MXPA99011387A

MXPA99011387A - Improvements to methods for preparing equine embryos for cryopreservation

Info

Publication number: MXPA99011387A
Application number: MXPA/A/1999/011387A
Authority: MX
Inventors: Legrand Emmanuel
Original assignee: Legrand Emmanuel
Priority date: 1997-06-10
Filing date: 1999-12-08
Publication date: 2000-12-06

Abstract

The invention concerns a method for preparing an equine embryo consisting in, previously to subjecting said embryo to the action of cryoprotectant(s), in subjecting it to a suitable treatment to eliminate the embryonary capsule or to increase said capsule permeability, in order to reinforce the subsequent action of said cryoprotectant(s). Preferably, the embryonary capsule is destroyed or simply made more permeable by a treatment based on collagenase or trypsin.

Description

IMPROVEMENTS TO METHODS FOR PREPARING EQUINE EMBRYOS FOR CRYOCONSERVATION The present invention relates to the field of embryo conservation by cold; it relates more particularly to an improvement of procedures for the preparation of equine embryos for their cryopreservation (freezing or vitrification), especially taking into account the prospect of a subsequent transplant. The freezing of embryos during embryo transfer presents numerous advantages. It allows, among other things, to suppress the obligation to synchronize the menstrual cycles between the donor and the recipient; facilitates the transport of embryos over large distances and also makes it possible to create embryo banks, particularly in the case of exceptional animals or animals in danger of extinction. After the first successes in the 1970s, the freezing of embryos has now become a routine technique in the case of ruminant domestic animals, especially in the case of bovines. Parallel work performed in the equine field allowed to reach the first births of foals from the transfer of frozen embryos in the early 1980s. From a biological perspective, the fertilization of the equine embryo occurs in the oviduct, in the union between the blister and the isthmus, usually 12 hours after ovulation. The embryo then develops in the oviduct; it goes from the unicellular zygote stage, from 100 to 160 μm in size to the "morula" stage, from 16 to 32 cells (from 150 to 200 μm). The mitotic stains are initially made simultaneously in the different blastomeres, and then in an increasingly asynchronous manner. The development phase of the equine embryo in the oviduct lasts approximately 5 days. The "blastocyst" stage occurs at the "morula" stage at approximately the time when the embryo passes into the uterus. It is at this stage that the differentiation between the two cell populations occurs: the trophoblast and the internal cell mass (MCI) that the fetus will provide. In this stage, the embryo is composed of a liquid cavity (the blastocele), surrounded by a cellular layer (the trophoblast), near which the internal cell mass (MCI) is located. To the periphery of the embryo can be observed two acellular structures: the zona pellucida and the capsule.

The zona pellucida has a thickness of the order of 15 to 30μm; it is constituted by an inner, dense layer, with a small number of thin channels and by an external layer that contains large lagoons. This zona pellucida is present in the oocyte and disappears towards the seventh or eighth day. The capsule is a characteristic of equine embryos. It forms approximately within eight hours after the entry of the embryo into the uterus between the trophoblast and the zona pellucida. This capsule is of a glycoprotein nature; its thickness increases regularly until a peak towards the eighteenth day; it then diminishes until its disappearance approximately on the twenty-second day, approximately at the moment in which the embryo is fixed in the uterus. The collection of the embryos for freezing must be carried out before they are fixed in the uterus. The appropriate moment is evaluated, after mounting or insemination, through a transrectal palpation and according to the data of an examination by means of ecotomography. This collection can be done very early after fertilization, at the level of the oviduct, but that then requires a traumatizing surgical operation for the animal. The other solution is to wait for the embryo to reach the uterus (after the sixth day of fertilization) and to work vaginally, through an adapted collection probe, in accordance with the technique of LAGNEAUX D. Et al. (1988"the transplantation embryo chez la jument" (the embryo transplant in the mare), CEREOPA, 14-day Ava 163-171). This area allows the introduction of a collection medium into the uterus (for example the medium is a phosphate-regulated saline solution).

(PBS) of Dubelcco +2 g / 1 of bovine serum albumin, available in I.M.V. (Instrument de Médecine Vétérinaire) B.P. 81, 61 L'AIGLE (France)), and then its recovery with the eventual embryo (s). The harvest medium is observed through a binocular magnifying glass, and in case of discovery of embryo (s), said embryo (s) is isolated in a dome containing the same medium (PBS + albumin) . Subsequently, the embryo "prepares" to support the subsequent freezing operation. This preparation consists in submitting the embryo to the action of cryoprotective agents intended to prevent the formation of intracellular crystals during the decrease in temperature. The cryoprotective agents employed can be chosen from glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, or 1,2-propanediol; they are used in concentrations of the order of 1 to 2 M during times of the order of 10 minutes to 30 minutes (these times and concentrations are adapted according to the effectiveness of the desired protection taking into account the possible toxicity of the products). Preferably, the embryo is subjected to different baths of cryoprotective agents in increasing concentrations. The embryo is then conditioned in a plastic receptacle with a small amount of medium corresponding to the last bath of cryoprotective agent and this receptacle is placed in the tub of a freezer that can be programmed at a temperature of the order of -7 ° C. After a time of equilibration in the order of 5 to 10 minutes, the crystallization of the content is induced by contact with a metallic rod previously cooled in liquid nitrogen (-196 ° C). After a new thermal equilibrium time, the freezer programmer lowers the temperature of said freezer to a level of -30 ° C / -30 ° C at a rate of -0.1 to 1 ° C per minute; at this temperature, the receptacle is submerged in liquid nitrogen and then stored in a tub. Thawing, before transfer, is obtained by immersing the receptacle for 1 minute in a water bath at a temperature of 37 ° C; The embryo is removed and the cryoprotective agent removal operation is carried out through a succession of dilutions in baths in decreasing concentrations. The embryo is then ready for transfer to the receiving mare. Classically, two techniques can be used: the first, a surgical technique, consists of implanting the embryo in the oviduct, the second, a non-surgical technique and that corresponds better to the objectives of commercial transfers, consists of placing the embryo in the uterus passing it through the neck with the help of a probe. The technique we have just written is derived from the technique used for many years in cattle, and allows, in bovines, obtain gestation rates from frozen embryos, greater than 50%. However, in the field of equines, the gestation rates obtained are often low, only in the order of 20 to 30%. Without being able to explain to date, the use of this freezing technique in equines frequently causes important necrosis of the Internal Cell Mass and the trophoblast of the embryo, which seem to cause the important number of premature abortions. The object of the present invention is to offer an improved embryo preparation technique for the purpose of its cryopreservation, which allows reducing the rates of necrosis related to these operations, and which in consequence allows the subsequent gestation to be favored during the transfer of the embryo. The method according to the present invention is based on the presence of the capsule surrounding the embryo during most of the time in which said embryo is in the uterus of the donor, and is based on the hypothesis in the sense that The presence of this capsule is detrimental to embryo preparation operations before freezing. To obtain an interesting recovery rate, the embryos are recovered relatively late; In general terms, they are in the blastocyst stage and surrounded by a capsule. After the classical operations of preparing embryos of equines for the purpose of their cryopreservation, the method according to the present invention plans, before subjecting said embryo to the action of cryoprotective agents, to subject said embryo to an appropriate treatment to suppress the embryo. embryo capsule and to increase the permeability of this capsule, with the purpose of reinforcing the subsequent action of said cryoprotective agent (s). In accordance with a first embodiment, the capsule is destroyed or simply becomes more permeable through an appropriate chemical or enzymatic treatment, for example, through collagenase or trypsin. The concentrations of the products used and the contact times (bath treatments) are adapted to at least partially lighten the capsule taking into account the eventual toxicity of the products so as not to alter the embryo. In the sites of action of trypsin and collagenase are different and therefore one can also consider the use of a combination of these two enzymes. According to another technique, the capsule is mechanically treated: it is partially or totally removed, or it is subjected to a simple drilling operation. This mechanical treatment is advantageously preceded by an appropriate chemical or enzymatic treatment to weaken said capsule. Example 1 The embryo is harvested 5 to 8 days after ovulation. After isolation, it is placed in a waiting bath (medium PBS + 2 g / 1 of ultrafiltered bovine serum albumin in a 0.2μm filter to avoid bacterial contamination). The embryo is then placed in a 2 ml dome containing 1 to 2 ml of a trypsin solution at a concentration of 0.2% (dilution in a PBS medium without calcium or magnesium), for 15 minutes and at room temperature (of the order of 23 ° C). The more or less important destruction or embrittlement of the capsule is obtained depending on the size of origin of said capsule. A fine or thinned "residue" of the capsule may persist around the embryo; it happens that this capsule is completely broken through the simple enzymatic action or that enzymatic action can be completed through a mechanical decapsulation carried out through a micromanipulation technique. To dilute the enzyme and suspend its action, the embryo is deposited in 3 or 4 successive baths of ultrafiltration medium PBS + albumin, for a total period of 10 minutes. After 10 min, the embryo is placed in the first bath of cryoprotective agent (glycerol 0.5 M for 15 min), then in the second bath (glycerol 1 M for 15 min) and finally in the third bath (glycerol 1.5 M) for 15 minutes). The embryo is then mounted in a receptacle and then subjected to the usual operations to obtain its freezing, in the manner described above. Thawing is obtained by immersing the receptacle for 1 minute in a water bath at a temperature of 37 ° C and after the removal operations of the cryoprotective agent is carried out: the embryo is placed in a first bath of 1 M glycerol + 0.25 M sucrose during 15 minutes, then in a second bath of 0.5 M glycerol + 0.25 M sucrose for 15 minutes, then in a third bath (0.25 M sucrose for 15 minutes) and finally in the PBS medium + albumin (15 minutes). Then the embryo is ready for the transfer. EXAMPLE 2 The previous treatment protocol is reproduced but 0.2% trypsin is replaced by type II collagenase in a concentration of 2.5%, for 15 minutes (the dilution is also carried out in a PBS medium without calcium and without magnesium). Results a) Cellular Necrosis Cellular necrosis refers to the structural modifications that occur after the death of a cell; it results from the progressive degradation of the cell by its own enzymes contained in the lysosomes. The percentage of necrotic cells is representative of the cellular damage caused by the manipulations and reflects the viability of the embryo. This histological examination is performed in accordance with the technique described by J.F. BRUYAS - 1994 - "Contribution to l'étude de la congélation des embryons équins: une approche métabolique et cellulaire" (contribution to the study of the freezing of equine embryos: a metabolic and cellular approach) - Mémoire de recherche Agrégation de pathologie de la reproduction (Memory of aggregation of pathology of reproduction) - Ecole Nationale Vétérinaire de NANTES, France. It is done before the freezing operation to determine the cellular damage related to the preparation treatment. The observed percentage of necrotic cells has been as follows: a) embryos treated by trypsin (according to example 1): 11% (one embryo analyzed) b) embryos treated by collagenase (according to example 2): 10.3% (an embryo analyzed) These results should be compared with the rates of necrosis of embryos that have followed the same preparation protocol, without enzymatic treatment: c) embryos without capsule: 13.9% (average of 6 embryos) d) embryos with capsule: 30.6 % (average of 8 embryos) it is observed therefore that the embryos whose capsule has been treated by an enzyme suffer less damage than the embryos that kept their capsule intact. These treated embryos behave like embryos without a capsule. b) In vivo tests Eight embryos were collected between the fifth and the eighth day after ovulation in accordance with the method of LAGNEAUX et al. Described above. The distribution of the embryos collected according to age is as follows: Age of the embryos Number of embryos 5.5 days 1 6.5 days 1 7.5 days 2 8.5 days 4 The sizes of the embryos harvested are the following: Embroidery No. size (μm) Embryo No. size (μm) 1 1020 5 187 2 714 6 1581 3 119 7 1105 4 442 187 These embryos were treated according to example 1 above. Embryos number 1 and number 2 were subjected to mechanical decapsulation after enzymatic treatment and the capsule of embryos number 6 and number 7 was fully used during the enzyme bath. We then had two batches of embryos: - a number 1 with embryos without capsule (embryos number 1, 2, 6 and number 7), and - a batch number 2 with embryos carrying a partially lysed capsule only (embryos number 3, 4, 5 and 8). After thawing, the transfer technique used has been the technique used by TAINTURIER D., BRUYAS JF, DUMONT P., FIENI F., ESCOUFLAIRE P. (1989, "The transplantation embryonnaire chez la jument" (the embryo transplant in the mare), Revue Med. Vet. 140, 1109-115), which consists in placing the embryo behind the neck with the help of a transplant syringe. The control of the pregnancy was carried out by means of ecotomography (scanner 100 of Pie Medical, Hospimédi 60790 POUILLY, FRANCE), at 12, 21 and 24 days. The results of the transfers appear in the following table: Lot No. of embryo DG14 DG21 DG28 1 2 2 3 2 4 + (a) (a 2 5 + - - 1 6 + + + 1 7 + + + 2 8 + + + DG = diagnosis of pregnancy (at 14, 21 and 28 days, respectively) (a): the receiving mare was injured after the diagnosis of pregnancy on day 14. The exams were then suspended, the gestation rates on days 14, 21 and 28 were as follows: DG No. of pregnancies / % No. of transfers A 14 days 6/8 75% A 21 days 3/7 43% A 28 days 3/7 43% In general terms, these rates are higher than the rates obtained with the protocols that were known to date are similar to the rates obtained after transfer of fresh embryos (58% on average in the last 10 years (CLEMENT F., HOFFERER S., VINCENT P., 1995"The transplantation embryonnaire chez la jument" embryos in the mare), Equ'idée mars-avrill 17, 56-62) Lot number 1 (embryos without capsule) DG No. of gestations /% No. of transfers At 14 days 3 / 4 75% At 21 days 2/4 50% At 28 days 2/4 50% Lot number 2 (embryos with partially lysed capsule) DG No. of pregnancies /% Transfer No. At 14 days 3/4 75% At 21 days 1/3 33% At 28 days 1/3 33% These results indicate that it is possible to obtain pregnancies with embryos that do not they have their capsule and there is no significant difference between the results of the transplanted embryos without capsule and the embryos transplanted with capsule. The gestational capsules according to age are the following: Age Day 14 Day 21 Day 28 5.5 days 0/1 0/1 0/1 6.5 days 1/1 1/1 1/1 7.5 days 2/2 0/1 0/1 8.5 days 3/4 2/4 2/4 these results show that it is possible to obtain a pregnancy from frozen embryos of an age of 7.5 days and more, using the procedure according to the present invention, which does not it had been obtained practically to date through the known procedures. The gestation rate according to size are as follows: Size day 14 day 21 day 2 < 200μ 2/3 1/3 1/3 between 200 and 500um 1/1 0/0 0/0 > 500 μm 3/4 2/4 2/4 > 1000μm 3/3 2/3 2/3 these results show that it is possible to obtain a gestation from frozen embryos having a size greater than 500um using the procedure according to the present invention, which had not been obtained at the date by the known procedures. The set of results obtained shows the importance of the embryo capsule in the process of cryopreservation of equine embryos. The method according to the present invention improves gestation rates from frozen embryos and allows to raise the possibility of commercial development of this technology. The treatment of the embryo capsule allows not to worry about the moment of the embryo's collection.

In particular, the harvest can be carried out quite late; It thus benefits from a high collection rate and the freezing operation is carried out in embryos in the "blastocyst" stage, which allows them to better resist the aggressions of the different treatments.

Claims

CLAIMS 1. A procedure for the preparation of embryos of equines for their cryopreservation, said procedure consists of preparing the embryo, after harvesting, subjecting it to the action of cryoprotective agent (s), and then conditioning it in a receptacle before subjecting it to the cryopreservation operation itself, characterized in that it consists, before subjecting said embryo to the action of cryoprotective agent (s), to submit it to an appropriate treatment to suppress the embryonic capsule, or to increase the permeability of said capsule, in order to reinforce the subsequent action of said cryoprotective agent (s).
2. A method according to claim 1, characterized in that it consists, after harvesting, of treating the embryo capsule through an enzymatic action before subjecting the embryo to the action of the cryoprotective agent (s) (es). ).
3. A method according to claim 2, characterized in that it consists, after collection, in treating the embryo capsule by immersing the embryo in a collagenase bath.
4. A method according to claim 3, characterized in that it consists, after harvesting, in treating the embryo capsule by 2.5% collagenase, for 15 minutes, at room temperature. A method according to claim 2, characterized in that it consists, after harvesting, in treating the embryo capsule by immersing the embryo in a trypsin bath. A method according to claim 5, characterized in that it consists, after harvest, in treating the embryo capsule by 0.2% trypsin, for 15 minutes at room temperature. A method according to any of claims 2 to 6, characterized in that it consists, after harvesting, in treating the embryo capsule through a mixture of collagenase and trypsin. A method according to claim 1, characterized in that it consists, after harvesting, in mechanically removing the embryo capsule before subjecting said embryo to the action of the cryoprotective agent or of the cryoprotective agents. A method according to claim 8, characterized in that it consists of mechanically removing the embryo capsule before having subjected the embryo to a suitable chemical treatment to embrittle said capsule. A method according to claim 9, characterized in that it consists in submitting said embryo to an enzymatic treatment adapted to embrittle the capsule in order to facilitate its mechanical removal.