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EP1255812A1 - Procede de production d'embryons non humains presentant une valeur genetique elevee et un sexe predetermine - Google Patents

Procede de production d'embryons non humains presentant une valeur genetique elevee et un sexe predetermine

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Publication number
EP1255812A1
EP1255812A1 EP01902306A EP01902306A EP1255812A1 EP 1255812 A1 EP1255812 A1 EP 1255812A1 EP 01902306 A EP01902306 A EP 01902306A EP 01902306 A EP01902306 A EP 01902306A EP 1255812 A1 EP1255812 A1 EP 1255812A1
Authority
EP
European Patent Office
Prior art keywords
spermatozoa
process according
comprised
semen
flow
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Withdrawn
Application number
EP01902306A
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German (de)
English (en)
Inventor
Andrea Galli
Riccardo Aleandri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Istituto Sperimentale Italiano " Lazzaro Spallanzani"
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Istituto Sperimentale Italiano " Lazzaro Spallanzani"
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Publication date
Application filed by Istituto Sperimentale Italiano " Lazzaro Spallanzani" filed Critical Istituto Sperimentale Italiano " Lazzaro Spallanzani"
Publication of EP1255812A1 publication Critical patent/EP1255812A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N5/0608—Germ cells
    • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation

Definitions

  • a flow-cytometer consists of: one or more laser beams that intersect the sample; a hydrodynamic system that conveys the cells sample to be analysed to a flow stream of a liquid, iso-osmotic with the sample; a series of light detectors (photodiodes and/or photomultipliers); a computer for data analysis in real time.
  • the analysis is based on the assessment of the light emitted from the cells under examination once they are intersected by the laser beam.
  • Said cells are previously stained with fluorochromes that, when exposed to coherent laser light, emit a particular fluorescence. Fluorescence is amplified by photomultipliers and computer analyzed, using software suitable for signal optimisation and interpretation.
  • the flow-cytometer due to its high sensitivity, measures the different amounts of DNA present in spermatozoa X in respect of spermatozoa Y (a difference that varies depending on the species) using dyes, such as Bisbenzimide Hoechst 33342 which binds to the adenine- and thymidine-rich DNA regions.
  • dyes such as Bisbenzimide Hoechst 33342 which binds to the adenine- and thymidine-rich DNA regions.
  • the following step i.e. sorting, allows the particles which are passing into the flow stream and which have been charged positively or negatively depending on their characteristics and to parameters previously set by the operator, to be separated.
  • the sperm cells with a different amount of fluorescence are physically separated by two differently charged plates which attract them on the basis of their charge and which divert them into two different collectors.
  • the collected and separated material may be re-analysed by letting it flow again in the stream.
  • the amount of light refracted at a 90 degree angle which includes fluorescence
  • the cells let in a flow stream acquire a rotational motion, which is directly proportional to the flow rate, evidence is provided that the thin portion of the spermatozoa head, when crossed by the laser beam, is predominantly turned towards the detector at a 0 degree angle, with production of a lower amount of light refracted at a 90 degree angle and, consequently, of a lower amount of fluorescence (Pinkel D. et al., 1982, ibidem).
  • the British patent GB 2,145,112 describes a sexing method based on flow-cytometry which exploits the spermatozoa electric charging, performed in a suitable sheath liquid. The method is set up for fresh semen.
  • the sexing process proposed therein is characterised by a series of modifications to the flow- cytometer (for example to the flow cell needle) which create a flow of oriented cells with the larger surface perpendicular to the laser beam.
  • the orthogonally illuminated instrument is modified so to acquire also the fluorescence from light refracted at a 0 degree angle (normally used for particles dimensional assessment only), by replacing the common photodiode, which cannot detect low fluorescence levels, with a photomultiplier.
  • Said modifications described in Johnson L.A. e Pinkel D., 1986, Cytometry 7:268-273, refers in particular the modification to the needle point which renders standardisation of the method more problematic. This is the reason why protocols have been drawn up in an attempt to minimise the detections variability.
  • the process of the present patent application describes the separation of frozen semen, according to a different DNA content carried by the X and Y sex chromosomes, by flow-cytometry.
  • the process object of the present application is characterised by specific and critical staining procedures.
  • Sperm separation and collection allow an efficient in vitro fertilization (IVF).
  • IVF in vitro fertilization
  • the process is useful in the zootechnical industry for the production of non-human mammalian embryos of predetermined sex and of high-genetic value.
  • the non-human embryos produced according to the process of the invention are endowed with a high-genetic value, as being obtained from gametes selected on the basis of the genetic characteristics of their donors.
  • the semen selected by the genetic characteristics of the animal, is frozen according to standardized procedures laid down by the European Union. It is then carried wherever the production of high-genetic-value embryos is industrially useful and economically profitable.
  • the frozen semen is promptly thawed and stained with a DNA specific fluorochrome such as Hoechst 33342 under conditions allowing the highest spermatozoa vitality. Then, it is separated by flow-cytometry into two spermatozoa subpopulations, one containing chromosome Y and the other containing chromosome X.
  • Figure 1 shows the cytograms obtained as FL2/FL1 ratio (FL2: 0 degree angle fluorescence; FL1 : 90° angle fluorescence), which were obtained to select the region with the highest fluorescence in FL1 and representing the population of spermatozoa which cross the laser beam with their larger surface. The region of interest has been boxed.
  • Figure 2 Histogram of fluorescence at a 90 degree angle, made on the region previously selected. The gate allowing the assessment only of the spermatozoa crossed by the laser beam in the larger sur ace and, therefore, with higher FL1 , has been marked.
  • Figure 3 Histogram of fluorescence at a 0 degree angle of spermatozoa with higher FL1 selected by gating. It is shown that the two spermatozoa subpopulations are physically separated.
  • the gate useful for sorting is the one that selects the two bimodal histogram tails of fluorescence in FL2: the tail on the left corresponds to the spermatozoa containing chromosome Y ( ⁇ DNA content, ⁇ fluorescence), and that on the right corresponds to the spermatozoa containing chromosome X (>DNA content, > fluorescence).
  • Figure 4 Evaluation of the efficiency of the spermatozoa sorting procedure by a genetic-molecular : PCR (polymerase chain reaction). Amplification was performed on sorted samples DNA and with standard samples of Y sorted gametes DNA opportunely contaminated with female DNA, to obtain a standard curve. DNA primers specific for the Y chromosome (BRY4a) were used. After amplification 15 ⁇ l of reaction product were electrophoresed for 1 h at 80 Volt on 2% agarose gels and stained with ethidium bromide. Gels were visualized with a UV ligth source, photographed with Polaroid film, and product bands quantified. The band intensities were used to quantify the relative amount of Y chromosomal DNA in each sample compared against the standard curve.
  • PCR polymerase chain reaction
  • A DNA extracted from female blood
  • B DNA extracted from female blood + DNA extracted from a pool of semen (corresponding to an enrichment of gametes carrying the Y chromosome of 10%)
  • C DNA extracted from female blood + DNA extracted from a pool of semen (enrichment of 20%)
  • D DNA extracted from female blood + DNA extracted from a pool of semen (enrichment of 30%)
  • E DNA extracted from female blood + DNA extracted from a pool of semen (enrichment of 40%);
  • F DNA extracted from a pool of semen (50% of Y);
  • G DNA extracted from sorted semen (X spermatozoa);
  • H DNA extracted from sorted semen (Y spermatozoa); I: negative control (water).
  • Object of the present patent application is a process for sexing frozen semen by flow-cytometry.
  • the new process is characterised by specific and critical staining, separation and collection conditions, which will be described hereinafter in detail.
  • the semen sexed under the conditions described in the present patent application is endowed with a fertilising capacity useful for an effective in vitro fertilization (IVF) for the production of non-human mammalian embryos of predetermined sex (e.g. of cattle, buffaloes, horses, sheep, pigs, rodents).
  • IVF effective in vitro fertilization
  • the invention describes a process for the production of non-human high-genetic value embryos of predetermined sex derived from gametes of breeding mammalian species, which comprises the following steps: a) thawing out a frozen solution of genetically selected semen and diluting it; b) staining the spermatozoa with a DNA specific fluorescent dye; c) diluting out the sample; d) sorting the stained spermatozoa population by flow cytometry into a spermatozoa subpopulation containing chromosome X and into a spermatozoa subpopulation containing chromosome Y; e) in vitro homologous fertilisation of oocytes with one or the other spermatozoa subpopulation; f) incubation of fertilised oocytes into a mammalian culture medium.
  • step a) of the process may be optionally preceded by a freezing step usually performed according to process well known in the art such as those officially acknowledged by the European Union.
  • the semen is derived from animals which have been selected on the basis of their genetic characteristics and/or on the genetic characteristics of their progeny, as for example semen derived from bulls which are genetic fathers of cows producing very high amounts of milk and/or which were found, when tested, to be free from genetic diseases like the biochemical defect BLAD (Bovine Leukocyte Adhesion Deficiency) and/or DUMPS ( Deficiency of Uridine Monophosphate Synthase) in Halstain breed.
  • BLAD Bovine Leukocyte Adhesion Deficiency
  • DUMPS Deficiency of Uridine Monophosphate Synthase
  • step a) of the process the frozen semen is promptly thawed, preferably in a thermostated water bath at 37°C for at least 30", then it is preferably diluted with a buffer solution at a preferred spermatozoa concentration comprised between 15 and 25x10 6 /ml, more preferably 18-22x10 6 spermatozoa/ml.
  • the dilution buffer is preferably chosen according to the extender used for semen freezing.
  • a citrate buffer is preferred when a solution such as the Laiciphos buffer containing milk-derived proteins is used as extender in the preparation of the frozen sperm, while a TALP (Tyrode's Albumin Lactate Pyruvate) Ca ++ and BSA free solution (whose composition will be specified ahead) is preferred when buffer solutions containing soya- (such as Biociphos) or egg yolk-derived proteins are used for the same purpose.
  • a commonly used citrate buffer has the following composition: 109 mM tribasic bihydrated sodium citrate and 2.4 mM citric acid, in water.
  • the process of the present invention utilizes thawed semen with a progressive motility (PM) not lower than 40% (per cent amount of spermatozoa with VAP (Velocity Average Path) > 25 ⁇ m/sec and STR (Straightness) > 0.8, according to WHO'S parameters (World Health Organization, 1992, Laboratory manual for the examination of human semen and sperm-cervical mucus interaction).
  • PM progressive motility
  • spermatozoa are stained in step b) of the process by incubation in a DNA specific fluorochrome dye solution.
  • the dye used is Bisbenzimide Hoechst 33342, used at conditions able to maintain the spermatozoa vitality at an optimal level.
  • This condition is achieved by the use of Bisbenzimide Hoechst 33342 in concentration of at least 50 ⁇ g/ml, preferably comprised between 70 and 180 ⁇ g/ml, even more preferably comprised between 80-120 ⁇ g/ml and for a time not longer than 30 minutes, preferably comprised between 10 and 20 minutes, more preferably 15 minutes, at a temperature ranging from 30°C to 38°C, more preferably 34°C-36°C, most preferred 35°C.
  • the staining is performed by adding the fluorochrome at a final concentration of 100 ⁇ g/ml at the solution containing the spermatozoa, for a time of about 15 minutes, at a temperature of 35°C.
  • sperm cells may be optionally washed with the same buffer used for the previous dilution.
  • the sample is then further diluted with a protein-free buffer or with isotonic solutions.
  • a protein-free buffer or with isotonic solutions According to a preferred embodiment of the invention, in step c) is used the same buffer as in step a), that are preferably a citrate buffer and/or an "enriched buffer solution", such as the TALP Ca ++ and BSA free solution.
  • the composition of the TALP solution for in vitro fertilisation is known in the art (see Ball GB et al., Biol Reprod, 1983, 28:717-725). In the process of the invention, this solution is called TALP IVF and is the buffer of choice for in vitro fertilisation.
  • the solution, as used in the method of the invention also for purposes different from IVF techniques, is modified and Calcium and/or BSA are not added. Other slight variations are introduced in the composition to maintain the optional osmolarity (comprised between 270-300 mOsm).
  • composition of the preferred TALP Ca ++ and BSA free version is here reported: NaCI 6000 mg, KCI 230 g, Na 2 HPO 4 40 mg, MgCI 2 -6H 2 O 310 mg, hepes 4760 mg, sodium lactate 60% 3700 ⁇ l, kanamycin 75 mg, sodium piruvate 110 mg, PVA (polyvinyl alcohol) 1000 mg, NaHCO 3 420 mg, NaOH 420 mg, sterile H 2 O 1000 ml; pH 7.4, with an osmolarity comprised between 270-300 mOsm, preferably comprised between 280-290 mOsm. It is known that other slight variations of this solution may be introduced.
  • sample is diluted at a preferred final ratio of 3:1 (sample:buffer).
  • buffer solutions may be used to dilute the sample, as i.e. PBS or Tyrode's, according to the preferences related to the extender used for initial semen freezing, with the provision that no proteic component is added.
  • preferred solutions are citrate buffer or TALP Ca ++ and BSA free buffer, by the use of which the total dissolution of the freezing extender is observed.
  • Sperm separation is carried out by flow-cytometry with orthogonal illumination on apparatuses modified according to Johnson L.A., Pinkel D. (1986) Cytometry 7:268-273, e.g. the Epics V (Coulter) instrument.
  • the method of the invention preferably employs the Becton-Dickinson FACS Vantage SE equipped with: i) a laser for the emission of UV light; ii) a MacroSORTTM head supporting a beveled needle as described in Johnson L.A., Pinkel D.
  • the instrument with orthogonal illumination is modified to acquire also the fluorescence from light refracted at a 0 degree angle (normally used for particles dimensional assessment only), by replacing the common photodiode, which cannot detect low fluorescence levels, with a photomultiplier.
  • the beveled needle allows to reduce or to eliminate the spermatozoa rotational motion, while the addition of a photomultiplier at 0 degree angle allows a more precise measurement of the fluorescence intensity.
  • the addition of the Turbo Sort Plus Option is however optional, although preferred, allowing only to fasten the sorting procedure and to obtain a high number of sorted sperm cells/second.
  • the efficiency of the instrument is further optimized by treatment of the sample according to the process.
  • the sample is made to flow in the cytometer (step (d) of the process), where evaluation of the DNA content of the cells and sorting is performed.
  • the solution used as entraining or "sheath" fluid is a protein-free buffer, preferably the same buffer used for the next step of in vitro fertilization (or a buffer fully compatible with it).
  • An "enriched buffer solution”, such as the TALP Ca ++ and BSA free solution is preferably used.
  • the composition of the TALP Ca ++ and BSA free solution has been reported above. This solution allows optimal results both in terms of flow stability and spermatozoa vitality and is usually the solution of choice (with the above mentioned modification on calcium and BSA concentrations) for the fertilisation step.
  • sheath fluid i.e. PBS, Tris etc.
  • sorting is performed by the flow-cytometer on the basis of the different amount of DNA present in spermatozoa carrying chromosome X with respect to spermatozoa carrying chromosome Y. This difference varies depending upon the mammalian species and is anyhow easily detectable by the instrument modified as said above.
  • the process of the invention is performed on bovine gametic cells.
  • Physical separation of the two subpopulations of gametic cells (spermatozoa), carrying one or the other sex-chromosome, also known as “sorting”, allows the cells which are charged positively or negatively depending on their characteristics and according to parameters previously set by the operator, to be collected separately.
  • the separation is carried out through the "gating" procedure, according to defined parameters and separates cells with a different amount of fluorescence: two differently charged plates attract the particles on the basis of their charge and divert them to two collectors.
  • the definition of the sorting parameters on the basis of the bimodality of the frequency distribution of the spermatozoa DNA content (Johnson L.A. ef al., 1989, Biol. Reprod.
  • the sorting process is carried out preferably using a flow of approx. 25,000/30,000 cells/sec, when the Turbo Sort is used .
  • the flux is approx. 1 ,500 cells/sec.
  • each gametic cell is detected by causing each single spermatozoon to pass through a channel illuminated by UV light, preferably set at a wavelength comprised between 333 to 364 nm; the signal obtained is diverted to two different photomultipliers (FL1 and FL2).
  • the fluorescence signal from the samples is preferably acquired and displayed by the software CellQuest run on MacOS 7.5.3. operating system.
  • the sperm, separated into the two populations X and Y in TALP Ca ++ and BSA free buffer, is preferably collected in protein-coated test tubes, such as those obtained by incubating them in a TALP Ca ++ free (standard) solution containing 5% BSA, for at least 8 hrs.
  • spermatozoa are collected, optionally concentrated, e.g. by centrifugation at 400xg, and used for the in vitro fertilisation (step (e) of the process of the invention) on primary non-mature (non-ovulated) oocytes.
  • the oocytes are obtained, e.g. by in vivo ovum pick-up or from freshly slaughtered animals and are matured in vitro using suitable media, such as the commercially available M199, added with hormones and foetal calf serum in controlled temperature and atmosphere conditions.
  • In vitro "matured" oocytes may be optionally treated with hyaluronidase to remove contaminating or "coating cells".
  • the in vitro fertilisation (step (e) of the process) is carried out according to procedures known in the art: a particularly preferred method is the one described by Galli C. et al. in Animal production and Biotechnology, Proceedings of the 19th International Symposium on Zootechnics, 1994, 185-189.
  • TALP IVF In Vitro Fertilsation
  • the preferred ratio between sexed spermatozoa and oocytes is 5000:1 (sperm/oocytes), in a final volume of 50 ⁇ l.
  • Incubation is performed for 18 hrs in an incubator at 5% CO 2 /air, according to methods well known in the art. After this incubation time the fertilized eggs are transferred into a cell culture medium such as SOF or M199, until the blastocyst stage is reached. However other commercially available media suitable for embryo development may be used for this incubation.
  • the non-human embryos may be used at the blastocyst stage for the implatantion into surrogated mothers or may be frozen and used in any other moment and in any other place of interest.
  • a further object of the present invention is represented by high-genetic value embryos, of predetermined sex, as produced in step f) of the process, obtainable by the process according to the invention. They may be used as such or after freezing. It should be noted that the values of these embryos are not limited to the single individual generated by the IVF but also, and more importantly, to the progeny that this single individual will generate and which will carry the genetic characteristics which have been selected for.
  • step f) is followed by an optional stage of freezing of the embryo, performed according to known protocols.
  • the convenience of the process of the invention is that all the sexing procedure and the in vitro fertilisation operations are performed into a laboratory fully equipped for both of them and they don't need to be performed on site in the breeding center usually equipped only for implantation of the embryo.
  • the fertilised bovine oocyte is co- cultured on a cell monolayer, in the particular and preferred case of bovine oocytes, derived from bovine tubal epithelium.
  • the embryo obtained may be frozen or directly re-implanted in substitute mothers for the later development growth or it may be analysed to confirm the efficiency of sperm sexing.
  • the method, object of the present invention is preferably applied to the bovine species, but can be advantageously applied also to any other breeding mammalian species and in particular to ovine, equine and rodent species, such as: sheeps, buffalos, horses, goats, pigs, rodents, mice.
  • the method of the present invention is effective for the production of embryos of predetermined sex of non-human mammalian species from frozen semen.
  • the semen is selected on the basis of useful genetic characteristic of the individual or of the progeny derived and has therefore a high-genetic value.
  • the process of the invention allows the choice of the individual carrying the useful genetic characteristics without time and geographical limitations, because of the use of frozen semen.
  • the possibility to sex frozen semen allows the more efficient production of progeny aimed at particular purposes in the zootechnical industry or addressed to particular production lineage (i.e. female for milk production or male for meat production).
  • the method of the invention allows a fertilisation efficiency still suitable for industrial purposes.
  • the embryos obtainable by the process of the invention have a high-genetic value, as they are derived from genetically selected individuals according to characteristics useful in the zootechnical industry and more importantly, they can be produced entirely in the laboratory where they can be frozen and used in the breeding centre.
  • EXPERIMENTAL PART The following examples show some of the practical applications of the process of the invention without representing any limitation. Instrumentation
  • the FACS Vantage SE (Becton-Dickinson) is equipped for the sexing procedure with the following accessories: 1 ) Water-cooled argon laser, Coherent Innova 300C, with 333.6-363.8 nm bandpass filters; 2) Detectors: the photodiode is replaced by a photomultiplier (PMT) for the detection of fluorescence at a 0 degree angle (FL2); No.
  • PMT photomultiplier
  • the aforesaid modifications to the FACS Vantage are described in: Johnson L.A. and Pinkel D. in Cytometry 7:268-273, 1986; the modified items are available on the market as FACSVantage (Becton-Dickinson) additional kits.
  • the modified instrument provides a cytogram which visualizes the different spermatozoa sub-populations obtained when the gametes, which have acquired a different orientation, flow through a laser beam. We were interested in the subpopulation that allows a more accurate analysis of the bimodality due to the different DNA content, since the spermatozoa are intersected by the laser beam in a perpendicular direction. Solutions used
  • Bisbenzimide Hoechst 33342 stock solution (5 mg/ml): H 2 O or Tyrode's solution 400 ⁇ l, Hoechst 33342 (Sigma) 2 mg.
  • the solution was prepared and divided into aliquots which were stored at -20°C.
  • the solution was prepared and aliquoted in 25 ml stock and stored at +4°C.
  • Lacmoid 1 % 100 mg Lacmoid, sterile H 2 O 5.5 ml, glacial acetic acid 4.5 ml. Filtered with 0.8 ⁇ m filter.
  • Example 1 Vitality and dilution tests on Hoechst 33342-labelled samples Tests were conducted with various concentrations of Bisbenzimide Hoechst 33342: final 5 ⁇ g/ml, final 10 ⁇ g/ml and 100 ⁇ g/ml, with incubation times of 15, 60, 120, 240 e 300 min. The vitality of spermatozoa thawed as described in Example 2 was assessed after different incubation times, at different final dye concentrations: vitality was the highest after a 15-min staining with 100 ⁇ g/ml Bisbenzimide Hoechst 33342.
  • the sample of stained spermatozoa was diluted with various solutions, such as PBS, Tyrode's solution, citrate buffer (109 mM tribasic bihydrated sodium citrate and 2.4 mM citric acid) and TALP Ca ++ and BSA free. Only these last two proved to be capable of completely dissolving the freezing extender, such to obtain an optical clearance allowing a better definition of the signal from the populations to be sorted by flow-cytometry.
  • various solutions such as PBS, Tyrode's solution, citrate buffer (109 mM tribasic bihydrated sodium citrate and 2.4 mM citric acid) and TALP Ca ++ and BSA free. Only these last two proved to be capable of completely dissolving the freezing extender, such to obtain an optical clearance allowing a better definition of the signal from the populations to be sorted by flow-cytometry.
  • the sorted sperm was collected according to different methods which were compared for the recovery efficiency: a) in test-tubes with extender based on Tris and egg yolk with or without BSA or in BSA-coated (saturated) test-tubes incubated overnight with TALP Ca ++ and BSA free pH 7.4, added with 5% BSA. The best results were obtained by collecting the spermatozoa in TALP Ca ++ and BSA free, pH 7.4, in test-tubes saturated with BSA, which maintains the spermatozoa vitality at an optimal level and does not interfere with the successive In Vitro Fertilisation steps. Conversely, the methods known in the art (e.g.
  • Example 2 Sperm sexing and separation of two subpopulations carrying chromosomes X or Y Preparation of thawed samples
  • the sexed semen was used for the following step on flow-cytometry only if its progressive motility was 40 % at minimum (measured as per cent amount of spermatozoa with VAP (Velocity Average Path) > 25 ⁇ m/sec and STR (straightness) > 0.8),
  • the semen was diluted in a citrate buffer (109 mM tribasic bihydrated sodium citrate and 2.4 mM citric acid) or in TALP Ca ++ and BSA free to a spermatozoa concentration of 20x10 6 /ml. Then an aliquot of Bisbenzimide Hoechst 33342 solution (5 mg/ml in Tyrode's buffer/H 2 O) was added to obtain a final concentration of 100 ⁇ g/ml. The sample was then incubated at 35°C for 15 min. For fluorescence assessment and for the successive spermatozoa separation, the sample was caused to flow in the instrument using TALP Ca ++ and BSA free, pH 7.4, as sheath liquid. Fluorescence analysis By means of an argon laser, Innova 300C, rated at 150 mW, fluorochromatised spermatozoa were excited by 333.6-363.8 nm UV light.
  • a citrate buffer 109 mM tribasic bihydrated sodium citrate and 2.4
  • the semen sexing was checked on the sorted material by cytometric re-analysis or by genetic-molecular methods.
  • the sexed material collected was re-analysed by flow cytometry.
  • computer software it is possible to overlap the histograms of fluorescence in FL2, obtained from spermatozoa X and Y.
  • the results obtained by this method indicate that in the sorted subpopulations the enrichment of spermatozoa containing the X or Y chromosome is always higher than 75%.
  • a semi-quantitative approach based on PCR was also used to confirm the efficiency of sorting using DNA primers specific for the Y chromosome (BRY4a, see Peura et al., Theriogenology, 1999, 35:547 -555).
  • the Y chromosome-specific primers corresponded to nucleotides 1159-1178 (5' primers) 5'-CTCAGCAAAGCACACCAGAC-3' and 1439-1459 (3' primers) 5- 'GAACTTTCAAGCAGCTGAGGC-3'.
  • Image analysis of the fluorescent intensity of PCR amplified Y chromosome-specific DNA was used to quantify the relative amount of y chromosomal DNA in each sample. Fluorescent intensity of each sample was compared with that of a reference sample prepared from pooled unsexed ejaculate (pooled sperm).
  • the standard curve consisting of 30 ng/ ⁇ l of female DNA (only XX) diluted with 30 ng/ ⁇ l pooled sperm DNA in different ratio (see Table 1 ). Table 1. Preparation of the standard for PCR amplification.
  • the 50 ⁇ l per reaction contained 30 ng of DNA template, 1x PCR buffer (Perkin Elmer), 200 ⁇ M each dNTP, 1 ,5mM MgCI 2 , 250 mM of each primer and 1.25 U of Taq Gold polymerase (Perkin Elmer).
  • the amplifications were carried out in a GeneAmp per System 9700 (PE Applied Biosystem).
  • the samples were denaturated at 95 °C for 10 minutes followed by 20 cycles consisting of denaturation for 15 seconds at 95 °C, annealing for 15 seconds at 65 °C and primer extension at for 72 °C for 1 minute. After the last cycle the samples were incubated for a further 5 minutes at 72°C.
  • 15 ⁇ l of reaction product were electrophoresed for 1 h at 80 Volt on 2% agarose gels, stained with ethidium bromide. Gels were visualized with a UV light source, photographed with Polaroid film, and product bands quantified.
  • Example 4 In vitro fertilisation and pre-implant growth of fertilised oocytes
  • the oocytes were added to TALP wash and freed from the spermatozoa and cells of the "corona radiata" by mechanical vortex motion.
  • a few of said fertilised oocytes were put on an ethanol-washed slide, closed with a cover glass to check whether fertilisation had taken place.
  • the slides were allowed to stand overnight in a fixative (ethanol/acetic acid 3:1 ). Then they were dyed by capillarity with 1 % lacmoid and promptly rinsed with 45% acetic acid. If fertilisation took place, the two male and female pronuclei and the penetrated spermatozoon tail can be viewed through a microscope. Otherwise, i.e. in the case of non- fertilised oocyte, the maturation of same is proved by the presence of a metaphase with a polar globule.
  • the medium was replaced by adding the culture drop with 20 ⁇ l SOF, equilibrated in an incubator, and by sucking an equal amount of it. After two further days of culturing in SOF, this medium was replaced, according to the same procedure, by equilibrated medium M199. This medium will support the embryos development to the blastocytes stage, which will be reached on the two following days.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biotechnology (AREA)
  • Reproductive Health (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
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  • Microbiology (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un procédé de production d'embryons mammifères non humains de sexe prédéterminé et présentant une valeur génétique élevée. Le procédé utilise du sperme congelé, qui a été congelé conformément aux normes en vigueur dans l'Union européenne. Le sperme est prélevé chez des animaux choisis pour leurs caractéristiques génétiques, par les centres de collecte du sperme, puis il est décongelé et le sexe est déterminé. Les spermatozoïdes dont le sexe a été déterminé sont utilisés pour la fécondation in vitro des ovocytes d'une espèce animale compatible afin de produire in vitro des embryons de sexe prédéterminé. Les conditions d'exécution du procédé permettent d'utiliser du sperme congelé.
EP01902306A 2000-01-14 2001-01-11 Procede de production d'embryons non humains presentant une valeur genetique elevee et un sexe predetermine Withdrawn EP1255812A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IT2000MI000030A IT1317724B1 (it) 2000-01-14 2000-01-14 Procedimento per la produzione di embrioni non umani di sessopredeterminato ad alto valore genetico.
ITMI200030 2000-01-14
PCT/EP2001/000281 WO2001051612A1 (fr) 2000-01-14 2001-01-11 Procede de production d'embryons non humains presentant une valeur genetique elevee et un sexe predetermine

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EP1255812A1 true EP1255812A1 (fr) 2002-11-13

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Country Link
EP (1) EP1255812A1 (fr)
AU (1) AU2001230176A1 (fr)
IT (1) IT1317724B1 (fr)
WO (1) WO2001051612A1 (fr)

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ATE383869T1 (de) 1998-07-30 2008-02-15 Xy Inc System zur künstlichen nicht-chirurgischen besamung von pferden
US7208265B1 (en) 1999-11-24 2007-04-24 Xy, Inc. Method of cryopreserving selected sperm cells
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CA2468774C (fr) 2000-11-29 2015-06-30 George E. Seidel Systeme permettant de realiser une fecondation in vitro avec des spermatozoides separes en population porteuse de chromosome x et en population porteuse de chromosome y
US7713687B2 (en) 2000-11-29 2010-05-11 Xy, Inc. System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
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ES2524040T3 (es) 2003-03-28 2014-12-03 Inguran, Llc Aparato y procesos para proporcionar esperma animal clasificado por sexo
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Also Published As

Publication number Publication date
ITMI20000030A1 (it) 2001-07-14
IT1317724B1 (it) 2003-07-15
ITMI20000030A0 (it) 2000-01-14
WO2001051612A1 (fr) 2001-07-19
AU2001230176A1 (en) 2001-07-24

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