WO1998048622A1

WO1998048622A1 - Suspended sperm composition and method of artificial insemination

Info

Publication number: WO1998048622A1
Application number: PCT/US1998/008651
Authority: WO
Inventors: Mark A. Haubrich
Original assignee: Haubrich Mark A
Priority date: 1997-04-30
Filing date: 1998-04-29
Publication date: 1998-11-05
Also published as: AU7266398A

Abstract

A sperm suspending composition that includes a thickening agent, and a method of artificial insemination using the thickened, suspended sperm. The present invention not only provides a thickening of the semen to suspend the sperm cells and render them immotile, it is also a process in which the sperm cells are deliberately concentrated and then mixed with the diluent inside the animal at the time of insemination. This process will help revive the sperm cell, and give it adequate liquid volume to be transported to the site of fertilization. The present invention relates primarily to the artificial insemination of swine, however, it is anticipated that this invention will be useful in any species where artificial insemination is taught.

Description

SUSPENDED SPERM COMPOSITION AND METHOD OF ARTIFICIAL INSEMINATION

CROSS-REFERENCE TO RELATED APPLICATIONS This application contains disclosure from and claims the benefit under Title 35 United States Code §119(e) of United States Provisional Application, Serial No. 60/044,301, filed April 30, 1997 and entitled "Suspended Sperm Composition and Method of Artificial Insemination".

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not Applicable

AUTHORIZATION PURSUANT TO 37 C.F.R. §1.71 (d) (e) A portion of the disclosure of this patent document, including appendices, may contain material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. BACKGROUND OF THE INVENTION Field of the Invention

This invention relates generally to artificial insemination compositions and procedures, and more particularly to a suspended sperm composition and a method of artificial insemination using the composition. Description of the Related Art

Presently known artificial insemination techniques involve the handling of 1 bottle, 1 cap and 1 catheter by the technician. Anywhere from 3-5 minutes is required to complete the actual insemination of the animal. Contemporary means of extending boar semen includes several steps. The technician collects between 150 ml-350 ml of semen from the average boar. The temperature of the semen is between 33 °C and 37 °C. The technician then heats 1,000 ml of sterile water to within ± 1 °C of the semen. This water is then added with a powdered medium or extender, which is mainly glucose which provides nutrients for the semen. This mixture is then slowly added to the semen to extend the semen life as well as the volume. The total volume of semen plus water and extender are then transferred to individual 100 ml containers; i.e., bottles, bags., etc., to be used one per animal, this mixture can be stored from 1-7 days. After entering the catheter into the animal, the operator must wait anywhere from 3-5 minutes for the insemination to be completed. During this time, much flow-back or expulsion of semen from the sow may occur.

In presently known methods of artificial insemination, the first thing one must do is to prepare an extender. An extender is a culture medium that allows you to extend the life (3-7 days), as well as the volume of the semen. Most extenders contain similar basic ingredients: sugars, which are nutrients for the sperm cells, buffers to control the pH of the solution, a preservative, and an antibiotic to curb bacterial growth.

To prepare a solution with the extender, one takes a suitable size beaker and measures out 1000 ml of sterile or distilled water, then adds a predetermined amount of the powdered extender. The beaker, with this solution, is then placed in a hot water bath for at least one-half hour to allow it to equilibrate and reach a final temperature of 36°-39° C. While the extender is equilibrating and heating, one collects fresh semen, from the boar, into an insulated collection thermos.

The average boar will produce 100-300 mis of raw semen. The temperature of the semen and the extender solution must match as closely as possible. Once the temperatures have been matched, the fresh semen is placed in a suitable size beaker. The extender solution is then added and mixed in thoroughly. The total volume of the semen and extender is then placed into individual containers of 80-100 mis and is stored at 15°-19° C until ready to use. It is very important to gently rotate the stored semen twice daily. The next step is to do the actual insemination of the animal. One takes a breeding catheter comprised of a hollow plastic tube approximately 45-60 cm in length, which has an approximate constant outer diameter of 0.6 cm. On one end of the catheter is affixed a tip which is designed to non-traumatically enter the vagina and, ultimately, the cervix of the recipient. There are many different styles of breeding catheters, but all are similar in length and diameter. The main difference between catheters, is the type of tip that is affixed to the end of the catheter tube. The two main categories of tips are: bulbed tips and spiral tips. Both types of tips work well for this application. Apply a small amount of non-spermicidal lubricating gel to the tip of the catheter, and place it at an upward angle in the vaginal canal. While gently pushing forward, turn the catheter counterclockwise until you feel the catheter tip pop into the cervical rings. Take the exposed end of the catheter and attach an individual container of the prepared semen to it. Expulsion of the semen from the container is due primarily to the recurring uterine contractions of the animal, but is necessary to hold the container in an inverted position above and behind the animal to facilitate drainage. After the container is empty, one may remove the catheter by gently pulling back and turning the catheter clockwise until it is completely removed from the animal. A single insemination may take 3-5 minutes or longer.

Problems associated with contemporary artificial insemination can and do occur in almost every step of the extension and insemination process. For example, during the preparation of the extender, one must carefully weigh out the extender powder on a gram scale every time a boar is collected or one can spend extra money to have it pre-measured for you. It is also known that because of the volume of liquid one deals with, the glassware used in this process is large and difficult to hold on to. When the extension process is complete, it is cumbersome trying to fill the 14-20, 80 ml containers from the large glassware.

Transportation of the semen from farm to farm or from the stud to the farm poses problems as well, mainly do to the temperature fluctuations, which can damage the sperm cells. The proper temperature for storing or transporting semen is 15 °- 19° C. Another concern of transportation is the cost of shipping the extended semen, which includes the actual freight cost, the cooler container, and hot or cold packs depending on the season or what part of the country it comes from or is going to. It is also known that if semen is transported great distances, the sperm cells can be damaged due to the sedimentation of cells and the extender.

Storage of the semen is one of the more critical issues in artificial insemination. Once the semen enters storage, it is very important to keep a constant temperature of l5°-19° C. This requires a semen storage unit. This unit is equipped with a heating element, a thermostat, and a cooling pump. It is also necessary to rotate the stored semen at least twice daily.

The actual insemination of the animal has its share of problems as well. One places the semen containers in a Styrofoam cooler to transport it to the site of use, which is usually a specified breeding room. Animals exhibiting signs of heat will generally receive two to three services per heat cycle. Once the female is detected to be in heat, she is placed in a pen, adjacent to a boar, for stimulation. One then takes a breeding catheter and places a small amount of lubricating jelly onto the tip. The catheter is then entered at an upward slant through the vaginal canal and into the cervix. Once it enters the cervix, the technician can feel the cervical rings clamp down around the breeding tip. This clamping down on the tip means the placement of the catheter is correct. The technician then takes one of the semen containers and attaches it to the exposed end of the catheter. The container is then held in an inverted position above and behind the animal until the entire contents of the container have been liquidated. One of the problems with this process is that even though the animal is in heat they may have a tendency to walk or run about the pen. This makes it quite difficult to hold the container correctly. It also may disconnect the container from the catheter. As stated earlier, the container is liquidated primarily due to the recurring uterine contractions. This presents another problem. Animals may have a large contraction and empty the container in seconds. Due to the capacity of the cervix, the animal may not be able to quickly absorb all that was taken in; thus, it is expelled from the animal and onto the floor. Other animals may have smaller contractions that are further apart. This means that the insemination may be lengthy. It has been proven in university studies, that the longer the insemination process takes and the more females that the technician has to breed, has a profound effect on the conception rate. There are some devices, such as a back strap, that can ease technician boredom and fatigue. However, there are problems associated with these as well. Animals may move about and dislodge the strap from their back or adjoining animals may tear them off. It is also necessary to bend over and secure the strap around the animals mid-section. This results in technician fatigue as well as soiling the strap with urine and manure. Animals may also swish their .tails about during the insemination. This can also dislodge the container from the catheter and drain the contents onto the floor. Those affiliated with artificial insemination know that the above described system works, but it needs to be improved upon.

Those concerned with these and other problems recognize the need for an improved artificial insemination technique.

BRIEF SUMMARY OF THE INVENTION The present invention discloses a sperm suspending composition that includes a thickening agent, and a method of artificial insemination using the thickened, suspended sperm. The present invention not only provides a thickening of the semen to suspend the sperm cells and render them immotile, it is also a process in which the sperm cells are deliberately concentrated and then mixed with the diluent inside the animal at the time of insemination. This process will help revive the sperm cell, and give it adequate liquid volume to be transported to the site of fertilization. The present invention relates primarily to the artificial insemination of swine, however, it is anticipated that this invention will be useful in any species where artificial insemination is taught. The composition of the present invention consists of fresh semen, a culture medium, and a thickening agent. The thickening agent is a gelatin product know as VEE GEE 250 BLOOM TYPE A. The extender is a well-known medium that is referred to as SPERM AIDE. It is distributed by International Boar Semen, 30473 260^th Street, Eldora, Iowa 50627.

Therefore, an object of the present invention is the provision of an improved artificial insemination technique.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Other objects, advantages, and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings, wherein: FIG. IA is a side elevation sectional view of an insemination device including a catheter and an elongated reservoir;

FIG. IB is a side elevation sectional view of an alternate insemination device; FIG. 1C is a side elevation sectional view of another alternate insemination device; FIG. 2 is an exploded side elevation sectional view of a dispensing cartridge for use in the artificial insemination method of the present invention;

FIG. 3 is a side elevation sectional view of a dispensing gun for use with the dispensing cartridge;

FIG. 4 is a side elevational view of a vinyl bag used to store cell restart solution; and

FIG. 5 is a side elevation sectional view of an insemination device suitable for use with highly concentrated cells.

DETAILED DESCRIPTION OF THE INVENTION Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views, FIG. 1 A shows an insemination device (10) including a reservoir (12) which is approximately 15 cm long and 2.5 cm in diameter, and is tapered to the front. A catheter shank (14), being approximately 26 cm in length with a 0.6 cm outside diameter, is attached by cooperating snap lock elements (16 and 18) located at adjacent ends of the reservoir (12) and the catheter shank (14). The opposite end of the reservoir (12) carries a positioning groove (20) having a diameter of approximately 1.3 cm to 1.9 cm in diameter. Removable rubber plugs (22) are fitted into openings at opposite ends of the reservoir (12).

FIG. IB shows an alternate insemination device (110) including a hollow tip reservoir (112) adapted for semen storage. A catheter tube (114), being about 45-60 cm in length, is attached to the reservoir (112) by cooperating locking elements (116 and 118) located respectively on an adapter tube filling port (113) and the catheter tube (114). The reservoir (112) may be bulbous or spiral and the end carries a positioning groove (120). Removable stoppers (122) are fitted into the ends of the adapter tube (113) and catheter tube (114). The end (111) of the reservoir (112) is snipped off prior to use in the insemination process.

FIG. 1C shows another alternate insemination device (210) including a solid bulbous or spiral end (211). A catheter tube (214) is preloaded by filling the reservoir (212) with semen. It is attached by locking elements (216 and 218) to the solid end (211). Removable stoppers (222) are used to close opposite ends of the catheter tube (214).

FIG. 2 shows a dispensing cartridge (30) having a tube (32) including a dispensing outlet (34) and a mating closure (35). The sanitary shield (36) is a standard shield used in the animal industry. N piston (38) is pushed into the opposite end of the tube (32) once it is filled with the modified semen, and a pop-on-cap (40) covers the end. The cartridge (30) is adapted for use with a dispensing gun (50) which includes a ram (52) advanced by a ratchet mechanism operated by squeeze handles (54). A ram head (56) is disposed to engage the cartridge piston (38) when the filled cartridge (30) is placed in the gun (50) with the end cap (40) removed. The insemination devices described above are adapted to dispense the suspended sperm composition of the present invention. In preparing the composition, the collection of semen is done by known procedures. N powered semen extender is used and the present invention includes the addition of a thickening agent, such as gelatin or a similar product, to the semen extender in a predetermined ratio. The modified extender is then added to 1,000 ml of water and heated to approximately the temperature of the semen. This heated material is then added to the semen. The result, when cooled to a storage temperature of 17°C-19°C, will be a viscous material. The modified semen is then packaged in containers of 10-1,000 ml for use in conjunction with a dispensing unit.

The following examples disclose specific formulations for suspended sperm compositions. However, it is to be understood that numerous variations of the formulations are considered to be within the scope of the present invention.

EXAMPLE 1 Sperm extenders allow the extension of the volume as well as the life of semen, such as boar semen. Although several sperm extenders are commercially available, all include similar basic ingredients. These ingredients include glucose which provides nutrients for the sperm, a buffer which balances the pH to match the semen pH, a preservative, and antibiotics to control bacterial growth. Example 3 discloses a formulation using a commercial sperm extender known as "Sperm AID Powder" distributed by International Boar Semen, 30473 260th Street, Eldora, Iowa 50627. EXAMPLE 2

The thickening agent used in the following example is commercially available gelatin know as "VEE GEE 250 Bloom Type A" manufactured by VYSE Gelatin Co., Schiller Park, Illinois 60176.

This is a product derived from processing pork skin and/or tallow. There are different strengths of this product, which will result in a more firm or a lighter setup. However, only the "VEE GEE 250 Bloom Type A" has been used in the formulation of Example 3.

This product is physically inert and has not caused any immune response in any of the test animals. In order to get the desired result from the gelatin, it must be heated to at least 46° C in order to break the protein bonds of the powder and allow it to set up in a gelatin form. Failure to meet the temperature requirement will result in inadequate gelatin, and power residue in the bottom of the container.

This product will set up at room temperature, but the cooler the temperature (not below 13 °-14° C) the faster the set. This product will start to break down with heat and moisture, but does not flow freely until about 32°-33 ° C EXAMPLE 3.

In a 2,000 ml beaker, 25 grams of VEE GEE 250 gelatin powder and 68.5 grams of sperm extender powder are mixed with 1,000 ml of sterile or distilled water. The beaker is then placed in a warm water bath of about 50°-60° C for one-half hour or until the mixture reaches 46 ° C.

Fresh boar semen is collected through conventional means. Boar semen is usually between 35°-38° C. It is imperative that the mixture of gelatin and sperm extender be within ± 1 ° C of the temperature of the fresh semen or it can shock and kill it. After temperatures have been matched, the mixture is slowly added to the semen and stirred to thoroughly mix.

The resulting suspended sperm composition is then packaged into containers of 10 ml- 1,000 ml, depending on design of the dispensing unit for the composition. The composition will "set up" in approximately one-half hour to one hour at 18°-19° C at which time it is ready for use. There are many advantages in using a thickened suspended semen composition. They include, but are not limited to: less expulsion of semen from the female; less time spent inseminating; less technician fatigue resulting in better animal conception rates; better resistance to temperature changes than less viscous material; better storage stability due to even dispersement of the nutrients and the sperm; no settling of the sperm or the extender; no rotation of the bottles; more cost effective operation; better transportation stability due to shock- absorbing characteristics of the more viscous material; and utilization by large operations as well as individual on- farm use.

The drawings illustrate different options for actually dispensing the composition into the female. As shown in FIGS. 2-3, a sanitary shield (36) is inserted into the cervix of the female, and the free end attaches to a cartridge which is carried in a dispenser gun (50) similar to a caulking gun. The technician then pumps the trigger the required number of times to deliver 60 ml-90 ml of the suspended sperm composition. The time required to dispense the composition is approximately 10-20 seconds per animal.

FIG. IA illustrates an insemination device (10) where the technician takes a catheter with an elongated reservoir (12) within its length and places the bulbed end with the positioning groove (20) into the cervix of the female. The free end is then taped or clipped to the rump of the female. In a short amount of time, the female's body heat will start to liquify the composition, and she will inseminate herself.

EXAMPLE 4 The following example provides an overview of VEE GEE 250 BLOOM

TYPE A gelatin.

Applicant has run experiments with many different types of gelatins. Some of the gelatins used include, plain Know Gelatin, VEE GEE 100 BLOOM TYPE A, VEE GEE 250 GLOOM TYPE A, VEE GEE 275 BLOOM TYPE A, AND VEE GEE 300 BLOOM TYPE A. Applicant has also run many experiments with varying amounts of the gelatins. VEE GEE 250 BLOOM TYPE A, at a rate of 23 grams/liter, has thus far proven to be the gelatin of choice.

After the semen has been mixed with the gelatin and allowed to cool to a temperature below 27° C, it is in a solid or semi-solid form. When the gelatin and the semen are entered into the animal, the gel will return to its liquid form and the cells are released. The movement of the cells through the reproductive tract is enhanced by the addition of the cell restart solution described in Example 6 below. The solid and semi-solid form is very important to the life span and to the quality of the semen, as it prevents the culture medium and the sperm cell from settling out. It also renders the sperm cell immotile which in turn reduces the metabolic activity, and cellular waste. By rendering the cell immotile, reducing metabolic activity, cellular waste, and activity, and settling, the following is achieved: storage of semen from 19°-27° C, the ability to store heavy concentrations of sperm cells in very little carrier, the need to rotate semen has been eliminated, easier, cheaper, safer transportation, drastic reductions in time spent preparing and inseminating, cost effectiveness, product utilized by large studs, as well as on farm use, and less technician boredom and fatigue.

EXAMPLE 5 Example 5 provides a description of the composition premix. The premix will be ready to use once the technician receives it, therefore, the following will be done at the manufacturing plant. Let it be understood that to be commercially viable, the manufacturer will produce the premix in large quantities. The following description of the process will be based on a smaller quantity for better understanding.

To prepare the composition premix, pour 1,000 mis of sterile or distilled water into a 2,000 ml beaker. Measure out 23 grams of the VEE GEE 250 BLOOM TYPE A and mix it with the water. Let the mixture stand for one-half hour to allow the gelatin crystals to become completely saturated. Place the mixture in a hot water bath for one-half hour and bring the temperature to 46° C. This will allow the protein bonds of the gelatin to be broken down. Remove the mixture from the water bath and let it cool to 40°-42° C, then add 59.5 grams of the SPERM AIDE extender and mix thoroughly. At this point, the "premix" is in a liquid form. It is then transferred to 300 ml tubes for storage and transport. Depending on the temperature, the premix will be in a solid or semi-solid form and should be stored between 10°-21 ° C. The storage of the premix should not exceed 30 days.

EXAMPLE 6 Another component that is part of this invention, is a cell restart. Cell restart is a solution that is friendly to the sperm cells and is used to "chase" the sperm cells through the reproductive tract. In this application, the cell restart is a physiological saline solution. However, other solutions that are friendly to the sperm cell can also be used. Cell restart is also prepared by the manufacturer and is ready to use. Cell restart is prepared by adding 9 grams of deiodized salt to 1 ,000 mis of sterile water. As illustrated in FIG. 4, prepared cell restart solution is packaged in soft vinyl bags (60) that have a small flexible tube (62) (60 - 90 cm long) protruding from one end. The bags (60) may range in capacity from 50-1,000 mis. The free end flexible hose (62) is attached to a syringe device which delivers a predetermined amount of the cell restart solution each time the trigger is squeezed.

FIG. 5 shows yet another alternate insemination device (310) specially designed to receive semen storage reservoir straws (312) and enter them into the animal. The straw (312) is received in a catheter tube (314) including a crimp (315) which prevents the straw (312) from sliding out through the solid bulbous or spiral end (311). Stoppers (322) close opposite ends of the straw (312). EXAMPLE 7

Although the following example sets forth specific dilution rates for the semen and premix, other dilution rates that do not significantly impair or reduce the number of viable cells within said dilution is considered to be within the scope of this invention.

After fresh boar semen has been collected, the premix is heated to match the temperature of the semen and is added at a rate of V_ ml of premix to 1 ml of raw semen. For example, if a boar produced 300 mis of raw semen, you would add 150 mis of the premix. The premix solution is placed into a machine, which fills the semen storage straws described above. The total volumetric capacity of the semen storage straw is 10 mis and contains 2-3 billion sperm cells. Due to different sperm cell densities in raw semen, it is understood that the volumetric capacity of the straw may be increased or decreased to accommodate the proper cell density. Within raw semen there is a threshold where there can be only so many sperm cells per ml. It is within the scope of this invention to be able to centrifuge the semen and spin off seminal fluid resulting in a very high concentration of cells per ml. This, of course, would significantly reduce the volumetric needs of the semen storage straw. Once the straws have been filled, they can be stored at typical room temperatures (19° - 27° C). EXAMPLE 8

N special breeding catheter illustrated in FIG. 5 is used to complete the insemination process. This catheter has a larger inner diameter than that of contemporary catheters, to facilitate the semen storage straw. It also contains a crimp around its outer diameter, located one inch behind the tip. This crimp partially closes off the inner diameter, which keeps the semen storage straw from progressing forward.

Take the semen straw, and peel off both ends. Take the breeding catheter and place the straw in the end opposite the catheter tip. Insert the catheter into the animal until it is locked in place in the cervix. Take the exposed end of the catheter and attach it to the syringe. The syringe is then squeezed to deliver the cell restart solution. It is recommended that at least 50 mis of the cell restart be used to supply an adequate amount of carrier to deliver the cells to the site of fertilization. The amount of cell restart solution and the time frame in which it is delivered is up to the technician doing the insemination. For example, if the technician delivers 40 mis of cell restart and notices flow back, he may remove the syringe and move on to another animal and return later to finish the insemination once the animal has absorbed the initial amount. Once the technician has delivered the desired amount of cell restart, the catheter is removed and the insemination is complete. Actual time spent inseminating is reduced from several minutes to just seconds.

Although only an exemplary embodiment of the invention has been described in detail above, those skilled in the art will readily appreciate that many modifications are possible without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.

Claims

CLAIMS What is Claimed is:

1. A suspended sperm composition comprising: sperm harvested from an animal; a sperm extender including a sperm nutrient; and a thickening agent.

2. The composition of claim 1 wherein the sperm is contained in a seminal fluid harvested from the animal.

3. The composition of claim 1 wherein the sperm is concentrated from a seminal fluid harvested from the animal.

4. The composition of claim 1 wherein the sperm nutrient is sugar.

5. The composition of claim 4 wherein the sugar is glucose.

6. The composition of claim 1 wherein the sperm extender further includes a buffer.

7. The composition of claim 1 wherein the sperm extender further includes a preservative.

8. The composition of claim 1 wherein the sperm extender further includes an antibiotic.

9. The composition of claim 1 wherein the thickening agent is a gelatin.

10. The composition of claim 9 wherein the gelatin is present in a concentration ranging from about 20 to 30 grams per liter.

11. The composition of claim 10 where the gelatin is present in a concentration of about 23 grams per liter.

12. The composition of claim 5 wherein the thickening agent is a gelatin.

13. The composition of claim 12 wherein the gelatin is present in a concentration ranging from about 20 to 30 grams per liter.

14. The composition of claim 13 wherein the sperm extender is present in a concentration ranging from about 50 to 80 grams per liter.

15. The composition of claim 14 wherein the gelatin is present in a concentration of about 23 grams per liter, and the sperm extender is present in a concentration of about 60 grams per liter.

16. A method of artificial insemination, comprising the step of: injecting the composition of claim 1 into the cervix of a recipient animal.

17. The method of claim 1 wherein the sperm is concentrated from a seminal fluid harvested from the animal.

18. The method of claim 16 further including the step of providing a quantity of a physiological saline solution as a carrier for the composition at the time of insemination.

19. The method of claim 17 further including the step of providing a quantity of a physiological saline solution as a carrier for the composition at the time of insemination.

20. The method of claim 16 wherein the recipient animal is porcine.