US 3791384 A
A method of preserving boar sperm by deep-freezing thawing and introduction into the uterus of the sow, in which method the ejaculate after being collected is centrifuged, and the sediment is diluted, frozen to a temperature of -180 DEG to -200 DEG C, at the time of use is thawed by being placed in such a quantity of diluent warmed to 40 DEG to 50 DEG C that a dilution of the sperm which is appropriate for effective bathing of the uterus is achieved, and the diluted fluid is introduced into the uterus. The ejaculate is inactivated after being collected and before being centrifuged, and after being thawed the sperm solution has added to it a solution which mobilizes the spermatozoa.
Description (OCR text may contain errors)
Waited States Patent Richter et a1.
ARTIFICIAL KNSEMINATION F SOWS Inventors: Ludwig Richter, Trappenkamp;
Albert Liedicke, Wahlstedt, both of Germany 11. Wilhelm Schaumann, Hamburg, Germany Filed: May 11, 1972 Appl. No.: 252,272
Foreign Application Priority Data July 15, 1971 Germany.... 2135318 Oct. 30, 1971 Germany 2154260 References Cited UNITED STATES PATENTS /1969 Rajamannan 195/1.8
3,472,735 /1969 Ashiya et a1. 195/1 .8 3,519,710 7/1970 Bass 3,431,172 3/1969 Rajamannan .1 195/1.8
Primary Examiner-Aldrich F. Medbery Attorney, Agent, or Firm-Beaman & Beaman  ABSTRACT A method of preserving boar sperm by deep-freezing thawing and introduction into the uterus of the sow, in which method the ejaculate after being collected is centrifuged, and the sediment is diluted, frozen to a temperature of 180 to 200C, at the time of use is thawed by being placed in such a quantity of diluent warmed to to C that a dilution of the sperm which is appropriate for effective bathing of the uterus is achieved, and the diluted fluid is introduced into the uterus. The ejaculate is'inactivated after'being collected and before being centrifuged, and after being thawed the sperm solution has added to it a solution which mobilizes the spermatozoa.
13 Claims, N0 Drawings ARTIFICIAL INSEMTNATION OF SOWS BACKGROUND OF THE INVENTION The invention relates to a method of preserving boar sperm by deep-freezing, thawing and introduction into the uterus of the sow, and fertilization of the egg cells. The invention is furthermore directed at creating a solution of boar sperm ensuring spermatozoon motility of at least 50 percent.
In stock-husbandry, artificial insemination is in continuously increasing use for economic reasons. Whereas in the case of cattle good results have already been achieved, in the case of sows artificial insemination with deep-frozen semen has succeeded only occasionally. The reason for this is seen in the phenomenon that bull sperm is less sensitive to preservation techniques by deep-freezing than boar sperm is, a far larger percentage of bull spermatozoa surviving the preservation process.
In the hitherto employed practice of preserving boar sperm, then of thawing it and of introducing it into the uterus of the sow, the following procedure followed the ejaculate, approximately 200 to 300 ml, was centrifuged, the residual fluid was poured away, thesediment was subjected to dilution, the duly diluted sperm was frozen, i.e., was lowered to a temperature of about -1 80 to 200 C, and it was maintained at this temperature until the sperm was to be utilized. Thawing then took place according to various methods, e.g. the frozen sperm was placed into so much diluent at 40 C that the result was a solution with a degree of sperm dilution in which proper distribution of the spermatozoa in the uterus is ensured. This solution was then introduced directly into the uterus.
SUMMARY OF THE INVENTION The object of the present invention is to provide a method according to which the preservation process, the subsequent thawing and the introduction into the uterus of boar spermatozoa can be carried out in such a manner that the sperm in practice suffers no damage. After the thawing process the sperm must give evidence of a motility of at least 50 percent and, once introduced into the uterus, must possess the requisite motility.
A further object of the invention is to provide a method in which the spermatozoa in the solution demonstrate effective forward motion for at least a 4-hour period when at 39 C and to provide a boar sperm solution having a spermatozoon motility of at least 50 percent and a keeping quality of at least 4 hours at 39 C.
According to the invention, there is provided a method of preserving boar sperm by deep-freezing, thawing and introduction into the uterus of the sow, in which method the ejaculate after being collected is centrifuged, and the sediment is diluted, frozen to a temperature of 1 80 to 200 C, at the time of use is thawed by being placed in such a quantity of diluent warmed to 40 to 50 C that a dilution of the sperm which is appropriate for effective bathing of the uterus is achieved, and the diluted fluid is introduced into the uterus. The ejaculate is inactivated after being collected and before being centrifuged, and after being thawed the sperm solution has added to it a solution which mobilizes the spermatozoa.
The sperm solution is therefore subjected to thawing when already rendered inactive, so that the spermatozoa undergo the deep-freezing process while inactivated. Only upon insemination are the spermatozoa mobilized by an analeptic solution.
The inactivation process is preferably effected by the addition of an aqueous solution containing glucose and a buffer system. According to a particularly advantageous form of embodiment of the invention, thawing is effected with a quantity of diluent insufficient to achieve the desired final dilution, and the final dilution is carried out by adding to the preliminarily diluted preparation the solution that mobilizes the spermatozoa. It has been proven that the most favorable results are achieved when the thawing process occurs with the diluent at 60 to C. The thawing is preferably undertaken with an inactivating solution at a temperature of 60 to 70 C. An aqueous solution containing sodium salts, a potassium salt, glucose, a buffer system and a sulfonamide may be employed for mobilizing the spermatozoa. Preferably, the final diluting process is only effected inside the uterus, the preliminarily diluted sperm being first introduced and then the solution for mobilizing the spermatozoa being syringed into position subsequently. The dilution of the centrifuged sperm before freezing can be undertaken by means of diluent for deep-freezing which comprises an aqueous solution containing fructose, citric acid, egg-yolk, glycerine and a buffer system. The freezing process will preferably be undertaken gradually, the sperm, treated by deep freeze diluent, being maintained at 5 C for only 1% to 1% hours then being caused to drip onto CO -type dry ice so that small pellets arise, and these are then surround by liquid nitrogen. When freezing the sperm in the form of pellets, according to a further form of embodiment of the invention, the dilution is carried out with a quantity of tablets such as is required for one insemination.
DESCRIPTION OF PREFERRED EMBODIMENT The invention will now be further described by way of example withreference to a particularly advantageous embodiment.
Immediately upon being collected, the ejaculate is diluted in the ratio of l 3 with inactivating solution warmed to about 36 C. The inactivating solution may possess the following composition, for example:
87.5 g dextrose 5.4 g dihydrate of ethylenedinitrotetra-acetic acid 5.4 g sodium citrate 7.3 g sodium hydrogencarbonate to 1,00 ml aqua bidest.
The duly diluted sperm is poured into centrifuging vessels and is allowed to stand at room temperature for lb to 2 hours. During this period the temperature of the solution will drop to room temperature, and the spermatozoa will lose their motility. Afterwards the centrifuge vessels are carefully positioned in a centrifuge, and centrifuged at approximately 2,500 rpm for 7 minutes. After removal of the centrifuging vessels from the centrifuge, the residual fluid is all poured away by inverting the vessels. The sediment is then thinned by a deep-freezing diluent in the ratio of l 3. Hereinafter the composition of a suitable deep-freeze diluent is indicated:
6.06 g tris( hydromethyl)aminomethane 2.50 g fructose 3.46 g citric acid 10.00 ml glycerine 25.00 ml egg-yolk to 200 ml aqua bidest.
The sperm and the diluent are thoroughly mixed by careful stirring with a glass rod. The diluted sperm is then lowered in temperature to about C in a refrigerator for from 1% to a maximum of 2 hours. After that, the diluted sperm is applied by means of a pipette to CO -type dry ice in the form of drops which within 5 minutes freeze into small pellets. These pellets are then surrounded with liquid nitrogen.
When the sperm is needed, according to the invention, it is thawed by putting 20 to 30 pellets which is the amount required for one insemination into 20 to 30 ml respectively (i.e., 1 ml for each tablet) of inactivating solution warmed to 60 to 70 C. All the pellets for one inseminating dose are placed simultaneously in the inactivating solution. The tablets thaw quickly if the vessel is gently shaken.
The duly thawed sperm, having undergone preliminary dilution, is then inseminated, i.e., introduced into the uterus. Following this, 75 ml of an analeptic solution which mobilizes the spermatozoa and which has been warmed to a temperature of approximately 40 C is syringed into position. The solution which mobilizes the spermatosoa will preferably possess the following composition:
24.3 g sodium citrate x 2 H O 2.0 g sodium bicarbonate 0.4 g potassium chloride 3.0 g glucose 3.0 g sulfanilamide to 1,000 ml aqua bidest.
After the mixing of the individual components above, the solution is heated for minutes to 90. Given sterile storage, the solution may be kept for a lengthy period.
The method according to the invention leads to an important advance in the art, for by its means it has become possible for the first time to carry out artificial insemination with pigs in an economical manner.
During trials, at each stage of the method, samples have been taken and the spermatozoa examined microscopically for viability. in the final phase the sperm solution indicated motility of 50 to 70 percent, whereas in methods used hitherto sperm solutions have been obtained with a motility of a maximum of 30 percent. The sperm solutions produced according to the invention were good fertilizing agents and led to litters of an average of 9 to 10 piglets.
it is assumed that the immediate inactivation according to the invention, i.e., the lowering of the metabolism of the sperm immediately after the spermatozoa are collected, provides greater powers of resistance in the face of treatment involving freezing. The mode of thawing is also essential to the invention. The tablets are not placed in a solution at body-temperature but in one raised to 60 to 70 C; since by placing the tablets, cooled down to about l90 C, into the solution the latter undergoes cooling, in the method according to the invention the result is a solution at approximately body-temperature, whereas with methods as hitherto known after thawing the resulting solution is at a temperature of about C. The following features are also completely new: both the process of preliminary dilu tion upon thawing, this only slightly thinning the sperm (about 1 ml to l pellet) and also the carrying out of the thawing process with a solution which inactivates metabolism, and the production of the final concentration by means of the mobilizing solution in the uterus itself. In this manner the spermatozoa remain in a less motile state until the time when they are to be actively involved and, to a great extent, are thereby protected.
The three solutions employed in the method according to the invention may naturally be varied in their composition from those given above. The essential point is that the inactivating solution should contain a suitable buffer system and dextrose; in the deep-freeze diluent it is important that the amount of egg-yolk should not be significantly exceeded. The mobilizing effect of the third solution is brought about by the increased quantity of sodium salt. The solution must also contain a buffer system, and it is practical to add a sulfonamide in order to guarantee freedom from germs to a considerable extent.
As has already been mentioned, the sperm solution must be introduced into the uterus of the sow at a temperature of 30 to 39 C. The spermatozoa in the solution obtained according to the above-described method have a viability lasting about 2 hours at this temperature. This time is, as a rule too short in practice, for the spermatozoa need a more lengthy period before they reach the Fallopian tubes. Between the infusion and the arrival at the Fallopian tubes 4, and in unfavourable cases 7, hours may well intervene.
According to a preferred form of the invention, the above-described method is improved in the sense of increasing the resistance of the recently thawed spermatozoa. According to this preferred form of the invention, the inactivation and the mobilization process are effected with the same solution, and the deep-freeze phase is carried out with a solution which contains no additional components apart from media protecting against the freezing state. The solution employed for inactivation and the mobilization process preferably contains glucose, sodium citrate, sodium bicarbonate and antibiotics, and the dilution of the centrifuged sperm before being frozen is undertaken with a deepfreeze diluent containing skimmed-milk powder, glucose, egg-yolk, glycerine, antibiotics, ethylenediaminetetra-acetic acid and potassium chloride.
Surprisingly, it has been demonstrated that for inactivation and mobilization the same solution can be employed. Through this circumstance the entire method is substantially simplified. One is merely required to make up a single solution and keep it in store. Cases of confusion are excluded. A still more substantial advantage which is achieved consists of the fact that the boar spermatozoa, after being mobilized, are resilient at 39 C for at least 4 hours. In practice, boar sperm solutions obtained in accordance with the invention were maintained for even in excess of 6 hours at 39 C and afterwards possessed a motility of approximately percent.
The precise composition of the solution employed as the inactivator and the mobilizing agent, hereinafter designated Diluent l, as well as the solution preferably employed for deep-freezing, hereinafter designated Diluent 2, are of the following compositions:
DILUENT l 140 g, but preferably g glucose g, but preferably 12 g sodium citrate l 3 g, but preferably 2 g sulfanilanide 0.5 3 g, but preferably 1.5 g potassium chloride 1.5 5 g, but preferably 2.5 g ethylenediaminetetraacetic acid 1.5 4 g, but preferably 2.5 g sodium bicarbonate antibiotics Diluent l is produced as follows: 2 l of aqua bidest. are boiled, the chemicals are added, are briefly brought to boil and allowed to cool. Thereafter 1.7 million international units of penicillin G sodium and 1.7 million i.u. of dihydrostreptomycin base are added, and the pH is adjusted by 4 percent NaOH to 6.8. The solution thus prepared is filtered.
DILUENT 2 base. From this solution 100 ml are decanted, and 18 ml of egg-yolk as well as 6.0 ml of glycerine are added.
- Hereinafter one example will be described of carrying out this preferred form of the invention. The-ejaculate collected from a boar was thinned with Diluent 1 in the ratio of 1 part of ejaculate to 3 parts of diluent. Afterwards the diluted ejaculate was transferred to centrifuge glasses of 80-ml capacity and was allowed to sediment for 24 hours at 15 C. The sedimented ejaculate was centrifuged at 1,700 rpm for 5 minutes. This centrifuging speed and the period indicated are not obligatory. It is self-evident that centrifuging can be at both faster and slower rates, but naturally with lower centrifuging speeds the time must be correspondingly extended. After the centrifuging the centrifuged material was poured away, and the sediment in each centrifuge glass has a double to triple amount of Diluent 2 added and mixed with the semen. The semen diluted in this way was within a period of 1% hours cooled to 5 C in a reagent bottle in a water-bath of 160 ml in a refrigerator. The semen diluted and cooled in this matter was deep-frozen by the pellet method on CO -type dry ice. The period of freezing extended over 5 minutes. The pellets were transferred to transparent plastic tubes and stored in liquid nitrogen.
When the sperm was required, for the thawing procedure the entire contents of a plastic tube were placed in 30 ml of Diluent l warmed to 50 C, and they underwent thawing and dilution by vigorous shaking in a matter of seconds. Following this, the thawed semen was mixed with Diluent 1 at the same temperature to produce a total of 100 ml. The sperm solution was transported in a vessel to the location where it was required. During insemination, first approximately 50 ml of Diluent l was infused through the catheter into the cervix and uterus. Then the sperm solution was introduced, and ml of Diluent l was thereafter infused.
The sperm solution obtained through this preferred form of embodiment of the method according to the invention may be kept, as already stated, at a temperature of 39 C for a considerable period. This temperature prevails in the uterus after the deep-frozen pellets have been thawed in diluent warmed to 50 C and expanded to ml with Diluent l at a somewhat higher temperature than that of the thawed sperm solution. It has been shown that sperm solution produced in this way possess spermatozoon motility of over 50 percent after the lapse of 6 hours, and indeed after 8 hours. The solution led to litters of 10 to 15 piglets.
By means of the invention, deep-frozen boar sperm pellets have also been created which are characterized by a content of immobilised boar spermatozoa capable of being mobilized embedded in a mass of frozen water which contains small amounts of glycerine, sodium citrate, potassium chloride, ethylenediaminetetra-acetic acid, sodium bicarbonate, albuminous substances and antibiotics. These pellets may be preserved for any desired length of time under liquid nitrogen. After the thawing of the appropriate number of pellets with Diluent l at a temperature of 50 C, a solution of boar spemi will be obtained having a spermatozoon motility of at least 50 percent, this being characterized by an average quantity of spermatozoa, as required for one insemination, in 100 ml of aqua bidest., plus small amounts of albuminous material, glucose, sodium citrate, sodium bicarbonate, ethylenediaminetetra-acetic acid, potassium chloride, antibiotics and sodium bicarbonate.
Other embodiment and modifications of the above described method are envisaged without departing from the spirit and scope of the invention as defined by the appended claims.
ll. The method of impregnating a sow comprising the steps of immobilizing the spermatozoa of boar semen with a first diluting spermatozoa inactivating solution, centrifuging the diluted semen to produce a sperm sediment, diluting the sperm sediment with a freezing diluent, slowing cooling the resultant sperm solution to approximately 5 C, freezing and storing the cooled sperm solution at a temperature of approximately C to -200 C, thawing the frozen spenn solution when needed, mixing the thawed sperm solution with a spermatozoa activating solution and utilizing said activated sperm solution within the uterus of a sow.
2. In the method of impregnating a sow as in claim 1 wherein said first diluting solution and said freezing diluent contain glucose and a buffer system.
3. In the method of impregnating a sow as in claim 1 wherein the thawing of the frozen sperm solution, mixing with an activating solution and utilization within the uterus of a sow comprises the steps of thawing the frozen sperm solution within a spermatozoa diluting solution, introducing the thawed diluted solution within the uterus of a sow, and introducing a spermatozoa activating solution into the uterus of the sow to activate the spermatozoa therein.
4. In the method of impregnating a sow as in claim 1 wherein said boar semen is diluted by said first solution immediately upon collection and said first solution is warmed to approximately 36 C.
5. In the method of impregnating a sow as in claim 4 wherein said first solution is slowly cooled to approximately room temperature prior to centrifuging.
6. In the method of impregnating a sow as in claim 1 wherein said resultant sperm solution is maintained at said approximate C from 1% to 2 hours and is then dropped in droplets upon CO dry ice to form solid pellets, said pellets being stored by immersion in liquid nitrogen.
7. In the method of impregnating a sow as in claim I wherein the step of thawing the frozen sperm solution comprises mixing the frozen sperm solution with a heated diluting solution.
8. In the method of impregnating a sow as in claim 7 wherein said heated diluting solution is of an initial temperature of approximately 60 C to 70 C.
9. In the method of impregnating a sow as in claim 7 wherein said thawing of said frozen sperm solution is made with an amount of heated diluting solution insufficient to obtain a desired final dilution, and the desired final dilution is obtained by introducing the thawed prediluted solution into the uterus of the sow and thereafter introducing a sufficient amount of said heated diluting solution into the uterus of the sow to obtain the desired final dilution of activated sperm solution.
10. In the method of impregnating a sow as in claim 7 wherein said heated diluting solution inactivates the spermatozoa at room temperature but activates the spermatozoa at the sows body temperature.
11. In the method of impregnating a sow as in claim 7, characterized in that said solutions used for spermatozoa activation and inactivation contain glucose, sodium citrate, sodium bicarbonate, antibiotics, ethylenediaminetetraacetic acid and potassium chloride, and said freezing diluent contains skimmed milk powder, glucose, egg yolk, glycerine, antibiotics, ethylenediaminetetraacetic acid and potassium chloride.
12. In the method of impregnating a sow as in claim 11 wherein said spermatozoa activating and inactiviating solutions contain about to g, preferably about 120 g, glucose, about 10 to 15 g, preferably about 12 g, sodium citrate, about 1 to 3 g, preferably about 2 g, sulfanilamide, about 0.5 to 3 g, preferably about 1.5 g, potassium chloride, about 1.5 to 4 g, preferably about 2.5 g, sodium bicarbonate, about 1.4 to 4 g, preferably about 2.5 g, ethylenediaminetetraacetic acid, about 1.7 millions i. u. penicillin G sodium, about 1.7 i.u. dihydrostreptomycin base in 2,000 ml water, and is adjusted be means of 4% NaOI-l to a pH of about 6.8.
13. In the method of impregnating a sow as in claim 11 wherein said freezing diluent contains about 100 to 120 g, preferably about 100 g, skimmed milk powder, about 20 to 30 g, preferably about 25 g, glucose, about 0.5 to 3 g, preferably about 1 g, ethylenediaminetetraacetic acid, about 0.5 to 1.5 g, preferably about 0.8 g, potassium chloride, about 1 million i.u. penicillin G sodium, about 1 million i.u. dihydrostreptomycin base, about ml egg yolk, and about 60 ml glycerine per 1000 ml of water.