WO2019165348A1 - Récipients pour le stockage de paillettes de sperme dans l'azote liquide - Google Patents

Récipients pour le stockage de paillettes de sperme dans l'azote liquide Download PDF

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Publication number
WO2019165348A1
WO2019165348A1 PCT/US2019/019358 US2019019358W WO2019165348A1 WO 2019165348 A1 WO2019165348 A1 WO 2019165348A1 US 2019019358 W US2019019358 W US 2019019358W WO 2019165348 A1 WO2019165348 A1 WO 2019165348A1
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WO
WIPO (PCT)
Prior art keywords
canister
liquid cryogen
layer
semen
thermally conductive
Prior art date
Application number
PCT/US2019/019358
Other languages
English (en)
Inventor
Stephen Paul HARSTON
Daniel Howard LIEBERMAN
Damian Madan
Elizabeth Jane MCCLURE
James Andrew Roecker
Shannon Weise Stone
Original Assignee
Tokitae Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/904,875 external-priority patent/US10512261B2/en
Application filed by Tokitae Llc filed Critical Tokitae Llc
Publication of WO2019165348A1 publication Critical patent/WO2019165348A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
    • A01N1/0257—Stationary or portable vessels generating cryogenic temperatures

Definitions

  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; a series of apertures in the circular side wall; and a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures.
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wail; and a layer of absorbent material affixed to the interior surface of the circular side wall, the absorbent material absorbent to a liquid cryogen.
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; and a solid mass of a size and shape to substantially fill the interior of the cylindrical cup, the solid mass including a plurality of cavities positioned vertically in the solid mass.
  • FIG. 1 depicts aspects of a Dewar.
  • FIG. 2 A shows a cross section of a Dewar.
  • FIG, 2B illustrates recommended practices with a Dewar.
  • FIG. 2C depicts poor handling practices with a Dewar.
  • FIG. 3 is a schematic of three canister embodiments.
  • FIG. 4 A is a cross-section depiction of a canister
  • FIG. 4B is a cross-section depiction of a canister.
  • FIG. 4C is a cross-section depiction of a canister.
  • FIG, 4D is a cross-section depiction of a canister.
  • FIG. 5 is a graph depicting test results.
  • FIG. 6 is a graph depicting test results.
  • FIG. 7 is a graph depicting test results.
  • FIG, 8 is a graph depicting test results.
  • FIG. 9 is a graph depicting test results.
  • FIG. 10 is a graph depicting test results.
  • FIG. 11 A is a graph depicting test results.
  • FIG. 11B is a graph depicting test results.
  • FIG, 11C is a graph depicting test results.
  • FIG. 11D is a graph depicting test results.
  • FIG. 12.4 is a graph depicting test results.
  • FIG. 12B is a graph depicting test results.
  • FIG, 13 is a graph depicting test results.
  • FIG. 14 depicts aspects of a canister.
  • Semen fertility the final variable in AI success, is most commonly affected by handling subsequent to semen packaging and freezing (B. W. Pickett,“Factors affecting the utilization of frozen bovine semen for maximum reproductive efficiency,” A.I. Dig., Vol. 19, No. 2, p. 8, 1971 which is incorporated by reference).
  • a vast amount of research has optimized the number of sperm packed per standard -sized straw, the additives that supplement semen, and the precise protocols used to properly freeze this mixture (E M. Walters, J. D. Benson, E. J. Woods, and J. K. Critser, “The history of sperm cryopreservation,” 2009 and W. V Holt,“Basic aspects of frozen storage of semen,” Anim. Ileprod. Sci., Vol.
  • Figure 1 depicts a representative example of a commercially-available Dewar (e.g YDS -3 3 L Dewar, Chart Industries, Garfield Heights, OH, and/or XTL3 3L Dewar, Taylor- Wharton Industries, Mobile AL).
  • a locking cover 100 reversibly covers the top opening of the container.
  • a cork 150 reversibly mates with the interior of the neck tube 120.
  • a handle 160 is affixed to the exterior of the container.
  • An evacuating nozzle 1 10 is positioned at the upper face of the container.
  • the neck tube 120 provides a conduit between the outer shell 140 and the inner vessel 180.
  • Multi-layer thermal insulation 170 is positioned between the outer shell 140 and the inner vessel 180 and surrounding the neck tube 120.
  • Adsorbent material 130 is positioned at the upper surface of the inner vessel 180, the adsorbent material surrounding the neck tube 120.
  • Multiple canisters 190 are positioned in the lower portion of the inner vessel 180 Each canister 190 includes a handle that projects upward and into the neck tube 120 The canisters are of a size and shape to hold standard-sezed semen straw's during storage and transport within the Dew'ar.
  • Figures 2A, 2B and 2C depict aspects of handling of containers and their enclosed semen straws within a Dewar.
  • Figure 2A depicts a cross-section view' of a Dew'ar including a cover 100 and a neck tube 120.
  • the device is positioned for storage of the canisters containing semen straws within the storage region of the inner vessel 180.
  • the neck tube 120 is affixed at its lower edge to an inner vessel 180.
  • An outer shell 140 surrounds the inner vessel 180.
  • Multiple storage canisters 190 are held within the inner vessel 180 for semen straw storage.
  • the inner vessel 180 w'ould include a cryogen, such as liquid nitrogen (LN) immersing the semen straw's within the canisters.
  • LN liquid nitrogen
  • Figure ZB shows a cross-section view of the Dewar to illustrate recommend ed practices to access semen straws within a canister.
  • the canister is brought up to the edge of the frost line 200 for access, but does not pass beyond the frost line 200.
  • Figure 2C depicts a cross-section view of a Dewar to illustrate common poor handling practices.
  • the canister is elevated above the frost line 200 of the Dewar while semen straws are removed, exposing the straw's to a potentially damaging temperature.
  • the canister found in most small portable Dewars used for AI consists of a cylindrical cup with a handle to allow' manipulation inside a Dewar and to rest on a hook at the neck.
  • a grate can be attached to the canister base to allow LN to flow out as it is raised.
  • Figure 3 is a cross-section schematic of Prototype Design 1 190A (left), prototype Design 2 190B (center), and prototype Design 3 190C (right).
  • FIG. 3 shows prototype Design 1 190 A to the left side of the Figure.
  • the embodiment includes a handle 300 affixed to the canister wall 315
  • Design 1 190A is shown with a cryogen 305, such as liquid nitrogen, within the canister, while a vapor guard 310 maintains the liquid nitrogen 305 within the canister.
  • a porous or permeable membrane 320 is positioned at the lower surface of the canister.
  • the prototyped canister Design 1 190 A retains a liquid cryogen, such as LN, during regular operation resulting in iow'er semen straw' temperatures during poor handling practices.
  • Design 1 190 A includes a sealed canister base to retain LN when the canister is raised.
  • the design features of Design 1 also include drain holes, drilled along the circumference, anywhere from one quarter to halfway up from the bottom of the canister, the drain holes spaced to control the level of liquid cryogen 305 within the canister. The holes are positioned to allow the canister to fill when the liquid cryogen 305 level in the Dewar is below' the top of the canister.
  • a canister of Design 1 190A will gradually sink when inserted into the Dewar as the cryogen fills the canister.
  • Features of Design 1 can include a vapor guard 310 that is fixed to the inside wall of the canister along the circumference to cover the drain holes and extend below the cryogen liquid level. The function of the vapor guard skirt structure is to minimi ze cryogen vapor from flowing out of the holes when a canister of Design 1 is withdrawn from the Dewar during use.
  • Design 1 includes a base fitted with a permeable membrane 320 designed to allow a liquid cryogen to slowly fill over several hours while restricting any meaningful amount of the liquid cryogen from exiting during a straw extraction event lasting up to a few minutes.
  • the permeable membrane includes a sintered metal. The use of such a membrane will extend also the effectiveness of this device when the liquid cryogen level in the Dewar drops below the elevation of the drain holes during storage or transport.
  • Figure 3 depicts prototype Desi gn 2 190B in the center of the Figure.
  • the embodiment includes a handle 300 affixed to the canister wall 315.
  • a liquid cryogen absorbent material 325 is positioned adjacent to the inner surface of the canister wall 315.
  • the absorbent material is absorbent of liquid nitrogen.
  • Some embodiments include a thermally conductive liner positioned adjacent to the surface of the liquid cryogen absorbent material within the interior of the canister.
  • the thermally conductive liner is fabricated from a material with thermal conduction properties sufficient to equalize the temperature between the top and the bottom of the container interior wall to match the cryogen within the container quickly, within a few seconds.
  • a thermally conductive liner includes apertures positioned and agled to direct cryogen vapor towards the interior of the canister and surround any enclosed semen straws within the center. The ciyogen vapor assists in maintaining the temperature of the stored semen straws at near cryogen temperature during a period of time when the canister is raised above the frost line.
  • the embodiment 190B includes a grated base 330 at the lower face of the canister.
  • the canister wall and base are lined with an absorbent material positioned to absorb liquid cryogen and provide additional insulation (see Figure 3).
  • the design permits the liquid cryogen to drain from a grated canister base as it is raised.
  • Some embodiments include a LN absorbent material.
  • Some embodiments include a LN absorbent material that includes a flexible aerogel.
  • Some embodiments include a LN absorbent material that includes a woven fiberglass material.
  • the LN absorbent material provides thermal isolation from the environment and blankets straws with cold nitrogen vapor as the liquid vaporizes.
  • the thickness of the material is designed to store sufficient liquid cryogen for a short exposure up to several minutes. In some embodiments, the absorbent material is between approximately 2 mm and approximately 7 mm in thickness.
  • the design including liquid cryogen absorbent material at the canister base allows liquid to fdl and drain during use.
  • Figure 3 illustrates prototype Design 3 190C at the right of the Figure.
  • the embodiment includes a handle 300 affixed to the canister wall 315.
  • the embodiment 190C includes a grated base 330 at the low'er face of the canister.
  • the embodiment 190C includes a solid mass 340 positioned within the canister A series of apertures 335 are positioned substantially vertically within the solid mass 340.
  • the apertures 335 include an opening at the top face of the solid mass 340.
  • the apertures 335 project vertically from the top face of the solid mass 340 into the solid mass 340 for a depth at least as long as a semen straw expected to be stored within the canister 315
  • the canister is designed to include solid mass with a system of holes along its length to insert several straws (see Figure 3).
  • the holes form apertures oriented along the long axis of the Design 3 canister.
  • the holes can be designed to sort straw's from different bulls.
  • the holes are sized, shaped, and positioned to store semen straw's in different sections that can be easily identified by a user based on one or more of these factors.
  • the solid mass can be fabricated, for example, from a heat-conductive material, such as a thermallv-conductive metal.
  • the solid mass of the canister is fabricated from aluminum.
  • the solid mass of the canister is fabricated from copper.
  • the solid mass of the canister is fabricated from stainless steel. The solid construction is designed to provide additional thermal mass relative to a standard canister, thus slowing the temperature rise during an exposure to remain below' -130 degrees C.
  • Figure 4 depicts additional aspects of canisters for storage of semen straw's within a Dewar.
  • Figure 4A, 4B, 4C, 4D depicts prototype schematics in cross-section.
  • Figure 4B, 4C and 4D depict additional aspects of Design 1, Design 2 and Design 3 as described above.
  • Figure 4A shows a depiction of a canister found in many small portable Dewars.
  • Figure 4A depicts a representative example of the standard canister design in a cross- section view.
  • the cylindrical cup 315 that holds semen straws is attached to a handle 300 that allows manipulation inside a Dewar and to rest on a hook at the neck.
  • a grate 330 or drain holes are incorporated into the canister base to allow liquid cryogen to empty as it is raised.
  • the canisters found in most small portable Dewars used for AI consist of a cylindrical cup with a handle to allow manipulation inside a Dewar and to rest on a hook at the neck.
  • a grate or drain holes 330 are incorporated into the canister base to allow liquid cryogen to flow out as it is raised.
  • a handle 300 is affixed to a top edge of the canister wall 315.
  • the bottom of the canister includes a grated base 330
  • Figure 4B depicts aspects of Design 1.
  • liquid cryogen enters the canister through drain holes 400.
  • the drain holes 400 are positioned above the base of the container at a position approximately one third to one half of the length of the container.
  • cold cryogen vapor e.g cold nitrogen vapor
  • a vapor guard 310 is included to minimize vapor exit via the drain holes.
  • the vapor guard 310 is formed as a skirt or flap structure affixed at its upper edge to a position above the drain holes 400, with the lower edge of the vapor guard 310 extending below the level of the drain holes 400. in some embodiments, the lower edge of the vapor guard fully covers one or more of the drain holes.
  • the lower edge of the vapor guard partially covers one or more of the drain holes.
  • a vapor guard can be fabricated from a thin metal sheet, for example fabricated from a stainless steel or aluminum sheet. In some embodiments, the vapor guard is fabricated from the same material as the canister walls.
  • the grate at the base of the canister is replaced with a solid metal surface 420 to prevent liquid cryogen draining.
  • the canister is fited with sintered metal 320 section that acts as a permeable membrane to allow liquid cryogen to slowly fill over several hours.
  • the speed of the cryogen moving through the sintered metal section can be modified depending on the embodiment based on factors including the type of cryogen, the permeability of the sintered metal secton, and the size of the sintered metal section within the solid metal surface making up the bottom of the container.
  • Figure 4C depicts aspects of Design 2. This canister is lined with a liquid cryogen absorbent material 325 to both provide thermal insulation from the environment and blanket semen straws with cold cryogen vapor as the liquid vaporizes.
  • a liquid cryogen absorbent material 325 to both provide thermal insulation from the environment and blanket semen straws with cold cryogen vapor as the liquid vaporizes.
  • the absorbent material is a material that absorbs liquid nitrogen.
  • the absorbent material is a felted material, such as fabricated from cotton or polyester.
  • the absorbent material is an acrylic felt material.
  • the absorbent material is a fiberglass mesh or weave material.
  • the absorbent material is an aerogel material.
  • the absorbent material is a cryogel material.
  • the absorbent material is a cryogen-permeable material with porosity.
  • the absorbent material can be, for example, between approximately 2 mm in thickness and approximately 7 mm in thickness, depending on the material used.
  • the absorbent material can be, for example,
  • the absorbent material can be, for example, approximately 3 mm in thickness.
  • the absorbent material can be, for example, approximately 4 mm in thickness.
  • the absorbent material can be, for example, approximately 5 mm in thickness.
  • the absorbent material can be, for example, approximately 6 mm in thickness.
  • the absorbent material can be, for example, approximately 7 mm in thickness.
  • a grated metal surface 430 is included as a permeable layer and positioned to protect the liquid cryogen absorbent material within the wall of the container.
  • the grated metal surface is fabricated from a material that is thermally conductive at cryogenic temperatures, for example stainless steel or copper.
  • the grated metal surface is sufficiently thermally conductive to equalize the temperature along the interior surface of the container when the container is removed from liquid cryogen within a Dewar for semen straw removal.
  • the grated metal surface equalizes the temperature quickly, within seconds, and maintains the equalized temperature during the period that the container is raised above the liquid cryogen.
  • the grated metal surface includes a plurality of holes positioned and angled to direct cryogen vapor into the interior of the container and to surround any portion of semen straws within the container above the liquid cryogen with cryogen vapor for maintenance of a temperature close to that of the liquid cryogen during a straw removal.
  • Some embodiments include the absorbent material and the grated metal
  • Some embodiments include the absorbent material and the grated metal surface along the walls only, and not along the bottom of the container.
  • the grated metal surface connects at the top edge with an angled connection to the outer wall of the container, the angled connection also thermally conductive to equalize heat throughout the length of the container i nterior from the top edge to the bottom.
  • Figure 4D depicts aspects of Design 3.
  • a solid cylinder 340 is designed to fit snugly within the canister to increase thermal mass.
  • the solid cylinder is aluminum.
  • a system of holes 335 is drilled along its length to accommodate semen straw's. The holes 335 can be sized and positioned to contain semen straw's in an easy position for handling and/or organization during storage.
  • Design 1 we sought to maintain a level of liquid cryogen within the canister by replacing the grate at the canister base with a solid metal surface 420 (see Figure 4B).
  • a canister that possesses drain holes 400 drilled along the circumference of the canister approximately half way (e.g. 5 cm) from its bottom to allow liquid cryogen to flow' between the canister and the Dewar
  • the drain holes allow' the canister to gradually sink when it is inserted into the Dewar.
  • a vapor guard 310 is positioned adjacent to the drain holes 400, the vapor guard 310 affixed to the interior of the canister wall 315 at a position above the drain holes 400.
  • Design 1 The intention of the features of Design 1 was to drive cold cryogen vapor over the top portion of the straw's that are not submerged in liquid cryogen. However, when we removed our canister prototype from the Dewar neck, we noticed that a significant portion of cryogen vapor escapes from the drain holes. To minimize this effect we modified the design to include a vapor guard on the inside wall of the canister along the circumference that covers the drain holes and extends below' the liquid cryogen level (see Figure 4B).
  • the canister with sintered metal 320 within the base that acts as a permeable membrane to allow liquid cryogen to slowly fill over several hours while restricting the loss of liquid cryogen during a straw ' extraction event lasting up to a few minutes (see Figure 4B).
  • the sintered metal permits some passage of the liquid cryogen but this occurs slowly enough so that the liquid cryogen is maintained within the canister during a relatively brief raising of the canister above the liquid nitrogen, for example during straw extraction.
  • the sintered metal does, however, permit the container to gradually refill with liquid cryogen when the container is repositioned within the liquid cryogen for storage.
  • the use of this prototype extends the effectiveness of this device when the liquid cryogen level in the Dewar drops below the el evation of the drain holes.
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; a series of apertures in the circular side wall; and a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to fit through the neck of a Dewar and to be retained within the inner storage region of the Dewar.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to retain a plurality of bovine semen straws.
  • a canister for liquid cryogen storage includes a series of apertures in a circular side wall, wherein the series of apertures are positioned around the circular side wail at positions approximately midway between the bottom edge and the top edge of the side wall.
  • a canister for liquid cryogen storage includes a series of apertures in a circular side wall, wherein the series of apertures are positioned around the circular side wall at positions approximately one third of the distance from the bottom edge relative to the top edge of the side wall.
  • a canister for liquid cryogen storage includes a series of apertures in a circular side wall, wherein the series of apertures are positioned around the circular side wall at positions between approximately midway between the bottom edge and the top edge of the side wail and approximately one third of the distance from the bottom edge relative to the top edge of the side wall.
  • a canister for liquid cryogen storage includes a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures, and wherein the flange affixed to an interior surface of the circular side wall is positioned to substantially cover the series of apertures in the circular side wall.
  • a canister for liquid cryogen storage includes a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures, and wherein the flange affixed to an interior surface of the circular side wall is positioned to partially cover the series of apertures in the circular side wall.
  • a canister for liquid cryogen storage includes a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures, and wherein the flange affixed to an interior surface of the circular side wall is affixed to the interior surface of the circular side wall at a position above the series of apertures.
  • a canister for liquid cryogen storage further includes a permeable membrane positioned within the bottom wall.
  • the permeable membrane can include, for example, a sintered metal.
  • the permeable membrane can include, for example, a membrane permeable to liquid nitrogen.
  • the permeable membrane can, for example, be permeable to liquid nitrogen at a rate sufficient to allow the cylindrical cup to fill in an hour when the cylindrical cup is submerged in liquid nitrogen.
  • the permeable membrane can, for example, be permeable to liquid nitrogen at a rate sufficient to allow the cylindrical cup to fill in a period longer than a minute when the cylindrical cup is submerged in liquid nitrogen.
  • a canister for liquid cryogen storage further includes a handle affixed to the canister.
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; and a layer of absorbent material affixed to the interior surface of the circular side wall, the absorbent material absorbent to a liquid cryogen.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to fit through the neck of a Dewar and to be retained within the inner storage region of the Dewar.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to retain a plurality of bovine semen straws.
  • a canister for liquid cryogen storage includes a layer of absorbent material affixed to an interior surface of a circular side wall, the absorbent material absorbent to a liquid cryogen.
  • the absorbent material can, for example, include a material that absorbs liquid nitrogen.
  • the absorbent material can, for example, include a felted material.
  • the absorbent material can, for example, include a fiberglass mesh material.
  • the absorbent material can, for example, include an aerogel material.
  • the absorbent material can, for example, include a cryogen-permeable material with porosity.
  • a canister for liquid cryogen storage further includes a cryogen-permeable region within the bottom wall.
  • the cryogen-permeable region within the bottom wall can include, for example, a group of apertures within the bottom wall.
  • the cryogen-permeable region within the bottom wall can include, for example, a sintered metal.
  • the cryogen-permeable region within the bottom wall can include, for example, a group of apertures within the bottom wall.
  • a canister for liquid cryogen storage further includes a permeable layer positioned adjacent to the layer of absorbent material at a surface of the layer of absorbent material opposite to the canister.
  • the permeable layer can include, for example, a layer of metal including a plurality of apertures.
  • a canister for liquid cryogen storage further includes a layer of absorbent material affixed to the interior surface of the bottom wall.
  • a canister for liquid cryogen storage further includes a handle affixed to the canister.
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; and a solid mass of a size and shape to substantially fill the interior of the cylindri cal cup, the solid mass including a plurality of cavities positioned vertically in the solid mass.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to fit through the neck of a Dewar and to be retained within the inner storage region of the Dewar.
  • the cylindrical cup includes: a cylindrical cup of a size and shape to retain a plurality of bovine semen straws.
  • a canister for liquid cryogen storage includes a cylindrical cup with a circular side wall and a bottom wall, and wherein the cylindrical cup includes a cryogen-permeab!e region within the bottom wall.
  • the cryogen- permeable region within the bottom wall includes a group of apertures in the botom wall.
  • a canister for liquid cryogen storage includes a solid mass of a size and shape to substantially fill the interior of the cylindrical cup, the solid mass including a plurality of cavities positioned vertically in the solid mass.
  • the solid mass is a thermal mass.
  • the solid mass is fabricated with metal.
  • the plurality of cavities positioned vertically in the solid mass can include, for example, a plurality of cavities of a size and shape to contain one or more semen straws.
  • the plurality of cavities can be, for example, positioned so that a user of the container can quickly identify and remove particular semen straws.
  • a canister for liquid cryogen storage further includes a handle affixed to the canister.
  • Prototype 2 based on Design 2, demonstrated improvement in maintaining temperature and reducing the loss of LN during testing, we tested its ability to protect against semen damage in comparison to the standard canister. Both a standard canister and Prototype 2, based on Design 2, were subjected to multiple ambient temperature exposures similar to those described herein. Semen stored in the Prototype 2 canister showed less damage by post-thaw aerosome integrity measurements (see Figures 12A & 12B). These data strongly suggest that a canister lined with an LN absorbent material can better maintain the fertility of frozen semen when subjected to repeated poor handling.
  • cryocapacitation a form of cryoinjury that can occur during the freezing process that largely affects sperm outer and acrosomal membranes (Bailey, J., Bilodeau, J. & Cormier, N., Semen cryopreservation in domestic animals: A damaging and capacitating
  • a canister for liquid cryogen storage includes: a cylindrical cup with a circular side wall and a bottom wall; a layer of absorbent material positioned adjacent to the interior surface of the circular side wall, the absorbent material absorbent to a liquid cryogen; a layer of thermally conductive material adjacent to the layer of absorbent material, the layer of thermally conductive material including a plurality of holes; and a connection between a top edge of the thermally conductive material and a top edge of the circular side wall.
  • Figure 14 depicts aspects of a canister embodiment of Design 2, above.
  • the canister is shaped as a right circular cylinder.
  • the canister includes a container 190 in the shape of a cylindrical cup with a circular side wall 1400 and a bottom wall 1450.
  • the container 190 includes an aperture 1430 at the top, the aperture of a size and shape to accept the insertion and removal of standard-sized semen straw's.
  • the interior 1410 of the container 190 is of a size and shape to retain and store a plurality of standard-sized semen straw's.
  • a handle is connected to the canister, the handle positioned for raising and lowering the canister during storage while minimizing the time involved in handling the canister as well as the potential hazard to a user of materials held at liquid cryogen temperatures.
  • the cylindrical cup is fabricated from a thermally conductive material which is durable in the temperature range of the liquid cryogen that the container will be submerged in during use.
  • the cylindrical cup includes a circular side wall containing a plurality of apertures between one third to one half the distance from the bottom wall to the top edge of the circular side wall.
  • the apertures are sized and positioned to permit liquid cryogen to enter the interior of the container when the container is partially submerged in a liquid cryogen.
  • the cylindrical cup includes a circular side wall that is solid.
  • the cylindrical cup includes a bottom wall that contains a plurality of apertures. In some embodiments, the cylindrical cup includes a bottom wall that is solid. In some embodiments, the cylindrical cup includes a bottom wall that includes a sintered metal section, the section configured to permit a reduced flow' of liquid cryogen into and out of the interior of the container. In some embodiments, the cylindrical cup includes a cryogen-permeable region within the bottom wall. The cryogen-permeable region can include a sintered metal. The cryogen-permeable region can include a group of apertures within the bottom wall.
  • the container 190 includes a layer of absorbent material 325 positioned adjacent to the interior surface of the circular side wall 1400, the absorbent material absorbent to a
  • the liquid cryogen expected to be used with the container is liquid nitrogen and the absorbent material is absorbent to liquid nitrogen.
  • the layer of absorbent material is a felted material.
  • the layer of absorbent material is a fiberglass mesh material.
  • the layer of absorbent material is an aerogel material.
  • the layer of absorbent material is a cryogen-permeable material with porosity. In some embodiments, the layer of absorbent material is positioned only adjacent to the circular side wall of the container, extending from the bottom to the top of the container. In some embodiments, the layer of absorbent material is further positioned adjacent to the interior surface of the bottom wall.
  • the container 190 also includes a layer of thermally conductive material 1440 adjacent to the layer of absorbent material 325.
  • the layer of thermally conductive material 1440 includes a plurality of holes in the material, the holes sized, shaped, oriented and positioned to direct cryogen vapor from the layer of absorbent material 325 into the interior 1410 of the container 190.
  • the holes are sized, shaped, oriented and positioned to direct the cryogen vapor from evaporated liquid cryogen within the layer of absorbent material radially into the center of the container interior.
  • this just-evaporated cryogen vapor is only a few degrees above the temperature of the liquid cryogen, and therefore the vapor flow maintains a near-liquid cryogen temperature for semen straws within the container when the container is raised from the liquid cryogen bath.
  • the expanding vapor within the container interior also creates a gas flow' up through the top aperture of the container, reducing the entrance of potentially warmer air from an exterior of the container when the container is raised. For example warmer air may enter a Dewar when the lid is removed, and this warmer air may be adjacent to the aperture of a container when it is raised within the Dewar as shown in Figure 2B and 2C.
  • the layer of thermally conductive material is a material thermally conductive at a boil point of the liquid cryogen.
  • the materi al can be stainless steel for use with liquid nitrogen as a liquid cryogen.
  • the layer of thermally conductive material includes a plurality of holes sized and positioned to direct cryogen vapor through the layer of thermally conductive material and into the interior of the container.
  • the holes may be positioned radially around the layer of thermally conductive material.
  • the holes may be oriented to direct the cryogen vapor across the layer of thermally conductive material at an angle
  • the holes may be oriented along azimuth and directed radially toward a center axis of the container.
  • the holes may be sized to permit a volume of cryogen vapor per unit time into the interior of the container, the volume and unit of time depending on the liquid cryogen and the expected use of an embodiment.
  • the container 190 includes a connection 1420 between a top edge of the layer of thermally conductive material and a top edge of the circular side wall 1400.
  • the connection 1420 is continuous with the layer of thermally conductive material 1440 and affixed at the top edge to the circular side wall 1400 of the cylindrical cup.
  • the connection 1420 is in thermal contact with both the top edge of the layer of thermally conductive material 1440 and a top edge of the circular side wall 1400.
  • the connection 1420, the layer of thermally conductive material 1440 and the circular side wall 1400 are fabricated from a contiguous piece of thermally conductive metal, such as stainless steel or copper.
  • connection 1420, the layer of thermally conductive material 1440 and the circular side wall 1400 are fabricated from differerent pieces of thermally conductive metal that are connected together with thermally conductive attachments during manufacture.
  • the connection 1420 can be angled relative to the planes of the layer of thermally conductive material 1440 and the circular side wall 1400 as well as the plane of the bottom wall 1450.
  • the angle of the connection 1420 can be positioned and oriented to provide convenient access to standard sized semen straws stored within the interior 1410 to a user moving quickly to access the stored straw's and return the unused ones to storage.
  • the connection can be solid, without apertures, which maintains the cryogen vapor at a level below ? the low ? er edge of the connection and in a level within the container to escape through the plurality of holes in the layer of thermally conductive material.
  • the connection is fabricated from a material thermally conductive at a boil point of the liquid cryogen.
  • the connection can be fabricated from stainless steel.
  • the connection can include a surface facing the aperture of the container which is sufficiently smooth to not hinder a semen straw being slide along the surface when being added or removed from the interior of the container.
  • the connection is fabricated from stainless steel, the stainless steel can be polished or otherwise made sufficiently smooth.
  • a container includes a bottom layer of thermally conductive material including a plurality of holes positioned adjacent to the bottom wall, the bottom layer of thermally conductive material affixed to the layer of thermally conductive material along the side walls.
  • a container in some embodiments, includes a layer of absorbent material positioned adjacent to the interior surface of the bottom wal! as well as a bottom layer of thermally conductive material including a plurality of holes positioned adjacent to the bottom wall, the bottom layer of thermally conductive material affixed to the layer of thermally conductive material along the side walls.
  • reagents used in this study were purchased from Sigma (St. Louise, MO). All frozen semen used in the study was purchased from Accelerated Genetics (Baraboo, WI). Unless otherwise indicated, semen within an experiment utilized straws from a single lot, which in some tests were from the Holstein Bull Michigan Frost (014H007313) and packaged in 0.5 ml French straws.
  • Example 1 Quantifying Semen Damage
  • Figure 5 depicts post-thaw characteristics of frozen semen subjected to repeated ambient temperature exposures.
  • Frozen straws from the same lot of semen were stored in the canister of a standard 3 L Dewar and subjected to repeated one minute ambient temperature exposures.
  • Measurements of membrane viability staining, sperm motility, and acrosome integrity are displayed in Figure 5.
  • Membrane viability and sperm motility data points were measured on three samples. Error bars represent standard deviation (SD). Each acrosome integrity data point was measured on samples from a single straw. Error bars are not included on the acrosome integrity data set.
  • the acrosome is a large vesicle located at the anterior region of the sperm head that contains hydrolytic enzymes and surface antigens necessary for the acrosome reaction, a necessary process in fertilization, where the sperm penetrates the zona pellucida of the egg (M Ikarva, N. Inoue, A. M. Benham, and M. Okabe,“Fertilization: A sperm’s journey to and interaction with the oocyte,” J. Clin. Invest., Vol. 120, No. 4, pp. 984-994, 2010 wiiich is incorporated by reference).
  • Dewar canisters contain multiple semen straws. If an A! technician uses poor handling practices then each time he or she removes a straw for use, the remaining straws within the inventory ' will experience multiple thermal exposures ⁇ see, e.g. Figure 2). Similar events could occur during the transfer of straws between Dewars, such as when importers transfer straws to distribution centers, distributers transfer straws to AI technicians, etc.
  • the acrosome is a large vesicle located at the anterior region of the sperm head that contains hydrolytic enzymes and surface antigens necessary for the acrosome reaction, a necessary process in fertilization where the sperm penetrates the zona pellucida of the egg (Ikawa,
  • Figures 7 and 8 show that poor handling damages semen.
  • frozen semen straws were subjected to repeated one minute ambient temperature exposures. Measurements of membrane viability staining, sperm motility, and acrosome integrity are displayed.
  • frozen semen was subjected to multiple ambient temperature exposures at 0.5, 1, 2, and 4 minute durations. The post-thaw acrosome integrity measurements are shown.
  • Frozen semen straws were thawed in a water bath set at 35° C for 30 seconds. Semen was then transferred to 1.7 mb microcentrifuge tube and incubated at 35° C for one hour.
  • samples were then cooled to 4° C and centrifuged 500 RCF for 5 minutes at 4° C. Supernatants were then discarded and the pellet was gently resuspended in
  • Cytofix (BD Biosciences, Franklin Lakes, NJ), and incubated for 15 minutes at 4° C.
  • Acrosome integrity was measured using fluorescence microscopy as previously described
  • thermocouples A bundle of three 36 AWG Type T thermocouples was inserted into a semen straw.
  • the thermocouple tips were positioned 34 mm, 79 mm, and 124 mm from the crimped end of the straw.
  • the bundle was secured to the crimped end of the straw using 19 mm Kapton tape, waxed lacing cord, and tapered round rubber plugs.
  • thermocouple was connected to a National Instruments data acquisition system with a Labview interface and sampled at 1 Hz. All thermocouples were two-point-calibrated using a water bath and LN bath.
  • Example 6 Prototypes
  • Prototypes based of all three designs were constructed and subjected to ambient exposure tests described below. In this study the holes on the Design 1 prototype were located at the canister midpoint. A vapor guard and permeable membrane are not included in this data set.
  • the Design 2 prototype used a standard canister fitted snuggly with a 9.5 mm thick eabosil infused blanket against the base and walls. LN was able to drain out when raised from the Dewar.
  • the Design 3 prototype fitted an aluminum block with twenty 3.2 mm thru holes into a standard canister.
  • Standard canisters of a YDS-3 Dewar (Chart, Garfield Heights, OH) were used as the platform for prototype construction.
  • the canister for Prototype 1 was modified by welding a circular piece of sheet metal to seal the canister bottom and drilling 3.175 mm diameter holes at the canister midpoint.
  • a 1.25 mm gap allowed the LN to flow through the holes at the midpoint.
  • Prototype 2 was fitted snuggly with a 5 mm thick Cryogel® (Aspen Aerogels, Northborough, MA) blanket against the base and walls.
  • Cryogel® Aspen Aerogels, Northborough, MA
  • Prototype 3 fitted an aluminum block with eight 7.2 mm thru holes into a standard canister.
  • each test was carried out by placing three straw's instrumented with thermocouples in a canister. Each instrumented straw' had three 36 gauge type T thermocouples that were positioned at the bottom, middle, and top of the straw. Each canister was raised by hand out of the Dewar into the environment and held for approximately one minute then re- submerged into the Dewar.
  • Figure 10 compares the thermal response of each design with the standard canister as a baseline. All three designs demonstrate an improvement delaying a temperature rise in the straws. Of note all three prototypes are very effective at keeping the mid and low'er portion of the straw near the LN temperature. Designs 2 and 3 appear to be slightly more effective at delaying the temperature rise in the top portion of the straw. Additional experiments are planned to confirm this finding.
  • Figure 10 shows a graph of temperature as a function of time for straws in the Design 1, 2, and 3 prototypes removed from LN in a 3 L Dewar to ambient temperature for approximately 1 min after which time, canisters are re submerged into LN. The standard canister is shown for reference.
  • thermocouple temperature sensors at the top, middle, and bottom of a semen straw. Each prototype contained three straws with thermocouples.
  • FIG 11 shows that prototypes reduce thermal fluctuations within semen straws during poor handling. Temperature measurements at the top (T), middle (M), and bottom (B) of a semen straw placed within Prototype (P) l representing Design 1, Prototype 2 representing Design 2 and Prototype 3 representing Design 3 (subfigures A, B, and C, respectively) removed from a full 3 L Dewar for approximately one minute then re submerged into LN are shown. Readings from similar experiments using a standard canister (SC) are shown for reference. D. The experiment in subfigure B was repeated with a Dewar that was 25% full of LN. All plots report values as mean readings from thermocouples located on three different semen straws from the same ambient exposure. Data are representative of three independent experiments.
  • Figure 11 is a set of graphs of testing results indicating that prototypes reduce thermal fluctuations within semen straws during poor handling. Temperature
  • FIG. 1 ID The experiment in subfigure 1 IB was repeated with a Dewar that was 25% full of LN. All plots report values as mean readings from thermocouples located on three different semen straws from the same ambient exposure. Data are representative of three independent experiments.
  • Design 1 prototype The ability of the Design 1 prototype to protect against semen damage was tested in a side-by-side experiment with a standard canister.
  • Figure 9 illustrates mass of liquid nitrogen loss after a canister was removed from a 3 L Dewar for one minute then reintroduced to the system.
  • Figure 13 show's post-thaw acrosome integrity measurements of frozen semen stored in the Design 1 prototype and a standard canister subjected to up to twenty 1 min ambient temperature exposures.
  • FIGS 12A & 12B Prototype 2 protects semen from poor-handling-induced damage. Frozen semen straw's were placed within either a standard canister or Prototype 2 in a full (A) or 25% full (B) Dewar and exposed to up to 40 one minute ambient temperature exposures. The post thaw acrosome integrity measurements are shown. Fitted lines are included as visual guides.
  • a canister for liquid cryogen storage comprising:
  • a flange affixed to an interior surface of the circular side wall at a position adjacent to the apertures.
  • a cylindrical cup of a size and shape to fit through the neck of a Dewar and to be retained within the inner storage region of the Dew'ar.
  • the series of apertures positioned around the circular side wall at positions approximately one third of the distance from the bottom edge relative to the top edge of the side wall.
  • the canister for liquid cryogen storage of clause 1 further comprising: a permeable membrane positioned within the bottom wall.
  • a canister for liquid cryogen storage comprising:
  • the canister for liquid cryogen storage of clause 16 further comprising: a cryogen-permeable region within the bottom wall.
  • the canister for liquid cryogen storage of clause 16 further comprising: a layer of absorbent material affixed to the interior surface of the bottom wall.
  • the canister for liquid cryogen storage of clause 16 further comprising: a handle affixed to the canister.
  • a canister for liquid cryogen storage comprising:
  • a solid mass of a size and shape to substantially fill the interior of the cylindrical cup the solid mass including a plurality of cavities positioned vertically in the solid mass.
  • a cylindrical cup of a size and shape to fit through the neck of a Dewar and to be retained within the inner storage region of the Dewar.
  • a canister for liquid cryogen storage comprising: a cylindrical cup with a circular side wall and a bottom wall;
  • the layer of thermally conductive material including a plurality of holes
  • a circular side wall containing a plurality of apertures between one third to one half the distance from the bottom wall to the top edge of the circular side wall.
  • a bottom wall containing a plurality of apertures.
  • the canister for liquid cryogen storage of clause 40, wherein the layer of thermally conductive material comprises:
  • the canister for liquid cryogen storage of clause 40, wherein the layer of thermally conductive material including a plurality of holes comprises:
  • a plurality of holes sized and positioned to direct cryogen vapor through the layer of thermally conductive material and into the interior of the container.
  • the canister for liquid cryogen storage of clause 40, wherein the layer of thermally conductive material including a plurality of holes comprises:
  • the canister for liquid cryogen storage of clause 40 further comprising: a cryogen-permeable region within the bottom wall.
  • the canister for liquid cryogen storage of clause 40 further comprising: a layer of absorbent material positioned adjacent to the interior surface of the bottom wall .
  • the canister for liquid cryogen storage of clause 40 further comprising: a bottom layer of thermally conductive material including a plurality of holes positioned adjacent to the bottom wall, the bottom layer of thermally conductive material affixed to the layer of thermally conductive material along the side walls.
  • the canister for liquid cryogen storage of clause 40 further comprising: a handle affixed to the cani ster.

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Abstract

L'invention concerne des conceptions de cartouches améliorées pour le stockage de paillettes de sperme animal dans des vases de Dewar contenant un liquide cryogénique. Dans certains modes de réalisation, les cartouches comprennent une couche de matériau absorbant le cryogène et une couche interne de matériau thermoconducteur comprenant des ouvertures orientées et positionnées pour diriger la vapeur cryogénique dans l'intérieur du récipient.
PCT/US2019/019358 2018-02-26 2019-02-25 Récipients pour le stockage de paillettes de sperme dans l'azote liquide WO2019165348A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022020618A1 (fr) * 2020-07-23 2022-01-27 Coopersurgical, Inc. Capuchons de récipient pour des applications de cryoconservation
US12038133B2 (en) 2021-07-22 2024-07-16 Coopersurgical, Inc. Canister caps for cryopreservation applications

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5419143A (en) * 1992-12-22 1995-05-30 International Cryogenics, Inc. Cryogenic apparatus for sample protection in a dewar
US6119465A (en) * 1999-02-10 2000-09-19 Mullens; Patrick L. Shipping container for storing materials at cryogenic temperatures
US8181813B2 (en) * 2007-03-29 2012-05-22 St Reproductive Technologies Llc Transportation and/or storage device comprising a double-walled insulating bulb
US20120297797A1 (en) * 2011-05-23 2012-11-29 St Reproductive Technologies, Llc Portable cryogenic container
US20180184645A1 (en) * 2015-06-02 2018-07-05 TOKITAE LLC, a limited liability company of the Stae of Delaware Containers for liquid nitrogen storage of semen straws

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5419143A (en) * 1992-12-22 1995-05-30 International Cryogenics, Inc. Cryogenic apparatus for sample protection in a dewar
US6119465A (en) * 1999-02-10 2000-09-19 Mullens; Patrick L. Shipping container for storing materials at cryogenic temperatures
US8181813B2 (en) * 2007-03-29 2012-05-22 St Reproductive Technologies Llc Transportation and/or storage device comprising a double-walled insulating bulb
US20120297797A1 (en) * 2011-05-23 2012-11-29 St Reproductive Technologies, Llc Portable cryogenic container
US20180184645A1 (en) * 2015-06-02 2018-07-05 TOKITAE LLC, a limited liability company of the Stae of Delaware Containers for liquid nitrogen storage of semen straws

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022020618A1 (fr) * 2020-07-23 2022-01-27 Coopersurgical, Inc. Capuchons de récipient pour des applications de cryoconservation
JP2023535177A (ja) * 2020-07-23 2023-08-16 クーパーサージカル・インコーポレイテッド 凍結保存用途のためのキャニスターキャップ
JP7386374B2 (ja) 2020-07-23 2023-11-24 クーパーサージカル・インコーポレイテッド 凍結保存用途のためのキャニスターキャップ
US12038133B2 (en) 2021-07-22 2024-07-16 Coopersurgical, Inc. Canister caps for cryopreservation applications

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