WO2004072220A2 - Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe - Google Patents

Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe Download PDF

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WO2004072220A2
WO2004072220A2 PCT/BR2004/000009 BR2004000009W WO2004072220A2 WO 2004072220 A2 WO2004072220 A2 WO 2004072220A2 BR 2004000009 W BR2004000009 W BR 2004000009W WO 2004072220 A2 WO2004072220 A2 WO 2004072220A2
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spermatozoa
semen
centrifugation
embryos
gradient
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WO2004072220A3 (fr
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Vera Fernanda Martins Hossepian De Alameda Lima
Mariney Flávia Pereira Di-Tanno RAMALHO
Carlos Alberto Moreira Filho
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Fundacão De Amparo A Pesquisa Do Estado De São Paulo
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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

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  • the invention allows the production by centrifugation in density gradient, in specialized companies for the production and commercialization of frozen semen, of semen doses enriched with X- or Y- chromosome bearing spermatozoa, without prejudice to the capacity of fertilization of said spermatozoa and which: a) are compatible with the semen freezing and packing processes adopted by said companies; b) may be commercialized for use in Al (Artificial Insemination) programs by using the traditional method (semen deposited shortly after the cervix) and under various reproductive handling conditions, obtaining pregnancy and birth rate of at least 75%; c) may be used for IVP (in vitro production of embryos), obtaining cleavage and embryo development rates above 75% and 35%, respectively; d) may be commerc ;iialized for use in conventional artificial insemination programs (births preferably of mascul ine or feminine gender) or those made after the hormone treatment of females to synchron ize the heat and/or multiple ovulations and subsequent harvest
  • samples of spermatozoa containing both X- and Y- chromosome-bearing spermatozoa can be separated to produce subpopulations enriched with X- or Y-bearing spermatozoa, which are substantially pure regarding the desired spermatozoa and substantially free from the other type of spermatozoa.
  • substantially free means herein a sample of semen enriched with X spermatozoa which, when used in Al or IVP, has only a small chance to produce the birth of a male, since the spermatozoa sample has less than 20% and preferably less than 10% of Y spermatozoa.
  • Density gradients of the invention have a defined composition allowing to keep the viability of said spermatozoa, so that they become resistant against the conventional dilution and freezing processes and may, after thawing, keep acceptable viability levels (motility, power, % intact acrosome, etc.) for commercialization intended for conventional artificial insemination programs (births preferably of masculine or feminine gender) or those made after the hormone treatment of females to synchronize the heat and/or induce multiple ovulations and subsequent harvesting of embryos (most of them of feminine or masculine gender) and later transfer to recipient females. Furthermore, the production of semen may be intended for in vitro production of feminine or masculine embryos and later transfer to recipient females.
  • the method of the invention may be used to separate spermatozoa from various species of mammals, including humans. Different technological routes are taken in the attempt to select gender in mammals, both for zootechnical interest species and for species in extinction, pet animals and assisted human reproduction. There are two alternatives for that: to separate X-chromosome-bearing spermatozoa from those Y- chromosome- bearing spermatozoa; or to sex pre-implanted embryos.
  • the separation of X-bearing spermatozoa from those Y- bearing spermatozoa is based on the detection of at least one of the following phenotypic (chemical or physical) differences between these two types of cells: a) pH sensitivity (EMMENS, 1960); b) electrical load of the membrane surface (GORDON, 1957; KANEKO et al., 1984a); c) morphology of the nucleus and head (SHETTLES, 1961); d) surface antigens (GOLDBERG et al, 1971 ; KOO et al, 1973); e) migration rate (ERICSSON et al, 1973; WANG et al, 1994); f) DNA content (GARNER et al, 1983); and g) density differences (WINDSOR et al, 1993).
  • Separation is made by adding to the semen antibodies against one of the genders, previously coupled to the beads.
  • Spermatozoa remain for 30 minutes in contact with the antibodies in a homogenizer with 3 RPM rotation at room temperature. After this period, spermatozoa are submitted to a magnetic field by 15 minutes and the supernatant removed constitutes the fraction enriched with X or Y spermatozoa. For example, if anti-Y antibodies were used, the fraction enriched with X will be recovered in the supernatant and vice versa.
  • the method described in that patent is widely known in immunology and therefore successful as for its efficiency, it had not yet been disclosed to separate these two spermatozoa populations.
  • the literature shows drastic reductions in fertility rates evaluated "in vitro" (penetration rate, cleavage rate, embryo development rate to blastocyst stage and number of blastocyst cells after spermatozoa sexing by cytometry in comparison to spermatozoa not submitted to sexing by this method (McNUTT and JOHNSON, 1996; RATH et al, 1997; BEYHAN et al, 1999).
  • JOHNSON (2000) underlines that the premature capacitation is clearly a characteristic of the sexed spermatozoa by the cytometer, which is a disadvantage for the spermatozoa intended to be frozen, but is an advantage for the spermatozoa to be immediately used after separation to make IVF, with no induction of the capacitation of spermatozoa before fertilization.
  • Another limitation is the reduced efficiency of thawing of sexed spermatozoa, since freezing prejudices the uniformity of nucleus staining, with coloring agent Hoechst 33342 (JOHNSON et al, 1994). This restricts the use of the best bulls (proven bulls) within each race in programs of animal improvement and progeny test using in vitro production of embryos.
  • motility of sexed spermatozoa by this method was of only 51% for X or Y spermatozoa subpopulation (JOHNSON et al, 1997). After the freezing/thawing process, mortality decrease to 45% (laser regulated for 100 mW; 351, 364 nm) and only 65% of sexed spermatozoa maintained acrosome integrity (SCHENK et al, 1999).
  • the present invention it is possible to produce from 8 to 15 million spermatozoa from a subpopulation (X or Y) per gradient tube, in 20 to 30 minutes of centrifugation, depending on the used gradient. Therefore, the volume of frozen semen produced depends on the centrifuge capacity and its increase depends on the purchase of other centrifuges. Thus, contrary to what occurs with the flow cytometry method, in the present invention, the quantity of semen doses produced may be largely increased with lower investment (purchase of a centrifuge) and with no purity loss of the separated spermatozoa.
  • semen doses enriched with X or Y spermatozoa produced by the invention may have increased precision (birth or IVP - "in vitro" production - of embryos of the desirable sex) if associated to embryo IVP methods and management techniques (Al time, nutrition, etc.) which may promote a deviation of the sex ratio.
  • Sedimentation or centrifugation procedures of spermatozoa are based on the density difference existing between X- or Y-bearing spermatozoa.
  • SUMNER & ROBINSON (1976) analysed by microinterpherometry the head of spermatozoa and verified that X-bearing spermatozoa have more DNA and nuclear protein than Y spermatozoa and this difference is proportional to the mass difference between the two types of cells.
  • Meistrich (mentioned by WINDSOR et al, 1993) calculated that the difference in bovine DNA content of X or Y spermatozoa results in a density difference of 7 x 10 "4 g/cm 3 , or 0.06% of the density over X spermatozoa.
  • the author emphasized that this difference makes the separation of the two types of spermatozoa possible, as long as gradients with high density resolution are used.
  • HEGDE et al (1977) separated human X or Y spermatozoa in a solution with 8% Ficoll and 33.8% sodium metrizoate. The final density of the solution was 1.08 g/ml. Equal volumes of this solution and semen were centrifuged at 100 X g for 20 minutes. Spermatozoa from the higher and lower portions were treated with Quinacrine to evidence F-body. The sediment before and after centrifugation was observed as containing 45.22 + 6.14% and 62.9 ⁇ 3.61% of Y spermatozoa, respectively, which was statistically significant.
  • Ficoll solution consisted of 5.7% (weight/volume) of Ficoll 400 and 9.0% (weight/volume) of sodium diatrizoate, pH 7.6 and density of 1.077 ⁇ 0.001 g/ml. After centrifugation at 250 X g for ten minutes, a significant difference of 53.0 ⁇ 1.5% and 40.6 ⁇ 1.8% of Y spermatozoa was obtained in the upper and lower portions, respectively.
  • Percoll was diluted to prepare solutions of about 40% to about 80% Percoll.
  • discontinuous gradients of Percoll were formed, by depositing 1.0 ml of each one of these solutions in tubes: a) 50 to 85% with 5% intervals (8 layers) at 15 °C; b) 30 to 80% with 5% intervals (8 layers) at 15 °C; c) 30 to 80%> with 5% intervals (11 layers) at 20 °C; d) 30 to 90% with 10% intervals (7 layers) at 20 °C.
  • SCHWIDERSKI et al (1991) and BLOTTNER et al (1993) used centrifugation in Percoll gradient to separate bovine X-or Y- bearing spermatozoa.
  • the gradient consisted of 10 layers of 0.6 ml of Percoll solutions with concentrations ranging from 22% to 48%. This method provided enrichment of more than 75% of X or Y spermatozoa in the lower and upper portions, respectively, as verified by in situ hybridization. After two centrifugations, spermatozoa from both subpopulations did not show any significant difference concerning morphology. The induction of acrosome reaction showed normal capacitation.
  • - second washing centrifugation made after the transfer of the recovered material from the bottom of the tube of the first centrifugation to a second tube over which the second gradient is deposited, with appropriate density to separate immature spermatozoa, cells and possible protozoa (page 7, line 26-31);
  • - third washing centrifugation made after the transfer of the recovered material from the bottom of the tube of the second centrifugation to a third tube over which the third gradient is deposited, with appropriate density to separate bacteriae, viruses and spermatozoon heads (page 8, line 4-10).
  • gradients composed of isotonic solutions made from substances comprising colloidal particles or gradients made with isotonic solutions from iodinated components were developed, allowing in a single centrifugation to separate impurities (slight particles, immature spermatozoa, cells, bacteriae, etc.) of viable spermatozoa and, among these, separate X from Y spermatozoa.
  • impurities light particles, immature spermatozoa, cells, bacteriae, etc.
  • the reduction in the number of centrifugations allows, not changing the precision for separation of spermatozoa X and Y, to reduce the injuries caused to spermatozoa by successive centrifugations and handlings and thus preserve their resistance to the freezing process.
  • the separation of X and Y spermatozoa only occurs in the fourth centrifugation, after three washing centrifugations to take out impurities.
  • the withdrawal of impurities, and the separation of X or Y spermatozoa are achieved in one single centrifugation.
  • Iodixanol is a non-ionic iodinated component developed for studies involving X rays (NOSSEN et al, 1990; BOLSTAD et al, 1991) characterized for being a dimeric form of Nycodenz and having the double of its molecular weight.
  • iodixanol presents advantages over Percoll and Nycodenz, since it is dense enough to produce gradients with higher densities than 1.32 g/ml and lower osmolarity at 300 mOsm (FORD et al, 1994; GRAHAM et al, 1994).
  • centrifugation of semen in iodixanol gradient to separate viable spermatozoa is increasingly used by assisted reproduction clinics for both artificial insemination and in vitro production of embryos (HARRISON, 1997; SMITH et al, 1997; YANG et al, 1998; MAKKAR et al, 1999; McCANN et al, 2000).
  • Iodixanol being an atoxic substance for therapeutical use in humans, its use in spermatozoa sexing protocols was not reported so far.
  • spermatozoa sexing depends on the establishment of a methodology which, is compatible with the freezing process, minimizes the loss of spermatozoa during the process and does not reduce their fertilization capacity. Considering these aspects, it is necessary to evaluate the quality not only concerning sexing accuracy, but also regarding the viability of the spermatozoa after thawing of frozen semen doses enriched with X or Y spermatozoa.
  • results after the insemination of cows with spermatozoa separated by centrifugation in the sexing gradients suggest that there was no interference of this process in fertility in vitro and in vivo.
  • Patent documents regarding this issue will be analysed below on their applicability and functionality: - Patents US 4,474,875 dated October 2, 1984 and US 4,327,177 dated April 27, 1982 (inventor: SHRIMPTON) separate X and Y spermatozoa based on their density difference, as well as the present invention, but use devices which could not be inserted among the equipments used in the semen freezing routine for commercialization and under conditions not allowing to keep sperm viability, such as: a) use of sedimentation for 24 hours in milk gradient; b) use of temperature between -5 and +2 °C; c) pH between 6 and 8; and d) use of means with density between 1.01 and 1.15 g/cm 3 at 0 °C.
  • the design of the device used in the invention is composed of eight columns receiving 480 million spermatozoa each, of which only 21 million spermatozoa (4.5% of the total) are recovered from the bottom of each column. It should be stressed that, commercially, 21 million spermatozoa correspond in average to one semen dose. The purity of separation (about 75% of females) was verified in only 13 pregnancies. However, the author does not report the total number of inseminated females, i. e. how many attempts were made to obtain these pregnancies, which shows that a quality control process allowing the use in the artificial insemination industry was not developed or performed.
  • the method of the patent was developed in such a way that sexed spermatozoa can be submitted to the control of sperm viability used in the routine of the companies producing frozen semen. Furthermore, a process to control the efficiency in separation of X or Y spermatozoa was developed, so that semen doses enriched with the X or Y subpopulations can be commercialized under the information of which should be the deviation in sex ratio after insemination.
  • the candidate method uses equipment and quality control test which can be inserted in the routine of semen freezing for commercialization and has the features to separate X or Y spermatozoa and keep sperm viability, since: a) centrifugation is performed for only 10 to 20 minutes in density gradient composed of culture medium chemically defined to preserve the stability and integrity of spermatozoa membranes; b) temperatures from 4 to 22 °C are used to preserve sperm viability; and c) pH is stabilized at 6.8 to 7.4 associated to the presence of glucose, preventing early capacitation and acrosomal reaction.
  • Examples are the breeds specialized for milk production, in which the maintenance of pregnancies and birth of male animals is one of the factors reducing productivity and increasing production costs. Genetic progress will be maximized in breeding programs to produce milk, in which sex ratio is controlled at the time of artificial insemination to obtain males or females when desired (VAN VLECK et al, 1987; HOHENBOKEN, 1999).
  • each cow can produce one or various heifers (by induction of multiple ovulations) in the first pregnancy and then be slaughtered after weaning said product (TAYLOR, 1985; HOHENBOKEN, 1999).
  • sexed spermatozoa for in vitro production of embryos (IVP) will minimize the cost of progeny test, since it will only allow the birth of calves of the desired sex for zootechnical evaluation (e. g. females for the progeny test of bulls of milk breeds; NICHOLAS & SMITH, 1983).
  • spermatozoa sexing depends on the establishment of a methodology that is compatible with the freezing process, that minimizes the loss of spermatozoa during the process and that does not reduce their fertilization capacity. Considering these aspects, it is necessary to evaluate the quality not only concerning sexing accuracy, but also regarding the viability of the spermatozoa after thawing of the frozen semen doses enriched with X or Y spermatozoa. These aspects are critical to make the production of semen doses enriched with X or Y spermatozoa by the artificial insemination industry feasible at a compatible cost with market reality.
  • IVF in vitro fertilization
  • the current market is bovine herds using Al and having compatible zootechnical control to the use of this technology.
  • the separation performed by the present invention uses the method of centrifugation in density gradient, which is based on the density difference between X or Y chromosome-bearing spermatozoa.
  • the process to select sex of mammal spermatozoa object of the invention comprises the following steps: a) centrifugation in density gradient composed of culture medium chemically defined to preserve the stability and integrity of spermatozoon membranes; b) utilization of temperatures from 4 to 22 °C to preserve sperm viability; c) pH stabilization within the physiological level for each species, preferably about 7.4; d) association with the presence of substances preserving sperm integrity, preferably using glucose to prevent early capacitation and acrosomal reaction, especially in bovines; and e) freezing of sexed spermatozoa in appropriate extender for this purpose, such as the use of Tris-egg yolk, for use in the in vitro production of embryos (IVP) or conventional artificial insemination (Al) (births preferably of masculine or feminine gender) or those made after the hormone treatment of females to synchronize the heat and/or multiple ovulations and subsequent recovery of embryos (most of them of masculine or feminine gender) and later
  • the semen obtained from sires according to routine procedures can be used "in natura” or frozen.
  • Percoll and Iodixanol (OptiPrep) gradients are used in the present invention.
  • Percoll must be used in the form of a stock solution (SS) prepared by diluting 9 parts of Percoll (standard density of 1130 g/ml) in 1 part of culture medium DMEM (1:9, v/v); pH 7.4; 280-290 mOsm/kg H 2 O.
  • SS stock solution
  • DMEM was prepared with ultrapure water, filtered in a membrane with 0.22 ⁇ pores and stocked at 4 °C for periods not longer than 10 days.
  • the pH was adjusted by using 1% glacial acetic acid or 1 N NaOH.
  • the OptiPrep solution is sold ready for use and there is no need to prepare stocks.
  • Crossbred females with ability to produce milk or meat were used for insemination. Two farms were chosen. These females were maintained in pasture and had access to mineral mix put in troughs distributed in fenced areas.
  • Percoll Gradient dilution, in different proportions, of SS (90% Percoll) in DMEM medium, containing 0.3 to 0.6% BSA; pH 7.2-7.5;
  • the discontinuous Percoll gradient was prepared by depositing 0.6 to 3 ml of each isotonic solution, from the denser one to the less dense, in conic tubes of polystyrene with the help of a pipette with adjustable volume.
  • the OptiPrep solution is sold ready for use and there is no need to prepare stocks.
  • OptiPrep gradient dilution, in different proportions, of OptiPrep in DMEM medium containing 0.3-0.6% of BSA, pH 7.2-7.5;
  • the discontinuous OptiPrep gradient was prepared by depositing 0,6 to 2 ml of each isotonic solution, from the denser one to the less dense, in conic tubes of polystyrene with the help of a pipette with adjustable volume. The following gradients were tested:
  • Gradient 18 (G18): three isotonic layers of OptiPrep with densities varying between 1.123 and 1.163 g/ml;
  • Gradient 19 (G19): ten layers of isotonic Percoll solutions with densities varying between 1.034 and 1.070 g/ml; 1.4. Centrifugation and recovery of spermatozoa in the sexing gradients
  • the spermatozoa sediment resulting from the centrifugation in Gradient 14 was recovered, washed in DMEM medium with 250 to 500 x g centrifugation for 10 to 20 minutes at the same temperature of the gradient and re- suspended in a Tris-egg yolk extender.
  • the sediment of spermatozoa resulting from the recentrifugation of Gradient 18 was re-suspended in a Tris-egg yolk extender.
  • Tris-egg yolk extender a Tris-egg yolk extender according to the routine protocol of the company to be used for in vitro production of embryo (IVP) or artificial insemination (Al) using the commercial method.
  • the quality control protocol for the fertility of spermatozoa submitted to sexing was composed by: a) evaluation of motility and power and Thermal
  • TRT Treatment Test
  • Semen doses produced from different gradients and their respective controls were thawed in water bath at 35 °C for 30 seconds. Semen from each dose was deposited in a centrifuge tube. Three washes were made in a TALP culture medium added by BSA-V, centrifuged at 250 g for 10 minutes, aiming at extender removal.
  • spermatozoa were analysed per slide. Despite being possible to identify eight different classes of spermatozoa: 1) live intact (LI); 2) live damaged (LD); 3) live with detached acrosome (LS); 4) live with acrosomal reaction (LR); 5) dead intact (Dl); 6) dead damaged (DD); 7) dead with detached acrosome (DS); 8) dead with acrosome degeneration (DR), in most of the analysed slides only spermatozoa of classes LI, LR and DR were found.
  • Semen doses submitted to centrifugation in density gradient and subsequently frozen were used for the in vitro production of embryos (IVP) using a methodology that eliminates factors that change the kinetics of blastocyst development and consequently sex ratio deviation towards masculine gender embryos.
  • Ovocytes obtained by follicle punction were maturated for 18 to 25 hours and washed three times in TCM-199 medium supplemented with 25 mM
  • HEPES 0.2 mM sodium pyruvale, 0.3% BSA fat acids free and once within IVF medium.
  • PCR Polymerase Chain Reaction
  • oligonucleotide primers amplifying two specific sequences of Y chromosome present in the genomic DNA of male bovines (XY).
  • Primers were synthesized by PROMICRO (Sao Paulo - SP).
  • the two pairs of primers chosen for the specific sequence of Y chromosome were: Primer 1 - 5' - CCT CCC CTT GTT CAA ACG CCC GGA ATC ATT - 3'
  • Primer 1 amplifies a 210 pb sequence for DNA of male bovines (BONDIOLI et al, 1989) and primer 2 amplifies a 250 pb sequence in males (SCHWERIN et al, 1991).
  • Each embryo was put in a tube with 0.2 ml capacity containing 10 microliter of ultrapure auloclaved water.
  • the microlube was immersed in liquid nitrogen for 20-30 seconds and stored at -20 °C until the moment of PCR.
  • Proteinase K (16 mg/ml) was added to each tube containing an embryo and incubated for 60 minutes at 37 °C. Subsequently, Proteinase was denatured at 98 °C for 10 minutes.
  • the contents of each tube were divided into two samples (A and B) which were subsequently submitted to PCR.
  • each procedure had a negative control (with no DNA template), a masculine control (DNA of male bovine), a feminine control (DNA of female bovine), Reaction conditions were as follows: 75 mM Tris-HCl, pH 9; buffer with 50 mM KCI, 2,0 mM MgCI 2 , 20 mM (NH 4 ) 2 SO 4 ; 1 U of Taq DNA Polimerase and 200 mM of dNTP.
  • the total volume in the reaction mixture per tube was 30 microliter. In the tubes containing sample A, 10 pmol of primers 1 and 3 were added. In those containing sample B, 10 pmol of primer 2 were added.
  • the amplification was performed in a Thermocycler PTC -
  • Samples B were submitted to 38 x 95 °C cycles for 60 seconds, 58 °C for 60 seconds and
  • Foetal sex was diagnosed by ultrasonography between 60 to 90 days after the artificial insemination, by using the Pie Medical Scanner 200 equipment, 5 MHz linear transducer.
  • the available methods are the Barr corpuscle analysis (or F-body), whose reliability is questioned, or hybridization with male-specific probe, by using the FISH method (KING, 1984), which execution is troublesome, especially for a large number of samples. Furthermore, it is possible to verify the separation of X or Y spermatozoa using the flow cytometer, but this method is available in few laboratories worldwide and for a very high cost.
  • Amelogenin constitutes the most abundant class of proteins, extremely important in the development of tooth enamel.
  • the amelogenin gene is located, only in bovines and humans, in the X and Y chromosome (LAU et al, 1989). In bovines, it is composed of six exons and five introns (GIBSON et al, 1991) and presents two different amelogenin transcriptions (class I and class II) which are products of genes located in the X and Y chromosome, respectively (GIBSON et al, 1991 , 1992).
  • a sequence of the amelogenin gene is amplified, it is possible to distinguish males from females, i. e. X and Y chromosomes, while using only a pair of primers, so that, during an amplification of said sequence, two very different fragments are observed, one from the amplification of the class I gene and the other from the class II gene.
  • the amplification product from the class I gene (X chromosome) and class II (Y chromosome) is distinguished by a 67 pb deletion in the class II amelogenin gene (Gibson et al, 1991). Description of the method to quantify X and Y chromosomes by PCR
  • DNA extraction from spermatozoa was made by the traditional method with phenol chloroform as per the protocol described by SAMBROOK et al (2001).
  • Semen was obtained from bulls in a collection regime in a commercial center and submitted to freezing according to routine of the company. Semen samples designated as sexed were previously centrifuged in density gradient with the purpose to select X spermatozoa. Samples of semen from the same ejaculation, but not submitted to X separation before freezing, were used as control.
  • Amplification products were analysed by the PCR real time equipment according to manufacturer recommendation.
  • the frequency of X spermatozoa over Y spermatozoa is inferred by the number of copies of amplified fragments originated from the respective chromosomes: a) fragment with 217 base pairs, originated from the amplification of class II amelogenin gene (located in Y chromosome); and a) fragment with 280 base pairs, originated from the amplification of class I gene (located in X chromosome).
  • IVP in vitro production of embryos
  • the method of the invention can be used in sexing of spermatozoa in various species of mammals, including humans; - the investment in the method of this patent is significantly lower than that for the production of spermatozoa sexed by flow cytometry (cost of US$ 280,000), since the refrigerated centrifuge costs in average US$ 15,000 and produces about 30 times more sexed semen doses than the flow cytometer in the same period of time and with the same efficiency; and
  • the present invention can produce 8 to 15 million spermatozoa from a subpopulation (X or Y) by gradient tube, with 20 to 30 minutes of centrifugation, depending on the used gradient. Therefore, the volume of frozen semen produced depends on the centrifuge capacity and its amplification depends on the purchase of other centrifuges. Thus, as opposed to what occurs with the flow cytometry method, in the proposed invention, the quantity of semen doses produced may be largely increased with lower investment (purchase of a centrifuge) and with no accuracy loss of the separated spermatozoa.

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Abstract

L'invention concerne un procédé de sélection du sexe d'un spermatozoïde mammaire et un procédé de contrôle de la qualité des doses de sperme congelé avec sexe. L'invention concerne la production par centrifugation en gradient de densité, dans des sociétés spécialisées dans la production et la commercialisation de sperme congelé, de doses de sperme enrichi avec un spermatozoïde porteur du chromosome X ou Y, n'affectant pas la capacité de fécondation desdits spermatozoïdes. D'après l'invention, des échantillons de spermatozoïdes contenant à la fois des spermatozoïdes X et Y peuvent être séparés afin d'obtenir des sous-populations enrichies de spermatozoïdes X ou Y, qui sont sensiblement purs par rapport aux spermatozoïdes souhaités et sensiblement exempts de l'autre type de spermatozoïde.
PCT/BR2004/000009 2003-02-17 2004-02-11 Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe WO2004072220A2 (fr)

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BR0300604A BR0300604A (pt) 2003-02-17 2003-02-17 Processo de seleção do sexo de espermatozóides mamìferos e método de controle de qualidade de doses de sêmen sexado congelado
BRPI0300604-2 2003-02-17

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WO2004072220A3 WO2004072220A3 (fr) 2004-10-07

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1695059A2 (fr) * 2003-11-26 2006-08-30 VLP Watertown Limited Partnership Methode de selection du sexe par les spermatozoides
EP2121901A2 (fr) * 2007-01-16 2009-11-25 Texas Tech University System Procede et appareil de selection du sexe
ITTO20100651A1 (it) * 2010-07-28 2012-01-29 Ist Sperimentale It Lazzaro Spallanza Procedimento ed apparecchiatura per la caratterizzazione e la separazione di spermatozoi con sensori micrometrici a leva sospesa
CN104962513A (zh) * 2015-06-30 2015-10-07 阜阳师范学院 狐狸x精子分离方法
CN109355250A (zh) * 2018-11-07 2019-02-19 临泉县欣达生态羊业有限公司 安徽白山羊的y精子分离方法
CN109370981A (zh) * 2018-11-07 2019-02-22 临泉县欣达生态羊业有限公司 安徽白山羊的x精子分离方法
JP2021519925A (ja) * 2018-03-30 2021-08-12 テキサス・テック・ユニバーシティー・システム 非侵襲的な胚の性判別のためのシステムおよび方法

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WO2002052244A2 (fr) * 2000-12-22 2002-07-04 Amersham Biosciences Ab Separation de spermatozoides x et y

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US3894529A (en) * 1969-04-10 1975-07-15 Bio Controls Inc Method and means for controlling the sex of mammalian offspring and product therefor
WO2002052244A2 (fr) * 2000-12-22 2002-07-04 Amersham Biosciences Ab Separation de spermatozoides x et y

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1695059A2 (fr) * 2003-11-26 2006-08-30 VLP Watertown Limited Partnership Methode de selection du sexe par les spermatozoides
EP1695059A4 (fr) * 2003-11-26 2007-11-14 Vlp Watertown Ltd Partnership Methode de selection du sexe par les spermatozoides
EP2121901A2 (fr) * 2007-01-16 2009-11-25 Texas Tech University System Procede et appareil de selection du sexe
EP2121901A4 (fr) * 2007-01-16 2010-03-24 Univ Texas Tech System Procede et appareil de selection du sexe
US9157063B2 (en) 2007-01-16 2015-10-13 Texas Tech University System Method and apparatus for gender selection based on pH
ITTO20100651A1 (it) * 2010-07-28 2012-01-29 Ist Sperimentale It Lazzaro Spallanza Procedimento ed apparecchiatura per la caratterizzazione e la separazione di spermatozoi con sensori micrometrici a leva sospesa
WO2012014142A1 (fr) 2010-07-28 2012-02-02 Istituto Sperimentale Italiano "Lazzaro Spallanzani" Procédé et appareil pour la caractérisation et la séparation de spermatozoïdes par des capteurs micrométriques à levier suspendu
CN104962513A (zh) * 2015-06-30 2015-10-07 阜阳师范学院 狐狸x精子分离方法
JP2021519925A (ja) * 2018-03-30 2021-08-12 テキサス・テック・ユニバーシティー・システム 非侵襲的な胚の性判別のためのシステムおよび方法
JP7367991B2 (ja) 2018-03-30 2023-10-24 テキサス・テック・ユニバーシティー・システム 非侵襲的な胚の性判別のためのシステムおよび方法
CN109355250A (zh) * 2018-11-07 2019-02-19 临泉县欣达生态羊业有限公司 安徽白山羊的y精子分离方法
CN109370981A (zh) * 2018-11-07 2019-02-22 临泉县欣达生态羊业有限公司 安徽白山羊的x精子分离方法

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