U.S. patent number 3,791,384 [Application Number 05/252,272] was granted by the patent office on 1974-02-12 for artificial insemination of sows.
This patent grant is currently assigned to H. Wilhelm Schaumann. Invention is credited to Albert Liedicke, Ludwig Richter.
United States Patent |
3,791,384 |
Richter , et al. |
February 12, 1974 |
ARTIFICIAL INSEMINATION OF SOWS
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.degree. to -200.degree.C,
at the time of use is thawed by being placed in such a quantity of
diluent warmed to 40.degree. to 50.degree. 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.
Inventors: |
Richter; Ludwig (Trappenkamp,
DT), Liedicke; Albert (Wahlstedt, DT) |
Assignee: |
Schaumann; H. Wilhelm (Hamburg,
DT)
|
Family
ID: |
25761438 |
Appl.
No.: |
05/252,272 |
Filed: |
May 11, 1972 |
Foreign Application Priority Data
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|
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Jul 15, 1971 [DT] |
|
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2135318 |
Oct 30, 1971 [DT] |
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2154260 |
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Current U.S.
Class: |
600/35; 604/515;
435/2; 62/1; 604/522 |
Current CPC
Class: |
A01K
29/00 (20130101) |
Current International
Class: |
A01K
29/00 (20060101); A61d 007/02 () |
Field of
Search: |
;128/1,260 ;195/1.8
;424/89 ;62/1 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Medbery; Aldrich F.
Attorney, Agent or Firm: Beaman & Beaman
Claims
I claim:
1. 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.degree. C, freezing and storing the cooled sperm
solution at a temperature of approximately -180.degree. C to
-200.degree. C, thawing the frozen sperm 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.degree. 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
5.degree. C from 11/2 to 2 hours and is then dropped in droplets
upon CO.sub.2 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 1 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.degree. C to 70.degree. 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 sow's 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
100 to 140 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% NaOH 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 180 ml
egg yolk, and about 60 ml glycerine per 1000 ml of water.
Description
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, the
sediment was subjected to dilution, the duly diluted sperm was
frozen, i.e., was lowered to a temperature of about -180.degree. to
-200.degree. 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.degree. 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.degree. 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.degree. 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 -180.degree. to -200.degree. C, at the time of
use is thawed by being placed in such a quantity of diluent warmed
to 40.degree. to 50.degree. 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.degree. to 70.degree.
C. The thawing is preferably undertaken with an inactivating
solution at a temperature of 60.degree. to 70.degree. 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.degree. C for only 11/4 to 11/2 hours then being
caused to drip onto CO.sub.2 -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 with
reference to a particularly advantageous embodiment.
Immediately upon being collected, the ejaculate is diluted in the
ratio of 1 : 3 with inactivating solution warmed to about
36.degree. 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 11/2 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 1 : 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 5.degree. C in a refrigerator for from 11/2 to a maximum
of 2 hours. After that, the diluted sperm is applied by means of a
pipette to CO.sub.2 -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.degree. to 70.degree.
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.degree.
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.sub.2 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 10 minutes to 90.degree.. 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.degree. to 70.degree. C; since by placing the tablets, cooled
down to about - 190.degree. 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 20.degree. C. The following features
are also completely new: both the process of preliminary dilution
upon thawing, this only slightly thinning the sperm (about 1 ml to
1 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.degree. to 39.degree. 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 deep-freeze 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.degree. 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.degree. C and afterwards possessed a motility of approximately
70 percent.
The precise composition of the solution employed as the inactivator
and the mobilizing agent, hereinafter designated "Diluent 1", as
well as the solution preferably employed for deep-freezing,
hereinafter designated "Diluent 2", are of the following
compositions:
DILUENT 1
100 -- 140 g, but preferably 120 g glucose
10 -- 15 g, but preferably 12 g sodium citrate
1 -- 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 1 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
100 -- 120 g, but preferably 100 g skimmed-milk powder
20 -- 30 g, but preferably 25 g glucose
0.5 -- 3 g, but preferably 1 g ethylenediaminetetraacetic acid
0.5 -- 1.5 g, but preferably 0.8 g potassium chloride antibiotics
egg-yolk, glycerine.
Diluent 2 is produced as follows: 1 l of aqua bidest is brought to
the boil, chemicals are added, briefly brought to boil and allowed
to cool. To the solution at room temperature are added 1 million
i.u. of penicillin G sodium and 1 million i.u. of
dihydrostreptomycin 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.degree. 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 11/2
hours cooled to 5.degree. 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.sub.2 -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 1 warmed
to 50.degree. 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 1 was infused through the catheter
into the cervix and uterus. Then the sperm solution was introduced,
and 20 ml of Diluent 1 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.degree. C for a considerable
period. This temperature prevails in the uterus after the
deep-frozen pellets have been thawed in diluent warmed to
50.degree. C and expanded to 100 ml with Diluent 1 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 1 at a temperature of 50.degree. C, a solution
of boar sperm 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.
* * * * *