EP2921134B1 - System zum Sammeln von tierischem Samen, und Sammelverfahren von tierischem Samen mit diesem System - Google Patents

System zum Sammeln von tierischem Samen, und Sammelverfahren von tierischem Samen mit diesem System Download PDF

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Publication number
EP2921134B1
EP2921134B1 EP15159227.6A EP15159227A EP2921134B1 EP 2921134 B1 EP2921134 B1 EP 2921134B1 EP 15159227 A EP15159227 A EP 15159227A EP 2921134 B1 EP2921134 B1 EP 2921134B1
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container
semen
dilution
collecting device
collecting
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French (fr)
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EP2921134A1 (de
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Eric Schmitt
Agnès CAMUS
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IMV Technologies SA
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IMV Technologies SA
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    • 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
    • A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
    • A61D19/021—Apparatus for collecting seminal fluids; Artificial vaginas

Definitions

  • the present invention relates to the general field of animal semen collection, including bovine semen, and more specifically to the systems for collecting this seed and the methods of collecting this seed using such collection systems.
  • the invention also relates to such systems comprising animal seed dilution media, especially bovine semen, cryoprotectants (or cryopreservatives).
  • the animal semen is analyzed in a specific room often called laboratory, then it is pre-diluted (first dilution) and then diluted (second dilution), before being preserved, for example by cryogenics, in unitary packages called glitter.
  • This first dilution can be done by mixing the collected seed with a predetermined dilution medium.
  • a predetermined dilution medium This may be, for example, a buffer solution comprising substances for preserving / protecting sperm membranes of the seed and / or antibiotics.
  • bovine seminal plasma contains certain proteins that bind to sperm membranes just after the ejaculation of the animal, which favors acrosomal reactions ( "Snapshots") and is therefore harmful to the conservation of spermatozoa.
  • a medium comprising, in addition to a tris-type buffer (for trishydroxymethylaminomethane or tris (2-amino-2- (hydroxymethyl) propane-1,3-diol) / citric acid / fructose, the composition of which is familiar to humans.
  • a tris-type buffer for trishydroxymethylaminomethane or tris (2-amino-2- (hydroxymethyl) propane-1,3-diol) / citric acid / fructose
  • a tris-type buffer for trishydroxymethylaminomethane or tris (2-amino-2- (hydroxymethyl) propane-1,3-diol) / citric acid / fructose
  • the US patent US 3,401,696 describes a system having a funnel for collecting animal semen, comprising a cylindrical body forming an artificial vagina, an end neck formed in one piece with the artificial vagina, with or without an intermediate portion, and a container in which is introduces the neck for the flow of the seed via a flow chamber formed in this neck.
  • the neck has a cylindrical wall and flexible fins radially projecting from said wall and configured to come into contact with an inner wall of the container for their assembly by force.
  • the British patent GB 827 895 describes an artificial vagina having a flexible receiving portion extended by a rigid sleeve, and a closure head screwed directly onto one end of the sleeve.
  • the head comprises a hollow longitudinal cylindrical wall which is introduced into the rigid sleeve when the head is screwed onto this sleeve.
  • the vagina further comprises a removable container received in the hollow wall of the head and therefore in the rigid sleeve, to directly receive the collected seed. This container may have on the side of its opening a conical skirt to prevent the collected seed from escaping the container.
  • WO 01/64936 describes a system and method for collecting animal semen, and also for preserving the seed through the use of a specific medium.
  • the system is provided with an artificial vagina, a Y-fitting removably attached to one end of the vagina, and two separate containers each detachably attached to a branch of the Y-fitting.
  • the containers have a diameter internal slightly greater than the outer diameter of the branches of the Y-connection for their attachment.
  • the container here initially has a volume of the specific medium for the conservation of the seed.
  • the invention relates to a system for improving the performance of known systems for collecting animal semen, particularly bovine semen, in a simple, convenient and economical manner.
  • the invention thus provides, in a first aspect, a system for collecting animal semen, particularly bovine semen, comprising a container configured to be mounted on an end of an artificial vagina separate from said collection system and having a configured internal space for collecting said seed, said container being further configured to include, prior to collecting said seed, a medium of first dilution of said seed and said system further comprising a collection device separate from said artificial vagina and said container and configured to be at least partially introduced into said container and to be fixed on said artificial vagina, said collection device being interposed between said artificial vagina and said container, said collection device comprising a flow channel of said seed extending into said internal space of said container and an outlet of said seed emerging in said internal space of said container; and said system being configured to prevent, at least substantially, said first dilution medium from proceeding in said flow channel of said collection device and from escaping said container.
  • the collection system according to the invention makes it possible to directly and immediately put the animal semen into contact with the medium of first dilution during the harvest of the seed, before proceeding to the analyzes.
  • the collection system according to the invention is configured so that contacting is performed without risk that the medium of first dilution can not go up (at least for the most part) to the artificial vagina where it would not allow have its function of first dilution of the seed.
  • the system according to the invention is thus advantageous in that it allows its user, often called the harvester, to easily mount the artificial vagina together with this system on the penis of the animal, in a position where the artificial vagina and therefore the collection system are returned (upside down); and this, without risk that the medium of first dilution can not escape from the container.
  • the system according to the invention is furthermore advantageous to use during the collection of bovine semen where the animal can be particularly brutal and inflict numerous shakes on the collection system.
  • Liposomes are artificial vesicles (or capsules) formed by concentric lipid bilayers, enclosing an aqueous volume. They are obtained from amphiphilic lipids, and especially from phospholipids having a polar (hydrophilic) "head” generally formed of a glycerol-3 phosphate residue esterified by a polar molecule, and of two hydrophobic "tails" (which consist of chains of two fatty acids).
  • Phosphatidylcholine (or lecithin or 1-oleyl-2-stearyl-phosphatidylcholine) is a phospholipid obtained from egg yolk by a reproducible method.
  • the process for obtaining liposomes is generally based on extrusion with a mini-extruder such as that of Avanti Polar Lipids; but any other method known to those skilled in the art is usable.
  • the vesicles formed are of spheroidal structure, generally of average size less than 200 nm in diameter.
  • Low density lipoproteins mainly comprise the following four lipids: sphingomyelin, cholesterol, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (16: 0-22: 6 PC; CAS NO.59403-54-2 ) and 1- (1Z-octadecyl) -2-docosahexaneoyl-sn-glycero-3-phosphocholine (P-PC) plasmalogen.
  • the process according to the invention comprises the steps of rinsing said collection device after collection of said seed and then removing the collection device from said container; and / or it comprises the steps of providing a device for diluting said seed and then mounting said dilution device on said container, in place of said collection device after removal of the latter from said container once the collection is made; and / or it comprises the steps of introducing a determined volume of a second dilution medium of said seed into said internal space of said container and into a space inside said dilution device, of fixing a lid of said dilution device on an end edge of the latter and then perform a second dilution of said seed.
  • the figure 1 illustrates an animal semen collection system 1, here in a rest configuration. This is a system 1 for collecting bovine semen.
  • This system 1 comprises a container 3 configured to collect the bovine semen, and a collection device 4 attached to both the container 3 and a cone of an artificial vagina 2 configured to be mounted on the penis of the animal (not shown) to stimulate it.
  • the cone of the artificial vagina 2 which is here only partially represented, here has a substantially frustoconical shape, the narrowest portion of which extends towards an end 9 of this cone of artificial vagina 2.
  • the container 3 is here a tube of generally cylindrical shape, having a side wall 10 extending between an end 11 closed by a bottom wall 14 and an open end 12, opposite to the closed end 11.
  • the side wall 10 of the tube 3 has an inner face 16 defining an internal space 15 to the tube 3, this internal space 15 being designed to collect, before collection, a predetermined medium of first dilution 5, and during collection, to collect the seed. animal.
  • the bottom wall 14 formed in the internal space 15 has a V shape, in the manner of a centrifugation tube and the tube 3 further comprises a recess 13 formed between the closed end 11 and the bottom wall 14 .
  • the tube 3 is here made of plastic material molded in one piece and its internal space 15 here has a volume of about 30 mL, and more generally a volume of about 20 mL to 200 mL.
  • the collecting device 4 extends substantially longitudinally between a first end 20 and a second end 21 opposite the first end 20.
  • the collecting device 4 comprises a first lateral wall 71 of generally substantially cylindrical shape, and a second lateral wall 72 extending in the extension of the first side wall 71 and also having a generally substantially cylindrical shape.
  • the first side wall 71 has a recess 29 forming an interior space and the second side wall 72 forms a flow channel 23 of the seed, which channel 23 is narrowed with respect to the interior space of the first side wall 71.
  • the recess 29 is in fluid communication with the flow channel 23.
  • the first side wall 71 has, at the first end 20, a first portion 22 of generally cylindrical shape and configured to be fitted with the end 9 of the cone of the artificial vagina 2.
  • the first side wall 71 has a second portion 54 of generally cylindrical shape extending from the first portion 22 and configured to be fitted with a side wall 10 of the tube 3.
  • the second portion 54 is connected to the first portion 22 by a shoulder 28 formed in the first side wall 71.
  • the shoulder 28 is configured so that the second portion 54 has an outer diameter greater than the outer diameter of the first portion 22.
  • the first side wall 71 has a third portion 55 extending into said internal space 15 of the container 3, from the second portion 54.
  • This third portion 55 has a generally frustoconical outer face and the collection device 4 is provided with a vent 17 formed in this third portion 55.
  • the vent 17 comprises a first chamber having a first internal diameter and opening out of the first side wall 71 of the collection device 4, and a second chamber having a second internal diameter, much smaller than the first internal diameter of the first chamber. and opening into the recess 29 formed in the first side wall 71 of the collecting device 4.
  • the vent 17 is thus configured to create fluid communication for air, which communication is reversible but facilitated from the second chamber to the first chamber due to the respective internal diameters (in other words facilitated by the recess 29 of the collection device 4 to the internal space 15 of the tube 3).
  • the vent 17 is also configured to prohibit fluid communication for animal semen, particularly from the first chamber to the second chamber (in other words prohibited from the inner space 15 of the tube 3 to the recess 29 of the device collection 4).
  • the second side wall 72 has a first portion 24 of generally cylindrical shape, connected to the third portion 55 of the first side wall 71 through an intermediate portion 27, and a tip 25 extending from the first portion 24 to the second end 21.
  • the second portion 24 is thus interposed between the intermediate portion 27 and the endpiece 25.
  • the tip 25 is frustoconical with its narrowest portion which is at the end 21, where a discharge port 26 of the seed is further provided.
  • the second portion 24 and the frustoconical tip form the flow channel 23 of the animal semen.
  • the intermediate portion 27 is configured to form an internal shoulder in the recess 29.
  • the collection device 4 is made of plastic material molded in one piece, and in particular here in a thermoplastic polymer, especially polyacetal or polyoxymethylene (POM).
  • a thermoplastic polymer especially polyacetal or polyoxymethylene (POM).
  • the collection device 4 is configured so that the flow channel 23 has an inner face 30 having a surface tension facilitating the flow of animal semen.
  • the first portion 24 has an outer diameter D 1 here approximately equal to 11 mm and an inner diameter D 2 here approximately equal to 8.6 mm.
  • the frustoconical tip 25 has meanwhile a minimum internal diameter D 3 here approximately equal to 5 mm.
  • This minimum internal diameter D 3 is representative of the smallest passage for the animal seed in the flow channel 23, at the discharge orifice 26 of this seed ( figure 2 ).
  • the outside diameter D 1 is between about 5 mm and about 17 mm
  • the inside diameter D 2 is between about 8 mm and about 20 mm
  • the minimum inside diameter D 3 is between about 5 mm and about 15 mm. mm.
  • the second cylindrical portion 54 of the first side wall 71 of the collection device 4 is partially force-fitted into the tube 3 so that the flow channel 23 of this collection device 4 extends into the internal space 15 of the tube 3 towards the bottom wall 14 of the latter.
  • this second cylindrical portion 54 has an outer face 31 which comes into contact with the inner face 16 of the side wall 10 of the tube 3.
  • the collection device 4 is fitted into the tube 3 in such a way that the frustoconical tip 25 of this collection device 4 and more precisely its discharge orifice 26, is opposite the first dilution medium 5 situated at the bottom of the tube 3, when the system 1 is in its rest configuration ( figure 1 ).
  • the tube 3 and the collection device 4 are configured so that the discharge orifice 26 and the high level of the first dilution medium 5 are separated by a distance denoted H 1 , here approximately equal to 4.7 mm, when the assembly formed of the collection device 4 and the tube 3 is in the vertical position. More generally, the distance H 1 is between about 2 mm and about 35 mm.
  • the cone of the artificial vagina 2 has a frustoconical portion 58 and a cylindrical portion 57 extending from the frustoconical portion 58, to the end 9 of the cone of the artificial vagina 2.
  • the cylindrical portion 57 is provided with a free edge 59 in the form of a bead.
  • the cylindrical portion 57 is partially force-fitted on the first portion 22 of the first side wall 71 of the collection device 4, at its first end 22, the free edge 59 in a bead bearing against the shoulder 28 formed on this first side wall 71.
  • the collection system 1 which is fixed via its collection device 4 to the cone of the artificial vagina 2, is in the extension of the latter and has a general inclination similar to that of the artificial vagina.
  • the system 1 When the harvester introduces the penis of the animal into the artificial vagina 2, the system 1 is disposed with an inclination for example of about 10 degrees to about 30 degrees above a horizontal plane (upside down or downward). other terms exhaust port 26 of the collection device 4 at the top). This at least substantially overturned position of the system 1 can be maintained until the ejaculation of the animal.
  • the arrangement of the collection device 4 and the tube 3 containing the first dilution medium 5 therefore advantageously allows, before collection during the assembly of the artificial vagina 2 and the system 1 on the penis of the animal, where the system can be returned (head down), and also during collection, to retain the medium of first dilution 5 in the tube 3 ( figure 2 ).
  • the collection device 4 and the tube 3 containing the first dilution medium 5 are configured to prevent, at least for the most part, this first dilution medium from running in the flow channel 23 of the collection device. 4 during assembly and during the collection of animal semen.
  • the system 1 changes position until it is inclined at about 10 degrees to about 30 degrees below the horizontal plane at the end of the collection.
  • the animal semen flows into the flow channel 23 of the collection device 4 by gravity until leaving this channel 23 through the discharge port 26 to immediately come into contact with the first dilution medium 5.
  • This arrangement of the collection device 4 and the tube 3 containing the first dilution medium 5 thus also advantageously allows just after the collection of the seed which is evacuated by gravity from the flow channel 23 through the orifice evacuation 26, to put this seed immediately in contact with the medium of first dilution 5.
  • the collection system 1 then comprises, in place of the collection device 4, a dilution device 6 of the animal seed having a general funnel shape having an open first end 45 facing the bottom of the tube 3 and a second open end 44 opposite the first open end 45.
  • This dilution device 6 is configured to be mounted on the container 3 after withdrawal of the latter from the container 3 after the collection of animal semen.
  • this mixture of seed pre-dilution 70 has a volume of from about 5 mL to about 15 mL for an initial volume of the first dilution medium of from about 2.5 mL to about 6 mL.
  • the initial volume of the first dilution medium may range from about 2 mL to about 60 mL so that the volume of the seed pre-dilution mixture may be from about 4 mL to about 120 mL.
  • the initial volume of the first dilution medium 5, also known as the predilution medium is preferably substantially similar to the volume of seed collected or collected.
  • the volume of such a medium can be about 6 mL whereas for young bulls, it can only be about 3 mL.
  • these volumes are a function of the animal to which the seed is to be collected and possibly the tube 3, the collection device 4 fixed on the tube 3 and the cone of the artificial vagina 2 fixed on the collection device 4.
  • these volumes may for example vary by a few milliliters for the bovine species depending on the young genomic bulls or service bulls. These variations may be more important for other species.
  • the dilution device 6 in the form of funnel has a side wall defining an internal space 44 to the dilution device 6 and having a first cylindrical portion 40 at its second open end 44, a second portion 41 substantially frustoconical and which s' extends from the first cylindrical portion 40 towards the first open end 45, a third portion 42 also substantially frustoconical and extending from the second frustoconical portion 41 towards the first open end 45, as well as a substantially cylindrical end portion 43 which extends from the third frustoconical portion 42 to the first open end 45.
  • the arrangement of the different portions of the dilution device 6 makes it possible to optimize its volume with respect to its height, as well as the flow of the fluids that it is configured to receive.
  • the end portion 43 is configured to be press fit in the side wall 10 of the tube 3 at its open end 12.
  • this end portion 43 has an outer face 47 which is configured to come into contact with the inner face 16 of the side wall 10 of the tube 3.
  • the first cylindrical portion 40 of the dilution device 6 here has an inside diameter much greater than that of the end portion 43 which is located opposite the first cylindrical portion 40.
  • the side wall of the dilution device 6 further comprises, at its second open end 44 and therefore opposite its end portion 43, an end edge 48 connected to the first cylindrical portion 40.
  • This end edge 48 is configured to form a protruding shoulder of the side wall of the dilution device 6.
  • the dilution device 6 further includes a cover 49 configured to be attached to the end edge 48 of the side wall.
  • the cover 49 has a circular shape so as to close the opening formed in the dilution device 6 at its second open end 44.
  • the cover 49 has a recess 52 at the bottom of which is formed a sealed wall 53 which is disposed in an internal space 46 of the dilution device 6 when the cover 49 is fixed on the latter, to ensure the seal between the cover 49 and dilution device 6.
  • the cover 49 further comprises a circular flange 50 configured to snap onto the end edge 48 of the side wall of the dilution device 6.
  • the circular flange 50 of the cover 49 is configured to leave a space between the latter and an outer face of the side wall of the device. dilution 6, under the end edge 48, to allow removal of the cover 49 of the side wall.
  • the side wall and the lid of the dilution device 6 are here made of plastic.
  • the collection system 1 formed by the tube 3 and the dilution device 6 fitted on this tube 3, as illustrated in FIGS. Figures 3 and 4 has a volume here equal to about 300 mL. More generally, the collection system 1 has a volume of between about 100 ml and about 1000 ml.
  • the collection system 1 is shown before the second dilution of the animal semen and more precisely the mixture 70 while on the figure 4 a diluted mixture of the animal seed 80 filled the volume of the collection system 1 illustrated.
  • the method of collecting animal semen comprises the step of providing the tube 3 as described above and then introducing into the internal space 15 of the latter a determined volume of the first dilution medium 5.
  • this determined volume of the first dilution medium may be in the range [2.5 mL; 6 mL].
  • this medium of first dilution may comprise a buffer solution composed for example of substances for the preservation / protection of the sperm membranes of the animal semen and / or antibiotics.
  • the method of collecting animal semen further comprises the step of providing a collection device 4 as described above and to introduce the latter at least partially into the tube 3 so that the flow channel 23 extends into the internal space 15 of the tube 3 and the discharge orifice 26 opens into this internal space 15.
  • the collection device 4 is fitted into the tube 3.
  • the discharge port 26 is facing the first dilution medium 5 and at a determined distance.
  • the method of collecting animal semen further comprises the step of providing an artificial vagina 2 as described above and fixing the collection device 4 on the cone of the artificial vagina 2 so as to interpose this collection device 4 between the cone of the artificial vagina 2 and the tube 3.
  • the collection system 1 is then in the extension of the artificial vagina 2 and is therefore inclined with respect to a horizontal plane (above or below, see above).
  • the collection system 1 and in particular the collection device 4 and the tube 3, are configured to prevent at least a major part of the first dilution medium from running in the flow channel of the collection device 4 during the collection of animal semen.
  • the method for collecting animal semen furthermore comprises, once the ejaculation of the animal has been completed and the seed has passed into the tube 3 and thus in contact with the pre-dilution medium 5, the step of removing the collection system. 1 of the cone of the artificial vagina 2, removing the cylindrical portion 57 of the cone of the first portion 22 of the first side wall 71 of the collection device 4.
  • the collection process then comprises the step of rinsing the collection device 4 after collection of the seed, for example by means of a second dilution medium, and then removing this collection device 4 from the tube 3.
  • the method for collecting animal semen also comprises the steps of providing a device 6 for diluting the seed as described above and then mounting this dilution device 6 on the tube 3, here by a force fitting, instead of the collection device 4 after removing the latter from the tube 3 once the collection.
  • the seed collection process comprises the steps of introducing a determined volume of a medium of second dilution of the seed into the internal space 15 of the tube 3 and into the internal space 46 of the dilution device 6, to fix the cover 49 on the end edge 48 of the dilution device 6 and then perform a second dilution of the animal semen and more specifically a dilution of the pre-diluted mixture of seed 70 to obtain the diluted mixture of this seed 80.
  • the method may further comprise the steps of taking a plurality of determined volumes of the diluted medium 80, filling flakes each with a determined volume of this diluted medium 80 and cryogenizing these filled flakes to maintain these mixing volumes. .
  • the first immediate dilution of the seed with the first dilution medium 5 directly in the tube 3, the rinsing of the collection device 4 mounted on the tube 3 and the second dilution by means of the dilution device 6 mounted on the tube 3 instead of the collection device 4 after removal from the tube 3 when the collection of semen is performed, allows both to simplify the animal semen collection process by minimizing the number of steps and to significantly limit the losses of volume of animal semen, pre-diluted mixture of this seed and diluted mixture of this seed; in comparison with the seed collection methods of the prior art where a plurality of transfer steps inducing volume losses are required.
  • the collection system 1 while being particularly simple, convenient and economical, thus makes the collection process itself particularly simple, convenient and economical and allows the filling of a higher number of flakes for the preservation of the animal semen collected.
  • the collection system 101 is here illustrated in the same configuration as the collection system 1 of the figure 2 .
  • the tube 103 here has a capacity greater than the tube 3 illustrated on the Figures 1 to 4 .
  • This tube 103 has here for example a volume of about 50 ml.
  • This tube 103 furthermore has a determined volume of a first dilution medium 105, for example between about 3 ml and about 15 ml.
  • This first dilution medium 105 is located at the bottom of the tube, against a V-shaped wall, at the closed end 111 of the tube 103.
  • the tube 103 further comprises a lid 163 pierced with an orifice 165, here central, and the lid 163 has a circular flap 164 which is applied on an outer face 166 of the side wall of the tube 103.
  • the collection device 104 of the collection system 101 has a cylindrical general shape, similar to that of the tube 103, having a side wall 160 extending between a first end 120 and a second end 121 opposite the first end 120. .
  • the collection device 104 here has a volume of about 15 ml.
  • the collection device 104 is further provided with a cover 161 mounted at its first end 120 and having a circular flap 162 provided to be applied on an outer face 131 of the side wall 160.
  • the collection device 104 is configured to be introduced into the internal space of the tube 103, on the side of the second end 121 of the collection device 104, through the orifice 165 formed in the lid 163 of the tube 103.
  • the opening 165 here has a diameter of approximately 15 mm and the collection device 104 is introduced into the tube 103 of a length here defined H 2 , between about 3 mm and about 10 mm.
  • the collection device 104 also has an outlet opening 126 formed in a substantially frustoconical bottom wall of this collection device 104.
  • the discharge orifice 126 here has a diameter of between about 3 mm and 10 mm.
  • this collection device 104 is configured to be attached to the cone of an artificial vagina.
  • the cover 161 may be removed from the collection device 104 or the cover 161 may include a passage opening for the passage of the end of the cone of the artificial vagina and / or mounting members thereon.
  • first dilution media 5 cryopreservatives, whose behavior in the presence of bovine semen are illustrated by the following examples will be described.
  • figures 6 , 7 and 8 Such a dilution medium has a volume of between about 2.5 ml and about 6 ml.
  • Buffered saline (White or "Bl") included tris (2-amino-2- (hydroxymethyl) propane-1,3-diol (173.0 mMol), citric acid monohydrate (70.0 mMol), fructose (56.0 mMol) and glycerol (6.4% vol / vol) The pH was adjusted to 6.2 and the osmolarity was approximately 1300.0 mOsm.
  • the low-density lipoprotein (LDL) cryoprotectant medium was extracted from egg yolk according to the protocol described by Moussa et al. (Theriogenology 57 (6): 1695-1706 ). After extraction, the resulting amount of this LDL sample was diluted in buffered saline (100 mL) resulting in the "comparable C1" solution: "Comp”. This solution, which is according to the invention, has been compared with the solutions according to the invention comprising liposomes.
  • the proteins, assayed by BCA method using a Pierce micro-kit from Thermoscientific, were present in the LDL sample in an amount of 8 g / L.
  • Liposomes were produced from an egg phospholipid extract in buffered saline by extrusion using a mini extruder from Avanti Polar Lipids. They were composed of 73% phosphatidylcholine, 11% phosphadiethanolamine and various minor compounds (neutral lipids, triglycerides and sphingomyelin). The extracts were hydrated in buffered saline overnight at lipid concentrations of 10 g / L for solution C1, respectively; 20 g / L for solution C2; 40 g / L for solution C3; and 60 g / L for solution C4. The solutions were then sonicated for 15 minutes (per Elmasonic S 30 H from Elma at 275 W power). Finally, the dispersions were extruded (20 passages per 100 nm membrane).
  • a characterization of the semen from Prim'Hosltein cattle was carried out by a preliminary protein determination.
  • the seed was immediately centrifuged twice at 10,000 x g to recover the seminal plasma in the supernatant.
  • the amount of protein in this bovine semen sample was 88.5 mg / mL. It was measured by BCA method (using a Pierce micro-kit from Thermoscientific).
  • sperm motility analyzes were performed on a CASA system for "computer assisted semen analysis" in English terminology, for example with a device known as IVOS designed and developed by Hamitlon Thorne. So-called intact acrosome viability assays of spermatozoa were performed by cytometry, for example with a device known as EasyCyte designed and developed by the applicant company and the company Millipore.
  • the figure 6 is a schematic diagram illustrating the percentage of motile sperm (MS) obtained after contacting each solution with the seed (the rate of 100% corresponding to the total sperm population).
  • the figure 7 is a schematic diagram illustrating the percentage of progressive spermatozoa (PS) obtained after contacting each of the solutions with the seed.
  • the figure 8 is a schematic diagram illustrating the percentage of viable spermatozoa with intact acrosomes (V / A (+)) obtained after contacting each of the solutions with the seed.
  • cryoprotective medium in particular comprising LDLs, can be used in the system according to the invention.
  • the latter comprises a flow channel formed for example by a cylindrical portion in which are formed walls to form a baffled system in the flow channel; thus preventing the first dilution medium from running in this flow channel.
  • the latter has a flow channel at the end of which is formed a valve allowing the passage of animal semen to the internal space of the container and preventing at least major part of the flow of the first dilution medium in the flow channel of the collection device.
  • the container has a rectangular shape and the collection device is formed by a plate at least partially introduced into the container and extending therethrough; with this plate which is provided with at least one orifice for the evacuation of the seed in the internal space of the container when this seed travels on the plate.

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  • Investigating Or Analysing Biological Materials (AREA)

Claims (23)

  1. System zum Sammeln von Tiersperma, insbesondere von Rindersperma, enthaltend einen Behälter (3), der dazu ausgelegt ist, um an einem Ende (9) einer künstlichen Vagina (2) angebracht zu werden, die vom Sammelsystem (1) separat vorliegt und einen Innenraum (15) aufweist, der dazu ausgelegt ist, um das Sperma aufzunehmen, wobei der Behälter (3) ferner dazu ausgelegt ist, um vor dem Aufnehmen des Spermas ein erstes Verdünnungsmedium (5) für das Sperma zu enthalten, und wobei das System (1) ferner eine Sammelvorrichtung (4) enthält, die von der künstlichen Vagina (2) und dem Behälter (3) separat vorliegt und dazu ausgelegt ist, zumindest teilweise in den Behälter (3) eingeführt zu werden, um an dem Behälter (3) befestigt zu werden und um an der künstlichen Vagina (2) befestigt zu werden, wobei die Sammelvorrichtung (4) zwischen der künstlichen Vagina (2) und dem Behälter (3) eingesetzt ist, wobei die Sammelvorrichtung (4) einen Strömungskanal (23) für das Sperma enthält, der sich in den Innenraum (15) des Behälters (3) erstreckt, sowie eine Spermaauslassöffnung (26), die in den Innenraum (15) des Behälters (3) mündet; wobei das System (1) dazu ausgelegt ist, um zumindest größtenteils zu verhindern, dass das erste Verdünnungsmedium (5) in den Strömungskanal (23) der Sammelvorrichtung (4) strömt und aus dem Behälter (3) entweicht.
  2. System nach Anspruch 1,
    dadurch gekennzeichnet, dass
    die Sammelvorrichtung (4) eine erste Seitenwand (70) mit einem ersten Abschnitt (22) enthält, der eine Zylinderform hat und dazu ausgelegt ist, mit dem Ende (9) der künstlichen Vagina (2) zusammengesteckt zu werden.
  3. System nach einem der Ansprüche 1 und 2,
    dadurch gekennzeichnet, dass
    der Behälter die Form einer zylinderförmigen Röhre (3) aufweist und die Sammelvorrichtung (4) eine erste Seitenwand (70) mit einem zylinderförmigen zweiten Abschnitt (54) enthält, der dazu ausgelegt ist, mit der Röhre (3) zusammengesteckt zu werden.
  4. System nach einem der Ansprüche 1 bis 3,
    dadurch gekennzeichnet, dass
    die Sammelvorrichtung (4) eine erste Seitenwand (71) mit einem dritten Abschnitt (55) enthält, der sich in den Innenraum (15) des Behälters (3) erstreckt und in welchem zumindest eine Öffnung (17) ausgebildet ist.
  5. System nach einem der Ansprüche 2 bis 4,
    dadurch gekennzeichnet, dass
    die Sammelvorrichtung (4) eine zweite Seitenwand (72) enthält, die sich ausgehend von der ersten Seitenwand (71) erstreckt und bezüglich dieser ersten Seitenwand (71) verjüngt verläuft und einen zylinderförmigen ersten Abschnitt (24) und ein Endstück (25) aufweist, das sich ausgehend von dem ersten Abschnitt (24) erstreckt, wobei das Endstück (25) und der erste Abschnitt (24) den Strömungskanal (23) der Sammelvorrichtung (4) bilden.
  6. System nach Anspruch 5,
    dadurch gekennzeichnet, dass
    das Endstück (25) eine Kegelstumpfform aufweist, die sich zu einem freien Ende (21) der Sammelvorrichtung (4) hin verjüngt, wobei die Auslassöffnung (26) an diesem freien Ende (21) ausgebildet ist.
  7. System nach einem der Ansprüche 1 bis 6,
    dadurch gekennzeichnet, dass
    die Sammelvorrichtung (4) aus thermoplastischem Polymer, insbesondere aus Polyoxymethylen, ausgebildet ist, wodurch der Strömungskanal (23) eine Innenseite (30) mit einer das Strömen von Sperma erleichternden Oberflächenspannung hat.
  8. System nach einem der Ansprüche 1 bis 7,
    dadurch gekennzeichnet, dass
    die Auslassöffnung (26) einen Innendurchmesser zwischen etwa 5 mm und etwa 15 mm aufweist.
  9. System nach einem der Ansprüche 1 bis 8,
    dadurch gekennzeichnet, dass
    es ferner eine Vorrichtung (6) zum Verdünnen von Sperma enthält, die dazu ausgelegt ist, an den Behälter (3) an Ort und Stelle der Sammelvorrichtung (4) angebracht zu werden, nachdem letztere nach erfolgtem Sammeln vom Behälter (3) entfernt wurde.
  10. System nach Anspruch 9,
    dadurch gekennzeichnet, dass
    der Behälter die Form einer zylinderförmigen Röhre (3) aufweist und die Verdünnungsvorrichtung (6) eine Trichterform hat, die einen zylinderförmigen Endabschnitt (45) aufweist und dazu ausgelegt ist, mit der Röhre (3) zusammengesteckt zu werden.
  11. System nach einem der Ansprüche 9 und 10,
    dadurch gekennzeichnet, dass
    die Verdünnungsvorrichtung (6) eine Seitenwand mit einem dem Endabschnitt (43) entgegengesetzten Endkante (48) und einen Deckel (49) enthält, der dazu ausgelegt ist, an die Endkante (48) der Seitenwand befestigt zu werden.
  12. System nach einem der Ansprüche 1 bis 11,
    dadurch gekennzeichnet, dass
    es ferner ein vorbestimmtes Volumen an erstem Verdünnungsmedium (5) enthält.
  13. System nach Anspruch 12,
    dadurch gekennzeichnet, dass
    es vor dem Sammeln von Sperma eine Ruhestellung einnimmt, bei der es dazu ausgelegt ist, dass die Auslassöffnung (26) der Sammelvorrichtung (4) dem ersten Verdünnungsmedium (5) in einem Abstand von etwa 5 mm bis etwa 35 mm gegenüberliegt, wenn sich dieses im Behälter (3) befindet.
  14. System nach einem der Ansprüche 12 und 13,
    dadurch gekennzeichnet, dass
    das erste Verdünnungsmedium (5) aus einer gepufferten Kochsalzlösung mit Liposomen besteht.
  15. System nach Anspruch 14,
    dadurch gekennzeichnet, dass
    die Liposomen im ersten Verdünnungsmedium (5) in einer Konzentration von 10 g/L bis 60 g/L vorhanden sind.
  16. System nach einem der Ansprüche 14 oder 15,
    dadurch gekennzeichnet, dass
    die Liposomen im ersten Verdünnungsmedium (5) in einer Konzentration von 30 g/L bis 40 g/L vorhanden sind.
  17. System nach einem der Ansprüche 14 bis 16,
    dadurch gekennzeichnet, dass
    die Liposomen 20 Gew.-% bis 100 Gew.-% Phosphatidylcholin enthalten.
  18. System nach einem der Ansprüche 14 bis 17,
    dadurch gekennzeichnet, dass
    die Liposomen 60 Gew.-% bis 80 Gew.-% Phosphatidylcholin enthalten.
  19. System nach einem der Ansprüche 12 und 13,
    dadurch gekennzeichnet, dass
    das erste Verdünnungsmedium (5) aus einer gepufferten Kochsalzlösung mit Lipoproteinen niedriger Dichte (LDL) besteht.
  20. Verfahren zum Sammeln von Tiersperma, insbesondere von Rindersperma, mittels eines Systems (1) zum Sammeln von Tiersperma nach einem der Ansprüche 1 bis 19, umfassend die Schritte:
    - Bereitstellen eines Behälters (3), der dazu ausgelegt ist, um an einem Ende (9) einer künstlichen Vagina (2) angebracht zu werden, die vom Sammelsystem (1) separat vorliegt und einen Innenraum (15) aufweist, der dazu ausgelegt ist, um das Sperma aufzunehmen;
    - Einbringen eines bestimmten Volumens an erstem Verdünnungsmedium (5) für das Sperma in den Innenraum (15) des Behälters (3);
    - Bereitstellen einer Sammelvorrichtung (4), die von der künstlichen Vagina (2) und dem Behälter (3) separat vorliegt und einen Strömungskanal (23) und eine Auslassöffnung (26) für das Sperma aufweist;
    - zumindest teilweises Einführen der Sammelvorrichtung (4) in den Behälter (3), um sie an dem Behälter (3) so zu befestigten, dass der Strömungskanal (23) sich in den Innenraum (15) des Behälters (3) erstreckt und die Auslassöffnung (26) in den Innenraum (15) des Behälters (3) mündet;
    - Bereitstellen einer künstlichen Vagina (2); und
    - Befestigen der Sammelvorrichtung (4) an der künstlichen Vagina (2) derart, dass die Sammelvorrichtung (4) zwischen künstlicher Vagina (2) und Behälter (3) eingesetzt wird; wobei das System (1) dazu ausgelegt ist, um zumindest größtenteils zu verhindern, dass während des Sammelns von Sperma das erste Verdünnungsmedium (5) in den Strömungskanal (23) der Sammelvorrichtung (4) strömt und aus dem Behälter (3) entweicht.
  21. Verfahren nach Anspruch 20, umfassend die Schritte des Spülens der Sammelvorrichtung (4) nach dem Sammeln von Sperma, dann des Entfernens der Sammelvorrichtung (4) von dem Behälter (3).
  22. Verfahren nach Anspruch 21, umfassend die Schritte des Bereitstellens einer Verdünnungsvorrichtung (6) zum Verdünnen von Sperma, dann des Anbringens der Verdünnungsvorrichtung (6) an dem Behälter (3) an Ort und Stelle der Sammelvorrichtung (4), nach letztere nach erfolgtem Sammeln von dem Behälter (3) entfernt wurde.
  23. Verfahren nach Anspruch 22, umfassend die Schritte des Einbringens eines bestimmten Volumens an zweitem Verdünnungsmedium für das Sperma in den Innenraum (15) des Behälters (3) und in einen Innenraum (46) der Verdünnungsvorrichtung (6), des Befestigens eines Deckels (49) der Verdünnungsvorrichtung (6) an einer Endkante (48) der letztgenannten, dann des Erstellens einer zweiten Verdünnung des Spermas.
EP15159227.6A 2014-03-18 2015-03-16 System zum Sammeln von tierischem Samen, und Sammelverfahren von tierischem Samen mit diesem System Active EP2921134B1 (de)

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US10195009B2 (en) 2019-02-05
FR3018679A1 (fr) 2015-09-25
EP2921134A1 (de) 2015-09-23
US20150265386A1 (en) 2015-09-24

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