Note: Descriptions are shown in the official language in which they were submitted.
CA 02337632 2006-05-05
r
f
CONTAINER ARRANGEMENT AND
METHOD FOR TRANSPORTING EQUINE SEMEN
FIELD OF THE INVENTION
The present invention generally relates to a thermally insulated transport
container
arrangement and method for using same to transport equine semen. More
specifically,
the present invention relates to an inexpensive, self contained, thermally
insulated,
disposable, refrigerated transport container arrangement that can greatly
extend the length
of time over which the spermatozoa of equine semen can be maintained motile
and fertile,
and thus render it possible to effectively transport this type of material
over long
distances.
BACKGROUND OF THE INVENTION
Transporting semen (germplasm), e.g., equine or canine semen, is beneficial to
breeders for several reasons. Shipping semen is less costly than transporting
female
horses or dogs to an unfamiliar facility. Stress and risk of disease are
minimized when
female horses or dogs can remain at home. Further, using artificial
insemination allows a
stallion to service more mares than he could using natural service, and allows
the stallion
to continue to show or perform during the breeding season. When shipped semen
is
handled properly, pregnancy rates approach those achieved using natural
service. Semen
destined for transport is collected from a male, examined and cooled for
shipment. The
development of conventional systems allowing controlled cooling has been
instrumental.
The advantages of using cooled semen make it a valuable addition to breeding
programs
and has been used to increase the genetic pool in many breeds.
However, numerous factors influence pregnancy rates achieved when mares are
bred with transported, cooled stallion semen. For instance, sperm are very
sensitive to
many environmental factors, including temperature, light, physical trauma, and
a variety
CA 02337632 2001-O1-15
WO 99/27884 PCTNS98/25310
2
of chemicals. Any factor that impacts the ability of sperm to resist
environmentally-
induced damage will adversely affect fertility achieved when using cooled
transported
semen. Semen must be handled from collection to insemination in such a manner
as to
not shock nor damage the sperm. If collection or storage devices are
contaminated by
bacteria, chemicals, or even soap residue, the survivability of the sperm
cells can be
severely diminished. Proper temperature control of semen prior to cooling and
prior to
insemination is crucial. For example, if semen is initially mixed with
extender that is too
cool or too warm, damage will likely occur.
Mares will ovulate 24 to 48 hours before the end of heat and pregnancy rates
from
cooled stallion semen are the highest when mares are inseminated within 24
hours
following semen collection. Some stallion semen is still highly viable up to
48 hours
following collection. Semen transported and stored for up to 72 hours may
appear to
have good motility, but fertilization capabilities are typically poor. Thus,
when it is
required to transport equine semen samples over long distances or even
overseas, it is
typically necessary to maintain the motility and fertility of the spermatozoa
for 48 hours,
at the very minimum, and ideally for more than 72 hours, in order that samples
reach its
destination and can be effectively used. However, until the development of the
present
invention, it has been virtually impossible, when using conventional
postal/courier
services, to achieve this. More specifically, although semen specimens can be
transported
for such prolonged periods of time if special motorized refrigeration units
are used, the
costs of such apparatus and the weight penalties incurred when air
mail/freight is
involved, are prohibitive. Thus, there has been a long felt need for an
inexpensive and
disposable container that is self contained (viz., passively cooled) and
sufficiently light to
enable ready dispatch by conventional delivery/mail services.
One example of a passively cooled, self contained transportation container,
that
has been proposed to transport equine semen is disclosed in United States
Patent
4,530,816 issued to Douglas-Hamilton on July 23, 1985. In this arrangement,
which has
been marketed under the name EquitainerT"', the specimen is enclosed in a
plastic bag and
placed in a metal cup in a manner wherein it is sandwiched between bags of
liquid, such
as water, which act as so called thermal ballast. The lower portion of the
container is
filled with a refrigerant can filled with ice or gelatinized ice. A thermal
insulating layer,
made of a vulcanized rubber, is disposed between the refrigerant (ice) and the
metal cup
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
3
(made of copper sheet 1/32" thick, for example) in which the sample and the
thermal
ballast bags are disposed. The '816 patent discloses that the optimum steady
state
temperature is close to, but slightly greater than, 0°C, and in a
temperature range of from
4°C to 10°C. When the semen specimen, which is enclosed in a
plastic bag, is placed in
the metal cup, it is allowed to cool at a rate of 0.3°C/min until the
temperature of the
specimen reaches about 5°C, it may be stored for about 30 hours.
Spermatozoa from most animal species are susceptible to irreversible damage if
exposed to a sudden drop in temperature, also known as "cold shock." It is
known that
stallion spermatozoa are more susceptible to cold shock than bovine, ovine, or
porcine
spermatozoa. Cold shock is generally considered to be the result of rapid
cooling from v
20°C to 8°C. It is known that semen can be cooled relatively
quickly from about 37° C
(99° F) down to about 20° C, but must be slow cooled at a rate
of .OS C°/min from 20° C
to 5° C (47° F). The above described and other shipping
containers have been developed
which purport to cool stallion semen at a correct, prescribed rate. Generally,
these
conventionally available containers generally cool semen over a 10 hour
period, and hold
the semen at 5° C (47° F). It is suspected that the cooling rate
achieved by these devices
is, in at least a certain temperature range, too rapid, and undesirable "cold
shock"
frequently occurs to a portion of a semen specimen. During the development of
the
present invention, however, it was discovered that, after the temperature of
collected
semen has fallen to about 15°C, the sensitivity of the semen to cold
shock is heightened.
In overlooking this fact, it is believed that conventionally available
containers undesirably
reduce the viability of semen samples transported/stored therein.
Of the factors believed to influence the length of time over which viable
semen
specimens can be successfully transported, water is particularly toxic and
exposure to
even small amounts of moisture is highly injurious to semen. The fact that the
preferred
refrigerant disclosed in the '816 patent is ice or a mixture of ice and
gelatin water, is also
suspected to have an adverse effect on the longevity of a semen sample
transported
therein, particularly after the ice melts and assumes a fluid liquid form. A
drawback with
this prior art arrangement is that it tends to promote the accumulation of
substantial
amounts of condensation, such as water, which not only aggravates the problems
associated with the spildextended semen (causing objectionable odors, and
providing an
environment in which various types of microorganisms may spawn and render it
very
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
4
difficult to maintain aseptic conditions), but, as mentioned above, also
effectively
functions as a powerful spermicide. Accordingly, during transport this
condensate can
seep into the container in which the semen is stored, and present a serious
risk of
catastrophic damage to the semen sample. It is also noted that plastic thermal
ballast bags
of the kind disclosed in the container of the '816 patent are filled with a
colored liquid
that has a high thermal inertia (high heat capacity) as does water. Leakage of
these
containers, which may occur during a rough transit, and/or poor
storage/handling by the
end user prior to disposition in the container, also presents the same problem
because
condensate tends to accumulate.
One attempt to improve upon the arrangement disclosed in the '816 patent took
the form of a foamed plastic container that was marketed under the name Equine
ExpressTM. This arrangement provided a simple plug-like door and a
commercially
available bottle type of refrigerant pack that was placed in the container on
top of a
thermal insulating layer which was interposed between the refrigerant pack and
the
samples. However, this arrangement proved to be unable to cool and maintain
the sample
in the required condition for more than about 45 hours, as is shown in test
results reported
herein.
Accordingly, there remains a need for a container that allows for transporting
samples of equine semen over long distances while at the same time maintaining
the
motility and fertility of the transported spermatozoa for at least 48 hours
without the
attendant disadvantages of conventionally available containers and methods.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an arrangement that
enables the
shipment of equine semen over long distances, including shipment overseas,
wherein
delays often occur, e.g., at customs and at any of multiple transfers between
different
modes of transport.
It is a further object of the present invention to provide an arrangement that
enables an equine semen sample to be maintained in a motile and fertile
condition for
spermatozoa for a prolonged period of at least three days from the time of
collection.
It is another object of the present invention to provide an arrangement which
enables the economic transport of a highly degradable material, such as equine
semen, in
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
S
a disposable, totally self contained, passively cooled, inexpensive container
that has
essentially no moving parts, and that is not equipped with elaborate
thermostatically
controlled cooling arrangements or the like, for periods of time approximating
at least
about three and a third days.
It is yet another object of the present invention to provide an arrangement
that is
both disposable, capable of receiving approval from the USDA-APHIS, and which
can be
properly sealed and used for intercontinental transit, for example.
The above and other objects are accomplished by a container arrangement for
transporting semen, such as equine semen, comprising:
~ at least one semen storage device or container for storing equine semen;
a rigid foamed plastic container comprising:
an interior with a bottom;
a floor located on the bottom of the interior;
at least one support member contoured to support the at least one semen
storage device;
an upper end; and
an upper edge located at the upper end and containing a recess therein
extending continuously thereabout for receiving at least one ridge extending
from a lid,
the lid for closing the upper end of the rigid foamed plastic container when
it is open,
wherein the lid is formed with at least one ridge that extends continuously
about a lower
surface of the lid proximate an edge of the lid, wherein the at least one
ridge is adapted to
be received in the recess of the rigid foamed plastic container in a manner
that forms a
seal when the lid is pressed onto the upper end of the rigid foamed plastic
container;
a refrigerant pack containing a solid foam coolant; and
a separating means disposed in the interior of the rigid foamed plastic
container
for separating the at least one semen storage device from the refrigerant
pack, and for
limiting the cooling rate of the at least one semen storage device, when
containing an
equine semen sample, to no more than about 0.20°C/min over at least the
temperature
range of about 38°C to about 5°C.
The present invention is also directed to a method for transporting semen,
such as
equine semen, comprising the steps of:
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
6
mixing an extender solution with a sample of equine semen to form an extended
semen mixture, wherein the extender solution has a pH and an osmolality, each
of which
has been previously adjusted to within a respective predetermined range;
introducing at least a portion of the extended semen mixture into at least one
semen storage device and capping the semen storage device;
placing the at least one semen storage device on at least one support member
provided therefor in the bottom of a rigid foamed plastic container, wherein
each support
member is contoured to accept at least one semen storage device;
placing a ported thermoregulating plate in the rigid foamed plastic container
in a
manner which partitions the interior of the container into a lower compartment
in which
the semen storage device is disposed, and an upper compartment in which a
refrigerant
pack containing a solid foam coolant is disposed, wherein the thermoregulating
plate is
formed of a rigid foamed plastic, and overlays the at least one support member
formed in
the bottom of the rigid foamed plastic container in a manner that closes each
port so as to
restrict fluid communication between the upper and lower chambers;
applying a lid to the rigid foamed plastic container in a manner that closes
an
upper end of the rigid foamed plastic container, wherein the lid comprises a
rigid foamed
plastic material, at least one sealing rib which extends about a lower surface
of the lid that
is snugly received in a recess which extends continuously about the upper end
of the rigid
foamed plastic container; and
enclosing the rigid foamed plastic container with the lid closed in place in a
corrugated cardboard box.
Further, the present invention is directed to a container arrangement
comprising:
a single corrugated cardboard blank adapted to be folded into a double-walled
box
having a single site of entry;
a plastic strip embedded in the cardboard blank and extending a full length of
the
blank;
first and second holes formed in the blank in a manner wherein the first and
second holes pass through the plastic strip, and are located proximate first
and second
ends of the cardboard blank; and
the first and second holes are located proximate one another when the double-
walled corrugated cardboard blank is folded into its box configuration in a
manner that
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
7
permits a lead to be passed therethrough to enable the double-walled
corrugated box to be
locked against unauthorized entry.
The combination of some or all of the above-mentioned features exhibits is
effective for storing/transporting semen for up to at least 77 hours. In
addition, upon
arrival at its destination, the amount of time that is required between
opening the foamed
plastic container and actually injecting the semen during an insemination is
minimal.
Thus, the present invention, ensures that the spermatozoa are delivered to the
actual point
of use with minimum degradation.
Additional objects and attendant advantages of the present invention will be
set
forth, in part, in the description that follows, or may be learned from
practicing or using
the present invention. The objects and advantages may be realized and attained
by means
of the instrumentalities and combinations particularly pointed out in
throughout this
description and the appended claims. It is to be understood that the foregoing
general
description and the following detailed description are exemplary and
explanatory only
and are not to be viewed as being restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in, and constitute a part of
the specification, illustrate embodiments of the present invention and,
together with the
description, serve to explain the principles of the present invention.
Fig. 1 is a schematic side sectional view showing an embodiment of the rigid
foamed plastic container of the present invention in a fully assembled state.
Fig. 2 is a perspective view showing details of the rigid foamed plastic
container,
which forms a vital part of the invention.
Fig. 3 is a plan view of the rigid foamed plastic container depicting the
layout of
the support/partition members, and showing a recess or channel which
cooperates with a
lid.
Fig. 4 is a side sectional view taken along section line IV-IV of Fig.3,
showing
details of the interior of the rigid foamed plastic container and the
support/partition
members, which are used to maintain at least one semen storage device in
position during
transit.
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/z5310
8
Fig. 5 is a perspective view of the lid that is used to close the rigid foamed
plastic
container depicted in Figs. 1-4.
Fig. 6 is a plan view showing the underside of the lid depicted in Fig. 5.
Fig. 7 is a sectional view taken along section line VII-VII of Fig. 6.
Fig. 8 is an end view of the lid showing the provision of recesses, which
facilitate
the opening of the rigid foamed container upon arrival as a destination.
Fig. 9 is a perspective view of a ported thermoregulating plate, which is
disposed
in the rigid foamed plastic container to define a lower storage chamber and an
upper
refrigerant chamber.
Fig. 10 is a plan view of the thermoregulating plate depicted in Fig. 9.
Fig. 11 is a sectional view taken along section line XI-XI of Fig. 10.
Fig. 12 is a plan view showing the configuration of a cardboard blank, which
can
be folded into a double-walled box structure that is used to house the rigid
foamed plastic
container-with-lid arrangement shown in Fig. 1.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
All patents, patent applications and literatures cited in this description are
incorporated herein by reference in their entirety. In the case of
inconsistencies, the
present disclosure, including definitions, will prevail.
The present invention is directed to a relatively inexpensive, passively
cooled,
transport container arrangement/system for overcoming the difficulty of
maintaining the
required motility/fertility/viability of spermatozoa for a time sufficiently
long enough to
permit the samples to be transported over long distances, including shipping
to overseas
markets. In other words, to ship such a sample to distal market sites, it is
necessary, even
with modern transport, to maintain spermatozoa motility/fertility for 72 hours
or more.
During the development of the present invention, it was realized that there
were several
factors that had a clear bearing on the required longevity, and that must be
considered to
achieve the required length of transportability time.
Among these different factors, the amount of time that is wasted at the
beginning
and end of the transportation of semen specimens was recognized to be
important. The
time required to move a semen sample to a bench, add extender prior to placing
it in a
CA 02337632 2001-O1-15
WO 99/27884 PCTNS98/25310
9
transport container, as well as the time for other operations relating to
unpacking and
preparation for insemination, must be taken into consideration.
Moreover, one of the measures that is taken into account in the practice of
the
present invention is the use of an extender, the pH and osmolality of which
are balanced
prior to mixing with a semen sample. This reduces the time for which the semen
is
exposed to detrimental influences such as light, atmospheric moisture, and the
like, and
enables the semen to be placed in a container or semen storage device as
quickly as is
practical. Unextended semen, maintained at it's collection temperature
(99° F) for over 1 S
minutes results in markedly reduced sperm motility at 24 hours. Therefore,
semen must
be collected, filtered, analyzed for volume, motility, and concentration,
mixed with a
suitable extender, and placed in the a shipping container within 15 minutes.
Use of an extender solution with semen processed for storage and transport is
critical in its survivability. Extender provides nutrients to the sperm cells
and contains
antibiotics to destroy harmful bacteria. Because of reduced viability, it is
believed that
mares should be inseminated with 1 to 2 billion sperm cells and a volume of
not more
than 40 ml of semen. If a stallion has a sufficient concentration, the
ejaculate may be
split and several shipments obtained from a single collection. A further
feature is the use
of an extender that contains both sucrose and glucose. While the exact degree
and
mechanism by which the use of this type of dual sugar extender is effective in
prolonging
the life of the sperm during transit has not been determined with any
certainty, is
preferred over conventional single sugar types of extenders. The amount of
extender used
is also important. The preferred amount of dilution with the present invention
is greater
than with the prior art, and can be as high as 6:1 or more (e.g., 10:1). By
way of example,
a dual sugar semen extender that can be used in accordance with the present
invention
may be formulated, without limitation, as follows:
Nonfat dry milk solids (from Sanalac, e.g.) 24.0 gram
Glucose 26.5 gram
Sucrose 40.0 gram
Potassium Penicillin G (Sigma # P7794) 1,000,000 units
Amikacin Sulfate (Sigma #A2324) 1 gram
Cell culture tested water (Gibco #15230-147) q.s. to 1000 cc
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
After thoroughly mixing the above ingredients, the pH and the osmolality are
adjusted using an aqueous solution of sodium bicarbonate until the pH falls in
the
approximate range of about 6.85 - 7.0 and the osmolality exhibits a value in
the
5 approximate range of about 350-365 mOsm/l. Alternatively, it is possible to
add a
suitable amount of dry sodium carbonate powder or crystals directly to the
above
formulation once the appropriate amount is accurately determined.
Examples of antibiotics that may be added to an extender used in accordance
with
the principles of the present invention include, without limitation,
penicillin G,
10 streptomycin, gentamicin sulfate, ticarillin, polymyxin B sulfate, etc.
Penicillin G
typically contains approximately 1600 units per milligram; thus, a typical
quantity is
about 625 mg. For each gram of solid material is used, approximately 1 cc of
water is
subtracted from that which is required to produce the final volume of 1000 cc.
The
inclusion of gram positive and gram negative antibiotics in the semen extender
solution
1 S enhances the success of the insemination that is carned out after
transportation to a
destination. As previously mentioned, both of these types of microorganisms
are found in
the reproductive tracts of male and female horses, and the proliferation of
such
contaminating bacteria during transit can have a detrimental effect on the
insemination, as
well as lead to an abortion inducting infection in the recipient mare. In
addition,
adjustment of the pH and osmolality of the semen extender solution prior to
mixing with
the semen has the clear advantage of reducing the amount of time over which
the delicate
semen sample is exposed to deleterious effects.
Possibly the single most critical factor affecting the motility, fertility,
viability,
and longevity of spermatozoa of semen samples is the cooling rate of a semen
sample
once placed in a transport container. In particular, the present invention is
based, in part,
on the observation that semen samples that are protected against cold shock,
and that are
stored at a reduced temperature, tend to survive longer than samples that are
maintained
at room temperature. Although it has been recognized that slow cooling rates
may be
important in maintaining the longevity of a semen sample, the phases of the
cooling have
heretofore not been addressed. Rapidly cooling in a temperature range of about
20 to
about 15°C has little detrimental effect on the motility/fertility of
spermatozoa. However,
below about 15°C, the rate of cooling has a marked injurious effect. In
fact, it has been
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
11
discovered during the development of the present invention that a cooling rate
of less than
about 0.20°CJmin after the temperature of the sample has lowered to
about 15°C, is
essential to prolonging the effective insemination life of equine semen
samples. Even
slower cooling rates after the temperature is reduced to about 15°C,
e.g., preferably no
more than about 0.10°C/min, most preferably about 0.01 °C/min,
provide better chances
for maintaining the motility/fertility of spermatozoa over long periods of
time. The
achievement of such a slow rate of cooling, followed by a prolonged
maintenance of an
optimum transport temperature (e.g., preferably about 5°C to about
10°C, most preferably
about 7.5°C to about 8.5°C) was found to be extremely difficult
to achieve in a passively
cooled environment, i.e., without the use of powered refrigerating devices.
Efforts to
insulate the sperm samples from the refrigerant in order to slow the cooling
rate, while
allowing the efficient use of the refrigerant over a prolonged period, often
met with
failure. The task was rendered even more difficult when attempting to achieve
an
economically viable cost per unit by using a simple, inexpensive, disposable,
1 S self contained, passive refrigerant package.
To overcome this problem, the present invention preferably uses a
refrigerant/cooling pack as a coolant. Although any suitable coolant or
refrigerant
capable of achieving a cooling rate within the above parameters may be used in
accordance with the principles of the present invention, a solid foam-type
coolant was
found to be preferred. This type of coolant permits the overall cooling rate
to be lowered
below that possible with phase change types of coolants/refrigerants such as
ice. An
example of a preferred coolant that can be used in accordance with the
principles of the
present invention is a foam brick coolant (MCC FPP31, commercially available
from the
Midlands Chemical Company). This preferred coolant was found to exhibit the
slowest
thawing, and is capable of remaining colder for longer periods of time
compared to a
variety of other types of refrigerants tested. Moreover, the preferred coolant
contains a
foamed material that is constantly solid, and does not undergo any perceptible
solid-liquid
phase change as heat is absorbed thereby. In other words, the preferred solid
foam
refrigerant is unlike frozen water, for which the change from a solid state
(i.e., ice) to a
molten state (i.e., water) follows a scenario that reflects certain physical
chemical
characteristics of water; namely, the solid-to-liquid transition of water
first exhibits a
powerful heat absorbing capacity as the ice converts to water, followed by a
secondary,
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
12
much reduced heat absorbing capacity as the cold water absorbs more heat. The
only
shortcoming found with the MCC FPP31 refrigerant pack was that it required a
slightly
longer period to freeze than other commercially available refrigerants tested.
While
capable of providing the critical cooling rate, the MCC FPP31 solid foam
refrigerant was
also found to exhibit another advantage. If the pack in which the foam brick
is enclosed
is compromised during transit, the problem of toxic liquid water running loose
in the
container is totally eliminated.
When the preferred coolant pack is combined with a thermoregulating plate that
separates the refrigerant from the semen samples, it is possible to enhance
the full
potential of the present invention. The plate may be ported so as to permit
relatively free
insertion of an operator's fingers to permit ready removal of the plate. In
addition,
ribbing, in the form of support/partition members, may be incorporated on the
inside
bottom of a rigid foamed plastic container to close off the ports and prevent
unrestricted
fluid communication between the respective chambers storing the semen samples)
and
the refrigerant pack. Any suitable material, thickness and density of the
thermoregulating
plate made of any suitable material may be used to control the amount of heat
that is
transferred to the solid foam refrigerant/coolant pack (to cause cooling), to
prevent the
spermatozoa from being cooled at an undesirably excessive rate. Because the
preferred
solid foam cooling pack exhibits a flatter curve of heat absorption versus
temperature,
i.e., it does not undergo a phase change with a concomitant large change in
heat capacity,
it was discovered that the ported thermoregulating plate can be thinner than
the plates
used to protect spermatozoa in prior art devices that utilized solid-to-liquid
phase change
coolants. A thinner ported thermoregulating plate also prevents over
insulation, which
otherwise tends to defeat effective cooling once the coolant has completely
converted to
the liquid phase, with its lower cooling capacity.
Further, the lid of the rigid foamed plastic container may be formed from any
suitable material, e.g., a rigid foamed plastic such as polystyrene or
polyurethane. In
accordance with the present invention, such a lid is formed with at least one
continuous
rib that extends about the periphery of the lower side of the lid. This rib is
arranged to be
received in a recess that continuously extends about an upper edge at the
upper end of the
rigid foamed plastic container. The sealing effect of this lid design is
believed to be of
importance in the overall effectiveness of the present invention.
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
13
Unexpectedly, the present invention eliminates the problem of condensation
formation within the container during transit. Without wishing to be bound by
the
following theory, it appears that the slow cooling rate that results from the
use of a solid
foam coolant (MCC FPP31 ), in combination with the sealing effect provided by
the
rib/recess at the lid/container interface, is responsible for the overall
success of the
present invention. In various other comparative arrangements that were tested,
it was
found that placing the coolant above the samples induced the problem of
condensation
formation, which tended to leak down into the area of the stored sample
containers. Of
course, such an arrangement produced the real danger that water (viz., the
condensate)
would seep into an imperfectly sealed containers) and cause at least partial
spoilage of
the samples) contained therein.
A further factor that is believed to contribute to the success of the present
invention resides in the semen storage device in which the semen samples are
stored
during transit. It is critical that the semen storage devices be both sterile
and free of any
chemicals or compounds that have spermicidal activity, or that otherwise could
have any
kind of negative effect on the longevity of spermatozoa stored therein. In
accordance
with the principles of present invention, preferred semen storage devices
include, without
limitation, sterile plastic syringes or free standing tubes made of
polypropylene with caps.
Use of such semen storage devices in accordance with the present invention
requires
selection of a size (volume handling capacity) that is sufficient to
accommodate a large
quantity of extender that may be added therein in proportion to the quantity
of semen
thereby permitting the mixing of an increased volume of extender with a given
volume of
spermatozoa/semen. The beneficial effects of using larger syringes and
relatively greater
volumes of sugar extender include, without limitation, (1) a smaller surface
area for a
given volume of semen, which attenuates the cooling rate of the contents, and
(2) the
immediate use of the material for insemination upon the opening of the
container.
Preferred sizes include, without limitation, from about 10 ml to about 225 ml,
with a more
preferred size being about 50 ml.
Further, it is known that exposure to radiation in the visible light,
ultraviolet or x-
ray ranges can have a detrimental effect on the viability of cooled semen. To
overcome
this problem, the semen storage device can be opaque or fitted with a
disposable,
removable, and form-fitting protective sleeve/tube that is capable of
preventing harmful
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
14
light, UV or x-ray radiation from penetrating the semen storage device and
reaching the
contents therein. The form-fitting sleeve may be made of any suitable material
or
combination of materials so long as it is capable of blocking harmful
radiation from
reaching the semen stored within the semen storage device. In a preferred
embodiment,
the form-fitting sleeve is a tube or cylinder that is disposed around the
semen storage
device and has a reflective foil layer affixed to the inner and outer sides of
the tube.
Unexpectedly, it has been discovered that use of a form-fitting sleeve is
advantageous
because it further attenuates/slows the cooling rate of the stored semen
during transport.
Further, an extender solution is preferably added to dilute a semen sample
contained the semen storage device of the present invention. As mentioned
above, use of
an extender enhances longevity of a sperm's motility, provides vital
nutrients, increases
the volume of the inseminate thereby allowing for the insemination of more
than one
mare, helps protects spermatozoa from extreme environmental changes that occur
while
cooling (including thermal shock and toxic materials that produced upon the
death of a
spermatozoa), and reduces bacterial contamination of the sperm. The amount of
dilution
with an extender is preferably about 3:1 to about 10:1, more preferably about
4:1, most
preferably about 6:1 (v/v). Within this range, the cascade effect, which can
kill an entire
initially viable semen sample, has been found to be greatly reduced or
eliminated.
Preferably, the extender contain at least two different sugars, which is
believed to have a
positive effect on spermatozoa longevity. Further, because both gram positive
and gram
negative microorganisms are found in the reproductive tracts of stallions and
mares, an
antibiotic may be added to the extender to promote successful insemination
carried out
after transport. It has been realized that semen contaminating gram positive
and gram
negative bacteria proliferate during storage and transport, and potentially
not only
diminish the viability of the spermatozoa, but also may lead to abortion
inducing
infections in recipient mares.
More specifically, the present invention resides in a container
arrangement/system
for transporting equine semen comprising:
at least one plastic syringe or polypropylene tube with cap containing equine
semen; a container made of a rigid foamed plastic material, the rigid foamed
plastic
container including a support structure for supporting the at least one
plastic syringe or
polypropylene tube;
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
a lid for closing an open upper end of the container, the lid being formed
with at
least one ridge which extends continuously about a lower surface of the lid
proximate an
edge of the lid, the ridge being adapted to be received in a recess which
extends
continuously about an upper edge of the rigid foamed plastic container in a
manner which
5 forms a seal when the lid is pressed onto the rigid foamed plastic
container;
a refrigerant pack containing a solid foam coolant; and
a separating means disposed in the rigid foamed plastic container for
separating
the at least one plastic syringe or polypropylene tube from the refrigerant
pack, and for
limiting the cooling rate of the syringe to no more than about
0.20°C/min, preferably no
10 more than about 0.10°C/min, most preferably about 0.01
°C/min, in a temperature range
of about 15°C to about 5°C.
In this embodiment, the separating means comprises a ported thermoregulating
plate disposed in the interior of the rigid foamed plastic container to
separate a lower
chamber in which the support members (which also serve as partition members)
are
15 formed and in which the plastic syringes) and/or polypropylene tubes) is
supported, and
an upper chamber in which the refrigerant pack containing the solid foam
coolant is
disposed, the thermoregulating plate having first and second ports formed
therein at
opposite ends of the thermoregulating plate, the first and second ports being
arranged to
overlay a support/partition member, and thus be closed in a manner which
restricts fluid
communication between the upper and lower chambers.
In this preferred embodiment, the semen sample is diluted with an extender
solution containing sucrose, glucose, dry skim milk, and preferably at least
two selected
antibiotics--one being effective against gram positive bacteria and the other
being
effective against gram negative bacteria. The semen sample is inducted into a
syringe
which preferably holds from about a 10 ml to about a 225 ml aliquot, more
preferably
about a SO ml aliquot, of extender solution. The semen sample is diluted at
the very least
with about 3 parts of extender, preferably at least about 4 parts of extender,
and more
preferably about 6 to about 10 parts of extender. Shipping volumes of extended
semen in
a semen storage device (e.g., plastic syringe or polypropylene tube with cap)
range in size
from about 10 ml to about 225 ml in the practice of the present invention. A
typical
volume is about SO to about 60 ml.
CA 02337632 2001-O1-15
WO 99/27884 PCTNS98/25310
16
In another preferred embodiment, the present invention is directed to a
container
arrangement for transporting equine semen comprising:
a thermally insulated rigid container made of a recyclable foamed plastic
material,
the rigid foamed plastic container including support members formed on a floor
thereof,
the support members being adapted to support at least one plastic syringe or
polypropylene tube with cap containing extended equine semen;
a lid for closing an open upper end of the rigid foamed plastic container, the
lid
being formed with at least one ridge which extends continuously about a lower
surface of
the lid proximate an edge of the lid, the ridge being adapted to be received
in a recess
which extends continuously about an upper edge of the rigid foamed plastic
container in a
manner which forms a seal when the lid is pressed onto the rigid foamed
plastic
container;
a ported thermoregulating plate disposed in the rigid foamed plastic container
to
separate the interior thereof into a lower chamber in which the support
members are
formed and in which the syringe or polypropylene tube is supported, from an
upper
chamber in which a refrigerant pack containing a solid foam coolant is
disposed, the
ported thermoregulating plate having first and second ports formed therein,
the first and
second ports being formed at opposite ends of the thermoregulating plate and
in positions
wherein, when the thermoregulating plate is disposed in the container in a
manner
wherein it rests atop of one or more support members, the first and second
ports are each
located over a support member so that fluid communication between the upper
and lower
chambers is restricted; and a double-walled corrugated cardboard box snugly
enclosing
the rigid foamed plastic container and lid.
In this preferred embodiment, the ported thermoregulating plate is selected to
attenuate the rate of cooling of the equine semen contained in the at least
one plastic
syringe or polypropylene tube with cap to no more than about
0.20°C/min, preferably no
more than about 0.10°C/min, most preferably about 0.01 °C/min
over a temperature range
from about 38°C to about 5°C, and especially from about
15°C to about 5°C.
The double-walled corrugated cardboard box tends to act as a wick to absorb
ambient moisture before it can reach the rigid foamed plastic container, and,
thus, in
effect, act as a vapor barrier to attenuate the invasion of water vapor into
the rigid foamed
plastic container. The double-walled structure also endows the corrugated
cardboard box
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
17
with a very high level of structural integrity, which is believed to
contribute substantially
to the commercial viability of the present invention. In other words, the box
is
inexpensive and disposable, but still is able to protect the insulating
container from
damage which is apt to occur during transit via commercial carrier/postal
services. The
lid used in the above arrangement is formed with a central boss portion that
is adapted to
fit snugly into a mouth of the upper end of the rigid foamed plastic
container. Further, the
first and second ports are so dimensioned as to permit the insertion of
digits, such as
human fingers, to additionally permit the thermoregulating plate to be quickly
and easily
lifted from the position in which it separates the rigid foamed plastic
container into upper
and lower chambers.
In a further preferred embodiment, the present invention resides in a method
for
transporting equine semen, the method comprising the steps of
mixing an extender solution having a pH and an osmolality which have been
previously adjusted to within respective predetermined ranges, with a sample
of equine
1 S semen;
inducting at least a portion of the mixture into a semen storage device
(syringe,
plastic tube/bottle with a cap) and then closing the semen storage device;
placing the syringe in the bottom of a thermally insulated container made of a
foam plastic material;
placing a ported thermoregulating plate in the foamed plastic container in a
manner which separates the interior of the foamed plastic container into a
lower
compartment in which the syringe is disposed, and an upper compartment in
which a
container containing a constantly solid, foamed refrigerant is disposed, the
thermoregulating plate being formed of a foamed plastic having a predetermined
thickness and density;
closing the ports in the thermoregulating plate by seating the
thermoregulating
plate on support/partition members which are formed in the inside bottom of
the foamed
plastic container, which are used to support the syringe in a stable position
in the
container, and which are located so that the ports are closed when the
thermoregulating
plate is in a seated position;
closing the container with a lid which closes an upper end of the foamed
plastic
container, and which is formed with at least one sealing rib which extends
about the lower
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
18
surface of the lid, the sealing rib being dimensioned so as to be snugly
received in a
groove which extends continuously about the upper end of the foamed plastic
container;
and
enclosing the foamed plastic container and lid in a corrugated cardboard box.
In this method, a suitable syringe is selected and has a surface area/volume
ratio
sufficiently low to attenuate the rate of cooling of the equine semen sample,
and provide
an increased volume for extender fluid to be mixed with the semen sample. The
extender
preferably comprises sucrose, glucose, dry skim milk, at least one antibiotic,
and cell
culture tested water, plus any chemicals (e.g., sodium carbonate or sodium
bicarbonate)
required to adjust the pH and/or osmolality of the extender fluid.
In still another preferred embodiment, the present invention resides in a
container
arrangement comprising:
a single corrugated cardboard blank adapted to be folded into a double walled
box
having a single site of entry;
a plastic strip which is embedded in the cardboard blank at manufacture and
which extends a full length of the blank; and
first and second holes formed in the cardboard blank in a manner wherein the
first
and second holes pass through the plastic strip and are located proximate
first and second
ends of the cardboard blank;
the first and second holes being located proximate one another when the
cardboard blank is folded into its box configuration so that a lead or other
attachment
means can be passed therethrough to enable the box to be locked against
unauthorized
entry, and also provide a user knowledge that the box has been entered without
permission. In one embodiment of the present invention, the cardboard may be
treated
with a water impermeable or resistive substance, e.g., wax, to create a vapor
barrier to
prevent undesired entry of moisture into the container arrangement.
This preferred embodiment of the present invention may further comprise:
a rigid foamed plastic container, including a unitary support structure for
supporting at least one storage vessel, and being so dimensioned as to be
receivable in the
double-walled box;
a lid for closing an open upper end of the rigid foamed plastic container, the
lid
being formed with at least one ridge that continuously extends about a lower
surface of
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
19
the lid proximate an edge of the lid, the ridge being adapted to be received
in a recess
which extends continuously about an upper edge of the rigid foamed plastic
container in a
manner which forms a seal when the lid is pressed onto the rigid foamed
plastic
container; a refrigerant pack containing a solid foam coolant; and
means disposed in the bottom of the rigid foamed plastic container for
partitioning
the interior thereof, separating the at least one storage vessel from the
refrigerant pack,
and for limiting the cooling rate of contents in the storage vessel to a value
that is
preferably no more than about 0.10°C/min, and more preferably about
0.01 °C.
Turning now to the figures, Figs. 1-12 show a preferred embodiment of the
present invention. Fig. 1 depicts the arrangement in a fully assembled state.
As shown,
this arrangement includes a foamed plastic container 100, which is closed by a
lid 102
that also is formed of foamed plastic. Foamed plastic container 100 and lid
102 are
enclosed within a double-wall corrugated cardboard container 106, only parts
of which
are shown for illustrative simplicity.
A ported thermoregulating plate 108 is disposed in foamed plastic container
100
so as to partition the interior of foamed plastic container 100 into a lower
storage chamber
100a, in which syringes 110 are stored, and an upper refrigerant chamber 100b,
in which
a container 112 of solid refrigerant (forming a coolant pack) is disposed. As
is best seen
in Fig. 9, ported thermoregulating plate 108 is formed with two ports or
openings 108a at
opposite ends thereof.
As is best seen in Fig. 3, the bottom portion of the interior of foamed
plastic
container 100 is provided with baffles/ribslsupports/partitions 100c, which
are spaced and
arranged to support syringes 110 in a rattle free state for smooth
transportation, as well as
one that interacts with ports of the thermoregulating plate in the manner
discussed below.
As is shown by broken lines (ports) 108a in Fig. 3, the ports are formed in
thermoregulating plate 108 in a location such that, when in position, each
port is located
atop a baffle/web/ribbing/supportlpartition that forms syringe receiving
recesses within
the bottom of foamed plastic container 100. This arrangement promotes
restriction for
fluid communication between upper and lower chambers 100a, 100b. Ports 108a
are
shaped so as to serve as openings into which digits, such as the fingers of an
operator, can
be inserted to facilitate removal of the thermoregulating plate when quick and
easy access
to one or more syringes in the lower chamber 1 OOa is desired.
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
In accordance with the present invention, thermoregulating plate 108 can, if
so
desired, be made slightly smaller than the interior dimensions of the interior
of container
100, thus permitting small clearances between the side walls of the interior
of the foamed
plastic container and the edges of the thermoregulating plate. This
facilitates access to
5 the syringes by promoting facile removal of the thermoregulating plate.
However,
excessive communication between upper and lower chambers 100b, 100a should be
avoided in order to decrease connective flows therebetween.
Container 100, lid 102, and thermoregulating plate 108 preferably may be
formed
of a rigid highly thermal insulating foam plastic, such as polystyrene or
polyurethane
10 foam, which is expanded to 2.0 Ibs./square inch. The thermoregulating plate
108 is
preferably about one-half to two inches thick. These values of thickness and
density were
found to provide appropriate insulation, and to optimize the upper/lower
chamber
division, upper/lower chamber fluid restriction, and ease-of use effects of
the plate.
As is shown in Figs. 1-4, container 100 is formed with a continuous channel
114
15 about its upper edge 100e. As shown in Figs. 5-8, lid 102 is formed with a
continuous
ridge 116, which is adapted to be snugly received in channel 114 when lid 102
is placed
on top of container 100. Lid 102 is further formed with a central boss portion
118, which
fits snugly into the open mouth of container 100.
This lid arrangement allows container 100 to be simply and easily closed off
with
20 only a minimum of external ambient moisture being subsequently permitted to
enter the
container.
In this preferred embodiment, the corrugated cardboard or fiberboard box 106,
in
which container and lid 100, 102 are disposed, is a double walled arrangement,
which
inherently adds to the thermal insulation to further isolate the container
from the effects of
ambient temperature and/or ambient temperature fluctuation. Furthermore, as
mentioned
above, this box also can act a wick to absorb moisture during transit.
Accordingly, when box 106 is closed, sealed, and ready for transportation, it
serves not only to protect container 100 against compressive forces, but also
adds
additional thermal insulation. Furthermore, box 106 provides a wicking effect
to absorb
water/moisture, which thus creates a type of vapor barrier or trap that
assists in
maintaining the interior of the container free of condensation.
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
21
The construction of cardboard box 106 is also unique in that it has a
configuration
such that, unlike conventional cardboard boxes, it has only one site or
opening via which
access to the interior thereof can be gained. In addition, cardboard box 106
has a security
plastic strip 106a that is buried from one end to the other of the cardboard
blank 106'
during its manufacture, as is shown in phantom in Fig. 12 before its being
folded along
chain lines 106b. When folded into its operative configuration, box 106 is
thus closed
and sealed in a manner which renders it essentially impossible for the
contents to be
tampered with without the recipient of the package being aware of same.
More specifically, two openings 106c are formed in two opposite ends of blank
106' within the end regions of embedded security plastic strip 106a. When
blank 106' is
folded along the dotted lines shown in Fig. I2 to form a box, openings 106c
are located
adjacent one another so that a lead, or similar member of a seal, can be
passed through the
openings. Upon the ends of such a lead being permanently connected to one
another,
such as by crimping, fusing, bonding, or the like, the interior of the box
cannot be
accessed, while the seal remains intact, without destroying at least a portion
of the
cardboard. Thus, this embodiment prevents anyone from secretly tampering with
the
contents of the box.
This box finds highly advantageous to use, according to the present invention,
in
that it is simultaneously lightweight and inexpensive, it provides increased
thermal
insulation, it acts as a vapor trap or barrier, and it can be conveniently
discarded or
disposed of after a single use. Furthermore, the double wall structure, which
results after
fully folding blank 106', results in a remarkably robust box that provides a
high degree of
structural strength and protection for the foam plastic container held within
during transit.
These properties prevent breakage/damage by the inevitable bumping and
jostling which
occurs during handling by commercial courier or postal services, especially
during
loading/unloading from one transport vehicle to another, such as at truck
terminals,
airports, train stations, and the like.
It will be noted that the use of the above described corrugated cardboard box
is
not necessarily limited to application with thermally insulated foam plastic
container 100,
and can be used to ship other items and products, e.g., electronics. In other
words, the
cardboard box of the instant invention can be purchased as a blank, and
subsequently
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
22
folded, loaded, and sealed in a manner which permits the dispatch of a variety
of items in
a secure and inexpensive manner.
In order to achieve successful shipping of delicate semen cargo, it is
preferred to
place a specimen of gel-free semen from all ejaculates in an incubator at
about 37°C
within about 2 minutes of collection. The sperm concentration is then measured
using a
densimeter. Immediately thereafter, the semen sample is mixed with a dual
sugar, pH
adjusted semen extender already in the syringes) and at room temperature by
drawing the
semen sample into the syringe(s). Each syringe is then capped with a sterile
cap 110a,
and placed in position in the bottom of container 100. Thermoregulator plate
108 is then
placed in position over top syringes 110 in the manner shown in Fig. 1.
Coolant pack 112
then is placed on top of thermoregulating plate 108 and lid 102 is fitted into
position to
close container 100. The closed container is then placed in the corrugated
cardboard
shipping container, which is then sealed closed, including use of security
plastic strip
106a with holes 106c.
Tables 1 and 2 show results of tests conducted on three prior art devices, as
well
as an embodiment of the present invention. In Tables l and 2, PRIOR ART 1,
PRIOR
ART 2, and PRIOR ART 3 represent the devices sold as EquitainerTM,
ExpectaFoal~,
and Equine Express, respectively. As will be appreciated by the skilled
artisan, the
unexpected and superior performance of the present invention over the
performance of
other prior art devices is self evident. In particular, the present invention
provided a
suitable slow cooling rate in the temperature ranges of about 16-14°C
and about 14-12°C,
as is clearly shown in Table 1. Furthermore, practice according to the present
invention
resulted in the greatest time to reach the lowest temperature, compared to all
other
arrangements tested (Table 2). Regardless of the extended time to reach the
ultimate
storage temperature, the time over which the semen could be maintained below
15°C (77
hours) was greater with the present invention than with the other arrangements
tested
(Table 2, last column), based on parameters such as the motility of
spermatozoa (total
motility, progressive motility, rapid motility, curvilinear velocity, straight
line velocity,
etc.).
CA 02337632 2001-O1-15
WO 99/27884 PCTNS98/25310
q
N
O
Z
,
m
i
U
d pp N I~ M N
O O O O
O 0 O O
i
w
w
H w
U
A o o ~ o
A ~ p
c c O
~l W N ,
O ~C
w
U
O U
0 ~ ~
~r ~ N ~' M
w ~ O O O
p
d a
H
z
0
U U
O ~ ~ N
Qi ef ~
O w O O O
'
z ~ ~
w
H
H z
U
0 N O
vp 0
w O G ~
O
d a
w V o
N .-.
w ""'' O .-~ .-. pp
0 0 0 0 0
W G7 N
W A
w z
0 0 0
U ~ A~,. 0.~ .~
23
CA 02337632 2001-O1-15
WO 99/27884 PCTNS98/25310
24
M
N
G
Z
d
y 4w
o w
a ~ ~ V .~
~
o O v~ N O
H ~ ~ ~ ~ M
C"" N
~ N k
-~ v~ E-iW
W W
H
A
A b
o z o o ~ ~ o
H -. -~ '
z H ~ ~ ~ N M vovl
w w
O 0 z H H
0 x
a
a o W
a y 3
O U o
o rn
z !r ~
o ~ ~
H o
A
W
0
W 0
1
w z
a A v~
U
W
w
N M
W
t~ O O O
U ti. a~.,
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
The foregoing description of preferred embodiments of the present invention
have
been presented only for purposes of illustration and description. It is not
intended to be
exhaustive or to limit the invention to the precise disclosed forms. Many
obvious
modifications and variations of the disclosed preferred embodiments will be
apparent to
5 practitioners skilled in this art. Similarly, any process steps described
might be
interchangeable with other steps in order to achieve the same result. The
embodiments
were chosen and described to best explain the principles of the invention and
its best
mode practical application to thereby enable others skilled in this art to
understand the
invention for various embodiments and with various modifications as are suited
to the
10 ~ particular uses contemplated. It is intended that the scope of the
invention be defined by
the claims appended hereto and their equivalents.
For example, any suitable alternate gram positive and/or gram negative
antibiotics
may be used in the semen extender solution, as well as any effective broad
spectrum
antibiotic(s). In addition, security plastic strip 106a of the cardboard box
could be
1 S replaced with a strip made of a different material. As yet another
example, the box could
be made of a material other than cardboard, or a combination of cardboard and
another
material. As a further example, the ribbing/support/partition members in the
bottom of the
foamed plastic container need not necessarily extend the greater part of the
length of the
bottom of the rigid foamed plastic container, provided that ( 1 ) the plastic
syringes) or
20 other semen storage devices) were held securely in position, and (2) the
ports of the
thermoregulating plate were appropriately "covered" to provide the requisite
restriction
for fluid communication between the upper and lower chambers of the bottom of
the rigid
foamed plastic container. In addition, the rigid foamed plastic container
could be made of
a materials} different from that of the instant invention, provided that
roughly similar
25 insulating/heat transfer characteristics obtained for the final product and
the above-
described relatively slower cooling rate is not comprised. As an even further
example, a
semen extender solution with a composition different from that described
herein (e.g.,
Kenny extender) could be used, provided the degree of dilution approximated a
value or
range of values that at least overlapped the range provided by the instant
invention. As
another example, storage and transport devices that are made of materials
different from
those disclosed herein would fall within the scope of the present invention,
provided that
CA 02337632 2001-O1-15
WO 99/27884 PCT/US98/25310
26
they effectively promoted semen storage, including being sterile and
containing no
spermicidal compounds. As a final example, use of the instant invention for
storage and
transport of equine semen samples need not be so limited. Other biological
(including
semen from other animal sources) and non-biological samples requiring similar
cooling
S rates and/or storage temperatures will benefit from practice of the
disclosure herein.
Furthermore, the principles of the present invention can be applied to create
similar
container arrangements, but with adaptations for the different cooling rate or
other
parameter that is required to meet the needs of the particular biological or
non-biological
product undergoing storage and/or transport.
