including protein components embedded within the bilayer.
  • the phospholipids usually exist in a fluid state, the fluid state being essential for the proper functioning of the membrane.
  • Porcine embryos develop from a single cell (fertilised egg), which divides during the early stages of cell development into a ball of cells without ever increasing its diameter. From the single cell to the blastocyst stage the embryo is covered by a protective layer known as a zona pellucida. The ball of cells in the early stages (from about the 16 cell stage) is referred to as a morula.
  • the protective layer is thought to protect the embryo against a variety of agents, including physical damage, and infectious agents, such as viruses, from penetrating into the cell mass of the embryo.
  • the porcine embryo develops from the morula to the blastocyst stage on day 4 to 5.
  • the blastocyst consists of a single peripheral layer of cells, the trophectoderm or trophoblast, and a group of cells inside the blastocoel cavity termed the inner cell mass cells from which the foetus develops.
  • the blastocyst begins to increase its mass, and this stage is known as an expanded blastocyst
  • the expanded blastocyst is still enveloped by the zona pellucida.
  • the zona pellucida thins as a result of enzymic digestion at about day 6 to 7 and the blastocyst "hatches" out from the zona pellucida.
  • Once fully hatched the blastocyst can become implanted and develop further, and at this stage is referred to as a hatched blastocyst.
  • the hatching and fully hatched blastocyst is far more susceptible to physical damage and to infection.
  • the term peri- hatching or peri-hatched has been coined to refer to embryos that are at any stage of development between and including expanded to fully hatched blastocysts.
  • Hayashi et al. (Vet Rec, 125; 43 (1989)) succeeded in obtaining 5 piglets from 11 expanded blastocysts which had been briefly frozen at -35°C with 1.5 M glycerol.
  • a live piglet was also produced by transferring 8 Meishan hatched blastocysts (day-7) frozen with 1.4M (10%) glycerol in PBS (Phosphate Buffered Saline) supplemented with 50% FCS and 10% egg yolk by slow cooling and seeding at -7°C with silver iodide (Fujino et al, Cryobiology 30; 299 (1993)). These embryos were transferred to a day-5 recipient with 4 unfrozen Large White hatched blastocysts to assist in the maintenance of pregnancy.
  • PBS Phosphate Buffered Saline
  • Blastocysts at peri-hatching stages must be chosen. They can be developed from earlier stages using in vitro culture with conventional embryo culture media such as Whittingham's M-16 (J. Reprod. Fert 14(suppl.)7 (1971)). Whitten's (Adv. Biosci. 6; 129 (1971)) or KRB (Davis and Day J. Anim. Sci. 46; 1043 (1978)). Addition of BSA to these media appears to be crucial to confer freezability (Nagashima et al Theriogenology 37; 839 ( 1992)).
  • step-wise dilution of the cryoprotectant using step-down sucrose from 0.3 - 0.5M to 0 M appears to protect the embryonic cells from osmotic shock.
  • porcine embryos are more susceptible to damage, following freezing and thawing, than other embryos.
  • Early stage porcine embryos have a large number of lipid droplets present in the cytoplasm of the cells. These lipid droplets can be stained by Sudan III (a lipid specific stain) and by Nile blue sulphate (a stain specific for neutral lipids). As the embryo develops towards the perihatching stage the size of the lipid droplets decreases, as does the total lipid content It is thought that the lipid droplets act as an energy store for use by the developing embryo.
  • the postulated effect of the lipid droplet on the freezability of embryos has been suggested to be either due to an effect that lipid droplets may have on the membrane of the embryo, or alternatively by some physical means, perhaps in preventing the cryoprotectant from penetrating into the embryo, or acting as nuclei for ice crystal formation. Which, if any, of these explanations is correct is however not clear, and before the present invention it was not clear that level of lipid was responsible for the poor survival of embryos after freezing.
  • the temperature below which porcine embryos at a stage earlier than peri-hatching do not survive is 15°C. This is also approximately the temperature at which lipid phase separations occur in mammalian cells. Lipid phase separations occur in plasma membranes, internal membranes and also the lipid droplets. Structural changes in lipid droplets also occur around this temperature - they coalesce and form larger droplets. Both changes may cause irreversible damage to the embryo at this stage. It has been further suggested that the high lipid content leads to uneven intracellular ice formation which may also be lethal (Niemann et al Theriogenology 1991 35; 109). SUMMARY OF THE INVENTION
  • the survival rate of porcine embryos to cooling and/or freezing can be greatly enhanced by reducing the level of lipid derived from lipid droplets in cells of the embryo.
  • the invention therefore could in a broad form be said to reside in a method of cryopreserving a porcine embryo comprising the step of reducing the level of lipid derived from lipid droplets in cells of the embryo, followed by the step of reducing the temperature of the embryo to a storage temperature suitable for storage of the embryo whilst the reduced level of lipid droplets is maintained, the level to which lipid is reduced such that significant damage is not sustained by said embryo when the step of reducing the temperature is performed.
  • lipid level may be a parameter that might be varied to best suit the technical means of reducing d e lipid level within cells of the embryo. It would seem however that removal of substantially all of the visible lipid as is possible by any appropriate technical means is desirable.
  • the invention could be said to reside in a method of cryopreservation of a porcine embryo, comprising the steps of removing substantially all visible lipid derived from droplets in cells of the embryo and the step of reducing the temperature of the embryo to a temperature suitable for the storage of the embryo.
  • the present invention has particular benefit in that it permits cryopreservation of porcine embryos at a stage of development earlier than the perihatching stage. It is to be understood that the temperature at which embryos are to be stored is most desirably that of liquid nitrogen. Alternatively where liquid nitrogen facilities are not available then the temperature attainable by any freezer that might be suitable for the purpose.
  • the lipid derived from lipid droplets might conveniently be removed from cells by centrifuging the embryo so as to polarize the lipid in the perivitelline space, between the cells of the embryo and the zona pellucida.
  • the lipid may be physically removed from the embryo. This physical removal can be achieved by piercing the zona pellucida of die embryo with a micropipette, contacting the lipid with the micropipette and applying suction to the lipid to remove lipid d rough the micropipette.
  • the micromanipulation is done with the assistance of a micromanipulator and a microscope.
  • the embryo is preferably treated with a substance such as Cytochalasin C or Demecolcine (which are termed cytoskeletal inhibitors) which relax the cytoskeletal elements to thereby minimise damage caused by piercing the cells.
  • a substance such as Cytochalasin C or Demecolcine (which are termed cytoskeletal inhibitors) which relax the cytoskeletal elements to thereby minimise damage caused by piercing the cells.
  • the lipid droplets may be removed at the single cells stage of the embryo. This facilitates the removal of the lipid from the cytoplasm. It is found mat cells still survive the physical removal of lipid droplets and can be frozen successfully immediately after such treatment
  • the cells of the embryo may also be cultured after removal of lipid before temperature is reduced to the storage temperature. Thus cells may be cultured for 14 to 18 hours before being cooled. Where lipid has been removed from single cells, such embryos are usually at the 2 or 4 cell stage. This extra treaunent is suggested to further enhance the survival rate of embryos. The reasons for this enhanced survival may be that the embryo has had time to recover from the trauma of lipid removal. Additionally further lipid removal may be achieved, because the lipid remaining after removal is further depleted because of the energy demand of the developing embryo.
  • microsurgical techniques may and can be destructive to embryonic cells. Such techniques can also damage the zona pellucida, a membranous structure protecting embryos from infection by pathogens. Accordingly viability of frozen embryos so treated is likely to be lower than might be desirable.
  • the lipid droplets are polarised away from the cells of the embryo and the embryo is frozen rapidly so that the lipid droplets are not able to redistribute back into the cells to a significant extent before freezing.
  • a convenient method for polarising the lipid within the embryo is by centrifugation at a suitable speed, so that the lipid droplets are polarised to the peri-vitelline space, and the temperature is reduced at a rate sufficient to pre-empt the redistribution of lipid back into the cytoplasm of the cells.
  • this method of cryopreservation may be part of a broader embryo transfer or breeding program and that the invention also resides in an embryo that has been cryopreserved by any of the methods outlined above. It is to be understood that the invention also encompasses animals produced from embryos that have been so cryopreserved.
  • An alternative method for depleting lipids from the embryos may be to collect embryos from sows at or close to ovulation, and culture such embryos. This approach is suggested as a possible means to increase the rate of lipid depletion, because culture of embryos is less efficient than development within a sow, and consequently more energy depleting. It may be possible to deplete the level of lipid droplets within an embryo by in vitro culture techniques without polarisation.
  • cryoprotectants Any one of a number of cryoprotectants may be used and the regime and rate at which the temperature is reduced can also be optimised.
  • the invention could be said to reside in an embryo which is cryopreserved using the method of cryopreservation of this invention.
  • the invention could be said to reside in a method of implantation of an embryo, comprising the step of cryopreserving a porcine embryo as described or defined in this specification, thawing the embryo and implanting the embryo into a recipient.
  • the invention is also to be understood to include the method of producing an animal using cryopreserved embryos of this invention, which embryos are then thawed and implanted into recipients and allowed to gestate to give rise to said animal.
  • the invention is also to be understood to encompass an animal produced by such method.
  • delipation is used, wherein the term delipidization has been used elsewhere the two terms are to be understood to have the same meaning.
  • delipation is to be understood not to mean total removal of lipid droplets within the cytoplasm of the embryos but rather to mean a reduction of the lipid, where the majority of lipid droplets are no longer apparent within the cytoplasm of embyronic cells.
  • Porcine 1 cell stage embryos treated with 7.5 ⁇ g/ ⁇ ml cytochalashin B were centrifuged to polarize the lipid droplets in the cytoplasm. The resultant lipid layer was then removed by micromanipulation using a bevelled micropipette. Sham operated embryos were treated with cytochalasin B and centrifuged but not delipated. The delipated embryos together with the sham operated and intact controls were then exposed to 4°C for 1 hr in PBS+10%FCS either within 2 h or after a 14 to 18h period of culture in Whitten's medium + 1.5% BSA. Both groups of embryos and the controls were subjected to in vitro culture to compare their survival. Embryos wre deemed to have survived if they cleaved following thawing
  • Control 11 0 cooled at 2-4 cell (0%) As shown in Table 1, most of the delipated embryos cleaved after cooling with development of the morula-blastocyst stage, whereas all of the control embryos lysed within 24 hours. The sham operated embryos appeared more tolerant to cooling but none cleaved more than once. In vitro culture for 14 to 18 hours of delipated embryos enhances survival.
  • a total of 45 pregnant crossbred gilts (Large White x Landrace) were aborted by intramuscular (i.m.) injection of 1 mg of the prostaglandin F2 ⁇ analogue cloprostenol (Estrumate; Pitman-Moore, New South Wales, Australia) between 24 and 40 days after mating, followed by a second injection of 0.5mg cloprostenol 24 h later.
  • 1000 IU of eCG Pregnecol; Heriot AgVet, Vic, Austraha
  • Ovulation was induced by i.m. injection of 500 IU hCG (Chorulon; Intervet, New South Wales, Australia) administered approximately 72 h after eCG.
  • Embryos were centrifuged in modified PBS (PB1) (Quinn et al J Reprod. Fertil 1982;
  • lipid droplets containing 10% foetal calf serum (FCS) and 7.5 ⁇ g/ml cytochalasin B (Sigma Chemical Co., St Louis, MO) in 1.5 ml micro-centrifuge tubes (Treff Lab, Switzerland) at 12000 xg for 8 min at room temperature in order to polarize the lipid droplets in the cytoplasm.
  • FCS foetal calf serum
  • cytochalasin B Sigma Chemical Co., St Louis, MO
  • the resulting lipid layer was then removed as much as possible (fully delipated) or approximately half was removed (partially delipated),by micromanipulation using a bevelled suction pipette (30 ⁇ m in diameter) attached to Narishige micromanipulators (MO- 1081 Tokyo, Japan) under a Nikon inverted microscope (TMD1 Tokyo, Japan).
  • Embryos were held in the same medium for micromanipulation as for centrifugation. Sham-operated embryos were treated with cytochalasin B and centrifuged, but were not penetrated with the suction pipette or delipated.
  • Embryos were chilled within 2 h after lipid removal or sham operation or were maintained in culture for 14-18 h and then chilled. Embryos were cultured in 50- ⁇ l droplets of Whitten's medium (Whitten Adv Biosci 1971; 6:129-141) supplemented with 15mg ml BSA (fraction V; Pentex, Miles IL) under paraffin oil in a plastic petri dish held under an atmosphere of 5% CO2, 5% O2, and 90% N2 in humidified air at 38.6°C.
  • Whitten's medium Whitten Adv Biosci 1971; 6:129-141
  • BSA fraction V
  • Pentex Pentex, Miles IL
  • Control 1-cell 19 0 (0) 0 (0)
  • Sham-operated 1-cell 19 1 (5.3)* 0 (0)
  • Experiment 3 The ability of delipated embryos to develop in vitro.
  • the developmental abiUty in vitro, without chilUng, of fuUy and partiaUy deUpated embryos was compared with that of intact (control) zygotes.
  • AU embryos were cultured from the 1-ceU stage for 144 h.
  • Table 4 represents the comparison between the development of fuUy and partiaUy delipated embryos and that of control zygotes after culture for 144 h. No significant difference was seen in development between partially and fully deUpated embryos. Moreover, the developmental abiUty of the deUpated embryos appeared to be comparable to that of the control zygotes, except that fewer of the partiaUy deUpated embryos developed to the blastocyst stage (25% vs 57%; p ⁇ 0.05; Table 3). The deUpated embryos cleaved and developed at almost the same rate as the controls. The mean ceU numbers of the deUpated embryos tended to be lower than those for control embryos, but this difference was not significant. CeU numbers in blastocysts derived from fully delipated embryos ranged from 16 to 68; this was comparable with the range for the control blastocysts of 23 to 75.
  • FIG. 1. is a comparison of the in vitro development of porcine embryos from which Upid was partiaUy and fully removed (deUpated). Note that polarized Upid droplets left in the partially deUpated embryo (a-d) have redistributed throughout the cytoplasm during culture and the blastomeres of the partiaUy delipated embryos are darker in color than those of the fuUy deUpated embryos (e-h). Photographs were taken immediately after lipd removal (a and e) and at 48 h (b and f), 72 h (c and g) and 120 h (d and h) in culture. Magnification is x200.
  • the cytoplasm of partiaUy deUpated embryos was darker in color than that of fuUy deUpated embryos throughout all stages of their development in vitro, indicating that the remaining lipid droplets had redistributed throughout the cytoplasm.
  • Control 30 26 25 24 17 34.0 ⁇ 18.38 (86.6)3 (83.3)3 (80.0)3 (56.6)b
  • This example of the invention is proposed to comprise three steps, including centrifuging embryos to polarise cytoplasmic Upid, equiUbrating the centrifuged embryos with cryoprotectant and freeze thawing embryos.
  • the embryos can be centrifuged at 1-cell to blastocyst stage approximately 12800 x g for 10 to 15 min to polarize cytoplasmic Upid.
  • Embryos can be centrifuged with or without cytoskeleton inhibitor (for example 5-10 ⁇ g/ml cytochalasin B, D). After centrifugation the Upid can visuaUsed microscopically as polarised in the peri vitelline space away from the blastinere and inside the zona peUucida.
  • cryoprotectant After centrifugation the embryos are equUibrated with cryoprotectant for 1 to 20 min.
  • cryprotectants that might be used include, 1-7 M DMSO, glycerol, ethylene glycol, propandiol with or without sucrose, toleharose or other sugars that are commonly used.
  • cryoprotectant 1-7 M DMSO, glycerol, ethylene glycol, propandiol with or without sucrose, toleharose or other sugars that are commonly used.
  • embryos can be equUibrated with cryoprotectant during the centrifugation.
  • the embryos equUibrated with cryoprotectant are loaded into a 0.25ml plastic straw with the freezing solution and cooled directly to -30 to -150°C, foUowed by plunging into Uquid nitrogen.
  • Embryos are thawed in a water bath at 10 to 37°C. Cryoprotectant is removed from embryos by stepwise dilution followed by thorough washing with culture medium.
  • Embryos at the 2 to 4 ceU stage were deUpated, by use of a micropipette after polarizing the Upid, as described earUer.
  • Delipated embryos were cultured for approximately 16 h and 33 were transferred each to two recipients both of whom tested pregnant at day 40 by ultrasound. Both pigs farrowed and produced normal sized litters of 8 and 10 piglets respectively.
  • Table 5 shows the results for each of the recipients separately and together. TABLE 5. DeUpated porcine embryos are able to develop to term

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Abstract

Des embryons porcins sont préservés cryogéniquement en utilisant un procédé pour réduire le niveau de lipides dans les cellules de l'embryo suivi d'une congélation des embryons. La réduction du niveau de lipides peut être obtenue soit en polarisant le lipide dans l'embryon par centrifugation de l'embryon, suivie d'une congélation rapide soit par enlèvement microchirurgical du lipide polarisé. De manière alternative, le niveau de lipide peut être réduit en cultivant l'embryon in vitro. La réduction du niveau de lipides avant la congélation permet un bon taux de survie et peut être utilisée sur des embryons porcins à zone intacte.
PCT/AU1994/000474 1993-08-13 1994-08-12 Conservation cryogenique d'embryons porcins WO1995005075A1 (fr)

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AU74532/94A AU7453294A (en) 1993-08-13 1994-08-12 Cryopreservation of porcine embryos

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AUPM055193 1993-08-13
AUPM0551 1993-08-13

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2306503A (en) * 1995-10-30 1997-05-07 Japanese Research Ass For Anim A method of directly freezing porcine embryo
WO2000030441A1 (fr) * 1998-11-24 2000-06-02 Bresagen Limited Cryopreservation d'ovocytes et d'embryons et methodes de production d'animaux par cette technique
WO2001093676A1 (fr) * 2000-06-16 2001-12-13 The United States Of America, As Represented By The Secretary Of Agriculture Cryopreservation d'embryons porcins
EP2331677A1 (fr) * 2008-08-29 2011-06-15 The Curators Of The University Of Missouri Procédé à haut rendement et non vulnérant de vitrification d'embryons de porc

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7399574A (en) * 1973-11-23 1976-04-08 Augspurger L L Embryo transplantation
JPS6335501A (ja) * 1986-07-30 1988-02-16 Snow Brand Milk Prod Co Ltd ラツト初期胚の凍結保存法
GB2219923A (en) * 1988-06-22 1989-12-28 Inst Kriobiologii I Kriomedits Method for low-temperature preservation of embryos
JPH02117601A (ja) * 1988-10-27 1990-05-02 Itochu Shiryo Kk 豚の凍結受精卵及び凍結保存方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7399574A (en) * 1973-11-23 1976-04-08 Augspurger L L Embryo transplantation
JPS6335501A (ja) * 1986-07-30 1988-02-16 Snow Brand Milk Prod Co Ltd ラツト初期胚の凍結保存法
GB2219923A (en) * 1988-06-22 1989-12-28 Inst Kriobiologii I Kriomedits Method for low-temperature preservation of embryos
JPH02117601A (ja) * 1988-10-27 1990-05-02 Itochu Shiryo Kk 豚の凍結受精卵及び凍結保存方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN, C-510, page 163; & JP,A,63 035 501 (SNOW BRAND MILK PRODUCTS) 16 February 1988. *
PATENT ABSTRACTS OF JAPAN, C-740, page 100; & JP,A,02 117 601 (ITOCHU SHIRYO K.K.) 2 May 1990. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2306503A (en) * 1995-10-30 1997-05-07 Japanese Research Ass For Anim A method of directly freezing porcine embryo
US5707339A (en) * 1995-10-30 1998-01-13 The Japanese Research Association For Animal Embryo Transfer Technology Method of directly freezing porcine embryos
GB2306503B (en) * 1995-10-30 1998-05-06 Japanese Research Ass For Anim A method of directly freezing porcine embryos
NL1002209C2 (nl) * 1995-10-30 1999-03-29 Japanese Research Ass For Anim Werkwijze voor het direkt invriezen van varkensembryo's.
WO2000030441A1 (fr) * 1998-11-24 2000-06-02 Bresagen Limited Cryopreservation d'ovocytes et d'embryons et methodes de production d'animaux par cette technique
AU2001268208B2 (en) * 2000-06-06 2005-01-27 The United States Of America As Represented By The Secretary Of Agriculture Cryopreservation of swine embryos
WO2001093676A1 (fr) * 2000-06-16 2001-12-13 The United States Of America, As Represented By The Secretary Of Agriculture Cryopreservation d'embryons porcins
US6503698B1 (en) * 2000-06-16 2003-01-07 The United States Of America As Represented By The Secretary Of Agriculture Cryopreservation of swine embryos
EP2331677A1 (fr) * 2008-08-29 2011-06-15 The Curators Of The University Of Missouri Procédé à haut rendement et non vulnérant de vitrification d'embryons de porc
EP2331677A4 (fr) * 2008-08-29 2013-07-24 Univ Missouri Procédé à haut rendement et non vulnérant de vitrification d'embryons de porc
US8852078B2 (en) 2008-08-29 2014-10-07 The Curators Of The University Of Missouri High-throughput and non-invasive method to vitrify porcine embryos

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