WO2009123889A1

WO2009123889A1 - Device for optimizing sperm quality

Info

Publication number: WO2009123889A1
Application number: PCT/US2009/038134
Authority: WO
Inventors: B. Ariel Cohen; Leon Glass
Original assignee: Cohen B Ariel; Leon Glass
Priority date: 2008-03-29
Filing date: 2009-03-24
Publication date: 2009-10-08

Abstract

A device having a collection container an outer wall, a bottom and sealable opening leading to an interior space defined by an interior wall and bottom and filler that allows creating and maintaining a homogeneous environment to maximizes the fertility of semen and/or the ability to produce gender bias on a repeatable basis.

Description

DEVICE FOR OPTIMIZING SPERM QUALITY

RELATED APPLICATIONS

[001] This Non-Provisional application claims priority under 35 U. S. C. § 119(e) on

U.S. Provisional Application No. 61/040,652 filed on March 29, 2008, the entire contents of which are hereby incorporated by reference.

BACKGROUND

[002] Treatment of mammalian semen to increase fertility and/or achieve a higher proportion of one gender over another can be advantageous. For example, a dairy herd would obtain economic benefit from its cows birthing a higher percentage of heifers relative to bulls. In such a situation, especially with low-beef value animals such as Holsteins, the expense of bull calves — and the cruelty these animals face when used in veal production — is reduced. In addition, replacement animals for the herds are produced more efficiently. The availability of replacement animals born at the farm eliminates the need to import replacements and the attendant risk of disease introduction into a herd. Additional advantages are found for businesses housing elite sires that produce dairy bull semen. Since these bulls are evaluated, i.e. "sire-proofed," for genetic quality only through their daughters, an elite bull can be brought into semen production more quickly if he produces daughters more quickly. This speeds improvement of the sire genotype, with the attendant competitive advantage, and further produces a savings in feed, vet, and other costs associated with bull farming. It also accelerates the improvement of the genetic base of dairy herds using semen from these processors, with the attendant economic savings to dairy farmer and semen processor alike, from animals with genetically improved performance traits. Similar situations exist for other types of animals raised for dairy and/or consumption such as goats, sheep, swine, etc.

[003] In another example, optimized sperm quality can lead to improvement and/or expansion of a particular population of animals. For instance, sperm collected from elite race horses or other champion animals, such as particular breeds of dogs and cats, is commonly used for artificial insemination to maximize the probability of maintaining particular features in the gene pool. Sperm quality is particularly important in the breeding programs directed to exotic and endangered animals where the number of captive individuals is limited. Here, both the ability to increase overall birth rates and increase the number of female offspring, thereby increasing the potential for rapid expansion of the population, are critical for success.

[004] In yet another example, the suffering and costs of human sex-linked diseases can be reduced through birth of females in affected human families. Female births are the only way to eliminate over 300 X-linked diseases, many of which shorten and impair quality of life and create staggering medical costs. Currently, the costs and suffering associated with these diseases can be minimized through pre-implantation genetic diagnosis. In this process, eggs are harvested by laproscopy following injections of hormones and fertility drugs. Eggs are fertilized in vitro and, after embryos have reached sufficient size, a single cell is microdissected from each embryo for genetic analysis. A suitable unaffected female embryo is chosen for implantation. Alternatively, sperm is collected and treated with mutagenic dye in preparation for fluorescent activated cell sorting (FACS). X-bearing sperm are obtained, however, they are so damaged that the sperm nucleus must be injected into an isolated egg in vitro using intracytoplasmic egg injection. Embryos are then cultured and implanted in recipients. Both of these techniques are expensive and raise unresolved questions about the effect of exposure to DNA-binding dyes with respect to their cytotoxicity and mutagenic potential (Downeyet al. (199I) J. Histochem. and Cytochem. 39: 485-489; Durand and Olive (1982) J. Histochem. and Cytochem. 30:11 1-116).

[005] The scientific literature describes several other methods for achieving gender bias through treatment of mammalian semen. Several methods are reported for generating sex bias by physical separation of sperm, all of which involve complex laboratory manipulations and equipment. Fluorescence activated cell sorting (FACS) resolves sperm into X (female) and Y (male) bearing pools, after cell labeling with mutagenic DNA-binding dyes to reveal chromosome content (Abeydeera et al. (1998) Theriogenology 50: 981-988; Cran and Johnson (1996) Human Reproduction Update 2: 355-363). Separation based on immunological methods and cell surface markers has been proposed (Blecher et al. (1999) Theriogenology 52: 1309-1321). Separation based on a miniscule size difference between X- and Y-bearing sperm has been attempted (Van Munster et al. (1999) Theriogenology 52: 1281-1293; Van Munster (1999) Cytometry 35: 125-128; Van Munster (2002) Cytometry 47: 192-199).

[006] Sex bias without physical separation of sperm into X and Y bearing classes has also been described. For example, stress (Catalano et al. (2006) Human Reproduction 21 : 3127-3131), good or poor physical condition (Trivers and Willard (1973) Science 179: 90- 92), feed composition (Alexenko et al. (2007) Biol. Reprod. 77: 599-604), temperature (Crews (1996) Zoological Science 13: 1-13) and other factors (Wedekind (2002) Animal Conservation 5: 13-20) have been shown to affect offspring sex ratio. Lechniak et al. (2003, Reproduction in Domestic Animals 38 (3), 224-227, which is hereby incorporated by reference in its entirety) has also shown that time-based sexing of semen can occur when semen is held for various times before use in insemination.

[007] Currently, ejaculate collection containers used on-site primarily consist of bags or standard tubes, for example, BD Falcon™ 15ml conical bottom sterile polypropylene tubes (cat # 352096) or BD Falcon™ 50ml conical bottom sterile polypropylene tubes (cat # 352098).

[008] With respect to sex biasing and fertility, the problem with all of the collection devices currently available is that any sex biasing that can be obtained from the semen collected is quite unpredictable. For example, while the average bias might be 60% female to 40% male, the actual values obtained per independent ejaculate collected using the current collection devices and sex biasing techniques can range from 50% (i.e. no improvement) to 90%. This variability leads to poor consistency and in turn poor performance as it is impossible to predict the actual sex biased outcome for a particular semen sample collected.

[009] The primary reason for this unpredictability is the variability in the sperm microenvironment within the semen ejaculate. In some cases, such as the horse, the variability is a result of the fact that different pulses of ejaculate have very different biological compositions and consequently there is a gradation within the chemical microenvironment. In other cases the variability is a result of temperature differences within the semen sample. In still other cases the variability arises, in part, from the decrease in sperm quality due to manipulation during and/or after collection.

[0010] One device, described in US 2006/0147894 A 1, uses a precooled jacketed collection container containing a high heat capacity material on the principle that exposure of the semen to low temperatures early on in the collection process impacts sex biasing. Another device is a swim-up column to harvest the most motile sperm, with the possibility of also generating a sex bias (U.S. Patent 5,908,380 to Zavos et al.). This is not practical on-site, however, due to extreme cell losses.

SUMMARY OF THE INVENTION

[001 1] A detailed study of the problems associated with obtaining a sex bias and/or fertility was conducted. In the course of this study it became apparent that a homogenous and stable environment for semen was important. For example, it was found that while the device disclosed in US 2006/0147894 A 1 was thought to improve obtaining a bias by rapidly cooling the semen, thereby correcting a temperature differential problem, the device itself actually exacerbates the problem. That is, while the outside walls of the tube holding the semen are chilled from contact with the pre-cooled high heat capacity material, because the diameter of the collection tube is relatively large and heat transfer within the semen sample itself is low and little to no mixing occurs, the internal temperature at the center of the semen collection is high. Also, the device creates a large temperature gradient between semen at the top and bottom of the collection tube due to exposure of the tube bottom to a region that is only thinly insulated from the 12⁰C bath into which the tube is immersed. Therefore the device actually produces a higher temperature differential than would be obtained normally. The substantial radial gradient that results leads to a decrease in consistency and increased variability and unpredictability since sperm populations with the same characteristics (e.g. X- bearing or Y-bearing) are biologically influenced at significantly different times. The instant device addresses and solves this problem.

[0012] The claimed device provides a collection container that creates a uniform microenvironment to ensure that all sperm are exposed to the same temperature and/or the same concentration of chemical agents in the sperm suspension; i.e. the same microenvironment. Such environmental uniformity enables greater synchronization of the biological processes that are triggered across all sperm at the time of ejaculate collection. Various agents can be used to accomplish this, from chemicals such as sugar polymers, lipids and enzymes to physical agents such as time, temperature and mechanical agitation, among others. This results in a semen product having greater consistency of performance for use in artificial insemination (AI). Optimal sperm quality can be defined on the basis of numerous attributes such as motility grade, percent motile cells, percentage of intact acrosomes, or — in the case of time-based semen sexing — ability to produce the highest sex bias in offspring. It also permits recovery of all cells from the collection, and retains their integrity for use in on- site AI techniques that are typical on-farm and in-clinic.

[0013] The claimed device includes a container for use in semen collection that rapidly homogenizes and stabilizes the environment of the semen with respect to pH, chemical environment and/or temperature and other parameters. The collection container of the device comprises an outer wall, a bottom and a top opening that leads to an interior space defined by an interior wall and bottom made of a biocompatible material that contains filler which is biocompatible with semen. The filler located in the interior space is designed to maximize mixing and/or self-mixing, thereby increasing the homogeneity of the semen collection. The device's collection container can be in the shape of a tube, a cup, a cube, a cone, a cylinder, combinations thereof or any three dimensional shape.

[0014] In one embodiment the filler takes the form of a fin and is attached to the interior wall or is seamless with the interior wall. In another embodiment the filler takes the form of a spiral that is attached to the interior wall or is seamless with the interior wall. In yet another embodiment the filler takes the form of a baffle and is attached to the interior wall or is seamless with the interior wall such that there is a continuous interior airspace from the top opening to the bottom of the interior.

[0015] Another embodiment has filler that is configured into straw-like cylinders encased in a housing. Yet another embodiment has filler in the form of at least one ball which can be solid, fluid filled or layered, and/or encaged to allow movement of the semen around and between the ball. In yet another embodiment the filler is a biocompatible liquid.

[0016] The filler is made of glass or plastic or metal, or combinations thereof, is separate from, attached to or coincident with the collection container, and is biocompatible with semen.

[0017] The collection container of the device can be used alone or in conjunction with an artificial vagina (AV) or other appropriate ejaculate collection device. The collection container of the instant device is used at ambient temperature, at the body temperature of the pertinent species, at 4⁰C, or at any temperature between 4⁰C and the ambient and/or the body temperature, such as, for example, 4⁰C - 4O⁰C, 15⁰C - 4O⁰C, 18⁰C - 25⁰C, or 25⁰C - 3O⁰C. The device is equilibrated to the desired temperature prior to addition of ejaculate. Once ejaculate is added, ejaculate can be optionally mixed, either immediately or at one or more times during the equilibration process. The collection container and the device are then incubated at an appropriate temperature such as a temperature falling within the range of, for example, O⁰C - ambient temperature, O⁰C - 3O⁰C, O⁰C - 2O⁰C or 4⁰C - 15⁰C. Semen is then allowed to complete equilibration before storage or further processing. After completion of equilibration, semen can be optionally mixed prior to storage or further processing.

BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure IA-C. Side view of illustrative examples of embodiments of the collection container showing top opening, outside contour and cap.

[0019] Figure ID-F. Top view showing internal arrangements of filler.

[0020] Figure IG-I. Cut- A way view showing internal arrangements of filler. [0021] Figure 2. A. Side view of an embodiment of the collection container. B. Cut- Away view showing fin- type filler attached. C. Top view showing fin-type filler attached.

[0022] Figure 3. A. Side view of illustrative example of embodiment of the collection container and cap. B. Top view of filler having straw-like cylinders encased in housing. C. Cut- Away view of filler having straw-like cylinders encased in housing showing slating top surface of housing.

[0023] Figure 4. A. Side view of illustrative example of embodiment of the collection container and cap. B. Cut- Away view of one configuration of headspace and filler having straw-like cylinders encased in housing showing flat upper surface of housing. C. Top view of filler having straw-like cylinders encased in housing.

[0024] Figure 5. A. Partial cut-away view of illustrative example of embodiment of the collection container with filler of one type of fluid filled balls. B. Side view of one type of fluid filled ball. C. Cut-away view of one type of a fluid filled ball.

[0025] Figure 6. A. Side view of one type of encaged ball. B. Angle view of one type of encaged ball with support struts. C. Cut- A way view of ball in cage.

[0026] Figure 7. A-D. Perspective view of illustrative examples of embodiments of collection container within the device. E. Perspective view of embodiment showing housing and support filled with phase change material. F. Cross-section view of device showing housing, collection container and filler.

DETAILED DESCRIPTION OF THE INVENTION

[0027] As used herein, the phrase "phase change materials" is used to describe substances with a high heat of fusion and/or high heat capacity which is capable of storing and releasing large amounts of energy.

[0028] As used herein, the term "plastic" refers to synthetic or semisynthetic organic solid material made up of repeating monomers formed from at least some combination of the following elements: carbon, hydrogen, oxygen, nitrogen, chlorine, sulfur and silicon.

[0029] The device is designed for smooth integration into current methods of semen processing. It permits immediate mixing and/or self-mixing, the ability to create a sex bias, preserves cell numbers and maintains fertility in standard techniques of artificial insemination, while avoiding exposure of sperm DNA to mutagenic agents.

[0030] The device comprises a collection container that is specially designed to facilitate sexing by providing a very uniform environment to all sperm. Variation in the microenvironment to which different sperm are exposed creates changes in the rate and/or timing of biological processes in those subpopulations. By creating a highly controlled environment, introducing uniform conditions for exposure to elements such as pH and concentration of solutes such as bicarbonate and calcium ions, as well as rapidly equilibrating the temperature of the collected sperm, the device facilitates maintaining and/or enhancing fertility and creation of a sex bias that preserves cell number and integrity while eliminating mechanical separation. Since the rate of many biochemical processes are strongly affected by both temperature and solute concentrations, uniform temperature control and solute concentration across all cells is of great benefit. This aids in synchronization of biological processes. It also creates a larger separation between populations of sperm cells whose characteristics (e.g. X-carrying vs. Y-caπying) produce an intrinsic difference in the timing and/or rate of shared biological processes that are different in the time or speed of their inception and/or progress.

[0031] The device's collection container is designed to create a more uniform environment post-collection than is traditionally provided for sperm. The collection container can be of any size or shape that is convenient for the species from which semen is collected and is designed to permit mixing and/or self-mixing and homogenization. In one embodiment the collection container is cylindrical in shape having a top opening (9), outer walls (1) and a bottom (10). The length of the sides of the container can be longer than the diameter of the opening (e.g. prism- or tube-like) or can be approximately the same length as the diameter of the opening (e.g. cube- or cup-like). Figures IA-C and 7A-D show seven separate illustrative embodiments, each of which has an outer wall (1), a top opening (9) and a bottom (10). The bottom can be flat (e.g. 10b), rounded (e.g. 1Od), conical (e.g. 10c) or some combination thereof (e.g. 10a). The area of the wall adjacent to the top opening and that is internal to the collection container, external to the collection container or both, can be smooth (3) or ridged (2). If ridged, the ridge can be a singular ridge or plurality of ridges that encircles the circumference of the opening or a single ridge in a spiral configuration, for example allowing a cap to be twisted in place. The collection container also includes a cap (4). The cap can be rotated along a spiral ridge or fitted over the ridge(s) to seal the opening. Alternatively, the cap can be fitted inside the container opening (9), or over the opening and the smooth wall adjacent to the opening (3). The volume of the container accommodates the ejaculate associated with the species from which semen is collected. Common container volumes are 15ml and 50ml, although smaller (e.g. .05 ml to 15 ml) and larger (e.g. 50 ml to 500 ml) volumes may be desirable. [0032] The collection container is made of biocompatible material that maintains sperm integrity and viability, and which is also compatible with molding practices and with sterilization of the container interior. Suitable materials include glass, glass or metal coated with a thin layer of biocompatible plastic, biocompatible plastics and combinations thereof. Examples of plastics include polycarbonate, ethylene vinyl acetate, polypropylene. Polyethylenes made using a Ziegler-Natta vinyl polymerization or a metallocene-catalyzed polymerization, such as linear low density polyethylene (LLDPE) or high density polyethylene (HDPE) are also suitable. Additional plastics that have the required inertness, deformability and resistance to puncture include polymers of low-density polyethylene where the polymer chains are branched. Starch-derived polymers, such as polylactide, are also suitable when sufficiently inert, flexible and puncture-resistant. Additional biocompatible additives and excipients can be added to the base material to improve manufacturing, strength, durability and exposure to extreme temperatures and temperature extremes. The collection container is durable enough to withstand standard sterilization procedures such as heat, ultraviolet treatment, gas, etc.

[0033] Some embodiments are compatible with currently used collection equipment such as an artificial vagina, etc. Other embodiments can be used alone.

[0034] The device's collection container contains components that facilitate generation of a uniform environment for sperm at the time and point of collection. These components permit and/or facilitate immediate mixing and/or self-mixing and homogenization of the chemical environment as well as the temperature of the collection. The components can be both physical and chemical. In one embodiment the components are fillers which are added to or are a seamless part of the container interior, where they function not only as physical mixers but as surfaces that aid in temperature equilibration. These fillers can also serve as carriers for chemicals that modify the semen collection by dissolving into it. Fillers take various forms.

[0035] In one embodiment the filler comprises vertical fins (5) in the container interior, which are used for mixing and/or self-mixing and temperature equilibration (See Figures ID, IG and 2). In another embodiment, the filler comprises fins arranged in a spiral pattern (6), such that the flow path of fluid contacting the fins would be along the fins themselves (see Figures 1 E and IH). This causes greater exposure of fluid to the tube surface and thus an enhanced rate of mixing. In yet another embodiment the filler comprises a series of horizontal baffles (7) (See Figures IF and II). Here, the baffles jut out from the inner side walls of the collection container into the interior space or they completely traverse the interior space. In the latter case the baffles contain openings so that the entire interior space of the container is fully connected from the bottom of the container to the top opening. In each of these embodiments the filler serves to immediately mix the semen as the semen collection passes along the filler.

[0036] In yet another embodiment, the filler is a series of circular, oval or polygonal straw-like containers (13) that have an opening (1 1) at only one end and which are enclosed in a housing (14) (See Figures 3 and 4). Typically the straw-like containers have a diameter of between 0.2-0.5mm, such that a cross section through the middle of the container would resemble a honeycomb, while a cross section from the upper portion of the container would consist of the container wall encasing empty space. The housing is sealed into the interstitial spaces between the straw-like containers and can be a solid mass or a shell filled with insulating materials (15). Suitable housing materials include plastics, metals, glass, foam and combinations thereof while suitable insulating materials include foams, phase change materials and combinations thereof . The housing is either removable from the collection container or is attached to the collection container or is a seamless part of the collection container. The top of the housing (16) traverses the area defined by the collection container opening. In addition, the housing is such that an air space or head space exists between the opening of the container and the middle of the top surface of the housing (12). Consequently, the conformation of the top surface of the housing can be flat or in the shape of a funnel or cone. In this embodiment, the collected semen are pooled and mixed within the head space before filling the straw-like cylinders. Further mixing and temperature equilibration can occur along the interior surface of the straw-like cylinder.

[0037] In another embodiment, the device comprises at least one collection container in the form of a tube, cylinder, prism, cone or cube or combinations thereof within an outer housing (see Figure 7). The housing can be in the shape of a cylinder, cone, cube, prism or other three dimensional shape or combinations thereof, some few examples of which are illustrated in Figure 7. The collection container can be removable, permanently attached to the outer housing or formed by the housing itself. The housing can be seamless or comprised of more than one part. This embodiment comprises a top (23), an external wall (25), an external bottom (26), and a collection container comprising an opening (9), an internal wall (27), an internal bottom (28), an outer wall (1) and a cap (4). The top (23) of the device can be sealed to the external wall (25) or can be removable. The opening (9) to the collection container can be in the same plane as the top of the device (23) or can be raised above. If the opening is in the same plane as the top of the device, the internal wall (27) that is adjacent to the opening can be smooth (3) or ridged (2). If the opening to the collection container is raised above, the internal wall adjacent to the opening or the outer wall adjacent to the opening, or both, can be smooth (3) or ridged (2). In either case, if ridged, the ridge can be a singular ridge or plurality of ridges that encircles the circumference of the tube or a single ridge in a spiral configuration, for example allowing a cap to be twisted in place. The collection container also includes a cap (4). The cap can be rotated along a spiral ridge or fitted over the ridge(s) to seal the opening. Alternatively, the cap can be fitted inside the collection container opening (9) or over the opening and the smooth wall adjacent to the opening (3).

[0038] The space between the outer wall of the housing and the internal wall of the collection container can be of variable size. The space can be filled with a gas, liquid, gel or solid material (see, for example, Figure 7 E). The space filling material has insulating properties. Appropriate materials include foam, organic phase change materials (e.g. paraffin and fatty acids), inorganic phase change material (e.g. UTEK® by ThermoSafe® product # 412), eutectic phase change material (e.g. PlusICE™) and combinations thereof. Fine particulate agents such as sands, talcs, powdered agents such as ground glass or other materials of this nature, with or without admixture with wetting agents such as polyethylene glycol (e.g., PEG-3350), polyvinyl pyrrolidine, or adhesive agents such as liquid glues can also be used.

[0039] The device itself may be made of a substance of suitable heat capacity and thickness so as to constitute the outer insulating agent, with an open internal space that comprises an interior tube, which may optionally be lined with a disposable removable liner that is single use, as described below. Alternatively, the device may be designed with an inlet and outlet for a water jacket, which is optionally within the housing, to create a controlled temperature drop in the interior vessel by changing the temperature of the water flowing through the jacket.

[0040] In embodiments where the collection container is removable or attached to the housing, the collection container can be supported from the bottom and/or from one or more side. The support can be in the form of a pedestal (29), a cylinder, a cone, a band, a ring (31), a solid or gel-like mass (32), a solid or perforated flat or curved disk (33) or other support, and combinations thereof (e.g. 30). Appropriate materials include foam, plastic, metal, wood and stone. The support can be solid or a shell filled with insulating material such as phase change material. The support is configured in such a way that the internal bottom of the collection container is provided a similar insulating environment compared to the internal wall that is adjacent to the housing.

[0041] In another embodiment, the filler is made of at least one ball (17). In some embodiments the ball is a solid mass made glass, plastic, metal, or combinations thereof. In some embodiments the ball is fluid-filled (for example, see Figures 5B and C). A fluid filled ball can be a hollow globe (18) made of glass or plastic or metal, where the metal surface is passivated with a suitable coating, filled with water or another agent (20). The thickness of the globe is such that heat or cold transfer from the fluid filled center to the semen in contact with the globe is of the desired rate, is rapid and is efficient.

[0042] In some embodiments a coating (19) is optionally applied to the outer surface of the ball or globe to increase semen stability. In another embodiment, the ball or fluid-filled ball is encaged to create space between adjacent balls and/or fluid-filled balls, such that semen/liquid can flow around and between the suspended balls (See Figure 6). The encagement can take the form of two halos aligned to prevent the ball from exiting the space defined by the halos (21). The halos can be free from each other or connected. The halos can also be free from the ball or attached to the ball by at least one strut (22) which holds the ball in the center of the space defined by the encagement. The halos can also be attached to the inner wall of the collection tube. In another embodiment, the ball or balls, without halo, can be attached to the container by at least one strut.

[0043] The size of the filler can be variable and adapted to the composition from which it is made. The filler can also be seamless with the collection container, attached to the collection container, separate from and added to the collection container and/or separate from the collection container but becoming attached to the collection container after addition thereto.

[0044] In one embodiment filler has a removable plastic covering that provides any necessary components or conditions for homogenization of the chemical environment. An example is a plastic liner that is placed inside the container in such a manner that semen is separated from contact with the filler, yet enjoys the chemical and/or temperature benefits of the filler. In one embodiment the removable plastic liner is a sterile form-fitting liner composed of a biocompatible material which preserves sperm integrity. After collection, the liner is drawn upward or emptied and any fins or baffles present, especially if spiral, effect a further mixing of the liner's contents that further contributes to maintaining a uniform environment for the semen from collection to packing into straws. [0045] The filler coming in contact with the collected semen is such that it does not interact with or adversely affect semen viability. In some embodiments the filler is made of glass, plastic or combinations thereof. Examples of plastics include polycarbonate, ethylene vinyl acetate, polypropylene and polyethylenes made using a Ziegler-Natta vinyl polymerization or a metallocene-catalyzed polymerization, such as linear low density polyethylene (LLDPE) or high density polyethylene (HDPE). Additional plastics that have the required inertness, deformability and resistance to puncture include polymers of low- density polyethylene, where the polymer chains are branched. Starch-derived polymers, such as polylactide, are also suitable when sufficiently inert, flexible and puncture-resistant. Additional biocompatible additives and excipients can be added to the base material to improve manufacturing, strength, durability and exposure to extreme temperatures and temperature extremes. The device and/or collection container is durable enough to withstand standard sterilization procedures (such as heat, ultraviolet treatment, etc.) and storage at temperatures at or above freezing.

[0046] The materials used for filler also include liquid, either encased in plastic or "free" that is added to the container to facilitate uniform rapid environmental homogenization of both chemical composition and temperature. Suitable liquids are those that support sperm function and integrity, and include buffering and supportive agents. Appropriate agents are commercial semen diluents and extenders, TALP, glycerol, egg yolk, bicarbonate ions, calcium ions or calcium chelators and monosaccharides or oligosaccharide polymers. Suitable monosaccharides include sialic acid, mannose, fucose or galactose, while appropriate oligosaccharide polymers are linear or branched chains of any compatible sugar that terminate at their non-reducing end in fucose, galactose, sialic acid or a combination of these sugars. In addition, these agents can be attached to the filler either covalently or non- covalently as a film. These agents can be mixed with substances or treated in ways that promote their adhesion to or timed released from the filler.

[0047] The collection container has a cap (4) that can aid in further mixing the collected semen, if desired, prior to further processing. Here, the cap is applied to the collection container and once it has sealed the collection container, the container is inverted, agitated or manipulated such that the collected semen may pool in a common area within the container. The dimensions of the cap can be such that the cap can accommodate the entirety of collected semen.

[0048] The device and/or collection container is equilibrated to a desired temperature prior to use, such as any temperature between 4⁰C and the ambient and/or the body temperature, or for example, between 4⁰C - 4O⁰C, 15⁰C - 4O⁰C, 18⁰C - 25⁰C, or 25⁰C - 3O⁰C, to name but a few. The equilibration can be done rapidly or over a prolonged period of time. The purpose of the equilibration is to activate or acclimate the filler material for efficient and effective homogenization such that once semen is in contact with the filler the semen environment is homogenized rapidly. A the time of use the collection container may have an overall and uniform temperature equivalent to the ambient temperature or falling within some range with a temperature not lower than O⁰C and a temperature not higher than the body temperature of the pertinent species. To illustrate only, and without further limiting the range endpoints, such temperature ranges can be, for example, O⁰C - ambient temperature, O⁰C - 4O⁰C, 15⁰C - 4O⁰C, 18⁰C - 25⁰C, or 25⁰C - 3O⁰C

[0049] The device and/or collection container can be attached to an artificial vagina or other device used in semen collection or can be used alone. If attached to a semen collection device, ejaculate enters the device and is directed to and enters the collection container where mixing or self-mixing can begin immediately. If used alone, ejaculate is collected directly into the collection container of the invention. After collection the semen complete equilibration to the desired chemical uniformity and temperature, are then optionally mixed again, and removed from the collection container. Collected semen is then ready for additional processing. Alternatively, the semen collection can be stored in the collection container until needed. If stored in the collection container, the container is capped and the device and/or collection container placed in a controlled environment. To maintain chemical uniformity of the collection, additional mixing by inversion or agitation can optionally occur throughout semen processing and storage. Temperatures for storage range from -196⁰C - 4⁰C, such as in liquid nitrogen, the vapor phase of liquid nitrogen, -2O⁰C or O⁰C, to name but a few. Storage can occur for short or extended periods of time.

EXAMPLES

Example 1- Preparation of mixing beads

[0050] The mixing beads are washed prior to use. One (1) ml of Fisherbrand Sparkleen 1™ powder detergent (Fisher Scientific cat # 04-320-4) is dissolved in 250 ml warm water. Twenty (20) beads are added to the wash solution and soaked for 1-2 hours. The beads are then rinsed repeatedly with tap water followed by two rinses with 100 ml of distilled water prior to drying. Example 2 - Preparation and use of device

[0051] At least one mixing bead is placed in the collection container of the device, the opening to the collection container is sealed and the device placed at 32⁰C for 60 minutes or more. If a water bath is used, the device is sufficiently submerged to ensure uniform heating without leakage into the collection container of the device. Just prior to use, the device is removed.

[0052] Using standard methods, the device can be attached to an artificial vagina and sperm collected in the collection container of the device. Alternatively, sperm from frozen straws can be used.

[0053] Within 2 minutes of collection or placement of sperm in the collection container, the device is sealed and inverted once, then immediately placed at 12⁰C for 15 minutes or more.

[0054] Cell evaluation assays and further processing is then conducted.

Example 3 - Cooling Kinetics

[0055] The device was equilibrated at 32⁰C in a water bath for 2 hours. At time zero, the device was removed from the bath and 7 ml of water at 35⁰C was quickly poured into the device to simulate a collection of bull sperm. The device was immediately transferred to a 12⁰C circulating water bath.

[0056] A temperature probe was immersed at alternating high and low locations and readings were taken over time. To standardize, the high reading was taken at a point 1 ml below the surface and the low reading was taken just above the mixing ball that was resting at the bottom of the device.

[0057] This experiment was repeated using the device described in US 2006/0147894 A 1 (the '894 device). In this case, the low reading was taken at the bottom of the device.

[0058] The results are presented below in Table 1 (the) and Table 2 (measurements one embodiment of the claimed device), where the squares represent values obtained for low location readings and the diamonds represent values obtained the high location readings. Table 1 - Kinetics of Cooling - '894 device

10 15 20 25

Time (in minutes)

Table 2 - Kinetics of Cooling - claimed device

Time (in minutes) →

[0059] As can be seen from the Tables, there is a considerable temperature differential within the '894 device that is absent from the claimed device.

[0060] The data shown in Table 1 were further analyzed to reveal the temperature gradient in the device. This was done by taking the average of two bracketing points (e.g. 2 and 4 minutes) from the temperature measured at the high location and subtracting the point from the low location which falls between the first two in time (e.g. 3 minutes) The time range from 3-9 minutes was evaluated. Results are presented in Table 3 below.

Table 3 - Magnitude and Variation of Temperature Gradient

[0061] As can be seen from Table 3, the '894 device has a significantly greater temperature differential within the collection chamber than does the claimed device. The ability of the claimed device to maintain a homogeneous environment maximizes the fertility of the semen and/or maximizes the ability to produce gender bias on a repeatable basis.

Claims

CLAIMSWhat is claimed is:

1. A device comprising a collection container comprising an outer wall, a bottom and sealable opening leading to an interior space defined by an interior wall and bottom and filler.

2. The collection container of claim 1, wherein said filler is biocompatible with semen.

3. The collection container of claim 1 or 2, wherein said filler is separate from the collection container.

4. The collection container of claim 1 or 2, wherein said filler is attached to the interior wall.

5. The collection container of claim 1 or 2, wherein said filler is seamless with the interior wall.

6. The collection container of any one of claims 1-5, wherein said filler takes the form of a fin, spiral or baffle.

7. The collection container of any one of claims 1-4, wherein said filler takes the form of a ball.

8. The ball of claim 7, wherein the ball is fluid-filled.

9. The collection container of any one of claims 1-8, wherein the filler comprises glass, metal, plastic or combinations thereof.

10. The collection container of any one of claims 1 -9 comprising the shape of a tube, cup, cylinder, prism or cube.

11. The device of any one of claims 1-10 further comprising a housing.

12. The housing of claim 1 1, wherein said housing comprises insulating material.

13. The housing of claim 12, wherein said insulating material is phase change material.

14. A method of using the device of any one of claims claim 1-13 comprising

a) equilibrating the device or collection container to a temperature between O⁰C and 4O⁰C;

b) adding semen to the collection container of the device;

c) optionally mixing the semen in the collection container of the device;

c) equilibrating the device or collection container and semen to a temperature between 4⁰C and 2O⁰C and;

d) optionally mixing the semen.

15. The method of claim 14, wherein said collection container is equilibrated with the device housing.

16. Use of the device for processing semen for artificial insemination.