DE2135318A1 - METHOD OF PRESERVING EBERSPERMA BY FREEZING, THAWING AND INTRODUCING INTO THE UTERUS OF THE SAU - Google Patents

Description

into the sow's uterus "

The ük'finäun ^ relates to a method for preserving Eberspenua by freezing, thawing and bringing in food in the uterus of the sow and yeast formation of the cells.

la der Tierzucht v / enuet ipt-n cicli aer Icünutliciien _3e ^ aua wirtöcLaftliciien 'iirviviunc ^ en iiiiaier meiir zu. v / y.hrend oei already change :; ^ ute successes have been achieved 3ind, i ^ e lfj, n_ oei üeuen die can ülione 'insemination with'L'ief / efriers s—

im-n only; _ele ₍ ;; eiitlicii. Tie Urr.aclie will --eoehen that spex'KU .jüberisijerma, by a far he Prwzentaa or .Bullerispermien the K

iii; i only practiced pra> .is to preserve excess - ι: I) C-LiU, i-uLtt.uen and ü) inbrin-: en in O. en Utcru.3 der: .iau,

209888/1275

BATH ORIGINAL

^ man do the following; The ejaculate, about 200 to 500 ml, was centrifuged, the JiTussirkf-it was poured off, the sediment was thinned, the thinned b'per..na was frozen, that is, a. to a temperature of about - 1c; o to -. "! was to the sperm used was then thawed by the frozen sperm warm in as much 4o ⁰ G" / "brought 2oo ⁰ O and genalten at this temperature, thinners given was 'That a solution resulted with a sperm dilution in which a good sperm distribution in the uterus is guaranteed. This loestiny was then placed directly in the uterus.

The successes that have been achieved with this procedure were so real that it was not useful in practice. When considering the thawed Eonservats the microscope presented SiCN nerauy that dux'ehschnittlicn 15 to 30 ■ ■} <> of the sperm, the G-efrierbehandlungeüberlebt had a part of it but had freeze damage. But only a survival rate of 50 to 70 γ> would make deep freezing economical, and there is still that. "

The invention is now based on the object of creating a method according to which the preservation, the

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BAD ORSGtMAL

subsequent thawing and insertion into the uterus from Bbersp ^ riuien can be carried out so that the sperm jji-tjctiucn takes no action. After thawing should the sperm have a mobility of at least 50 4 lines and, placed in the sow's uterus, the necessary Have mobility.

The problem is solved by a process of preservation of boar semen by freezing, thawing and introducing into the sow's uterus, in which the ejaku- J

50 ⁰ C warm thinner given: frozen 180 to-2oo ° 0 to, e ^ flat time for thawing in an amount 40 ° 0 oir - diluted UEAs sediment f.uf a temperature ΐοη;: let the nsch centrifuged Jititnahme that a sperm dilution suitable for the diameter of the uterus arises and this dilution is introduced into the uterus, as is characterized in that the ejaculate is inactivated after the intake and before centrifugation and the sperm solution is inactivated after thawing a sperm mobilizing solution is added.

The operative solution is thawed inactivated, so that the sperm pass through the freezing process in an activated manner. jili-üt after insemination, the sperm are labeled with a "'..ecklösun _f ';;" Juooilized.

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BATHROOM 0RK31NAL

The inactivation is preferably effected by adding an aqueous solution containing glucose and a buffer system. In a particularly preferred embodiment of the invention, thawing is carried out with an insufficient amount of water to achieve the desired predilution the final dilution prepared by adding the sperm mobilizing solution to the pre-dilution. Am gunntigsten had been found is made dai3 thawing at 60 to 7o ⁰ G diluent. Preferably, the defrosting with a 60 to 7o ° ö warm inactivation solution is made. An aqueous sodium salt, a potassium salt, glucose, a buffer system and a sulphonamide solution has proven to be an excellent solution to mobilize the sperm the sperm mobilizing solution is forwarded of the centrifuged sperm before freezing can be done with a freeze thinner, which is an aqueous solution containing fructose, citric acid, egg yolk, glycerol and a buffer system. The freezing is preferably carried out gradually by first keeping the sperm mixed with freezing _{thinner at + 5 ° C for 1} 1/4 to 1 1/2 hours, then dripping it onto CO 2 dry ice so that small pellets are formed

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209885/1275

then surrounded with liquid nitrogen. When freezing of the sperm in the form of pellets is obtained after a Another embodiment of the invention is the dilution with the amount of tablets required for insemination Manufactured.

The invention will now be described on the basis of a particularly preferred embodiment.

The ejaculate is immediately after removal with approx. 36 ⁰ G was-

mer inactivation si ösun · ^ diluted in a ratio of 1: 3. The " Inactivation solution can e.g. B. the following composition to have:

87.5 S -d-olucoae

5.4 β ethylenedinitrilotetraacetic acid dihydrate 5.4 g sodium citrate

7 »5 g sodium hydrogen carbonate
to 1000 ml of sqia bideat.

The diluted sperm is placed in centrifugation vessels and left at room temperature for 1 1/2 bia 2 hours. During this time, the temperature is reduced Room temperature and the ImmobiliBierun.fj & er sperm cells.

209885/1275

After that, the centrifuges are Dehält he carefully placed in a centrifuge and centrifuged 7 minutes at about 2500 Umdrenungen per minute, iiach removing the Zen trifugenbehälter from the centrifuge, the liquid, protruding durcn turning inside the container is completely drained. The sediment is then diluted with a deep freeze thinner in a ratio of .1: 3. The following is the composition for a suitable deep freeze thinner:

Of whether g tris (nydromethyl) aminomethane i £, 50 g jructose

3,4ö β citric acid
10.00 inl of G-lycerin
25.00 ml egg yolk
to 200 ml of aqua bideat.

The semen and the thinner are mixed in a liquid-like manner by pre-heating it with a lass stick. The diluted sperm is then cooled down in the refrigerator 1 1/2 to max & l 2 otuncLeii to approx. 5 Ο. Then the diluted L. pe rat is frozen by means of a pipette on COp-'Irockeneis in i'Or ^ i of ΐχ · ορ-ieii aui'jebraciit j which within ¹ J i-iinuton au kloinen Pej-leta. .These l-illets v / erdeu gut in flius «! ■ i; n nitrogen uitti: eia

-Y-

209885/1275

BATH ORIGINAL

If the L ^ oerma is needed, it is thawed according to the invention by putting 20 or 30 ml (1 ml per tablet) 60 Ms 70 ⁰ O ^ warm Ino.ktivierunrslosun be given all pellets each for a Besamun;. sdosis be added all at once vieinzrigslösung in the inactivation Lie tablets dissolve immediately leiciitem Sciiüttels the G-efäases on.!.

The thawed prediluted sperm is then inseminated, j d. ii. ate introduced into the uterus, Annchließend be a 75- nil sperm mobile isere ligand Wecklösung, which was also heated to a Teinperatur of about 40 ⁰ G, naciij-respült. The solution that mobilizes the opermia preferably has the following composition:

24.3 S sodium nitrate χ 2 H _? 0

«., 0 g sodium bicarbonate

0.4 g potassium chloride

3.0 β glucose

3.0 grams of sulfanilamide
to 1000 ml aqua bidist.

fr

ϊΤε-cn when the individual components are combined aie solution heated to 90 ° for 10 minutes. In sterile packaging, the solution can be kept for a long time.

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The method according to the invention leads to a very considerable advance, because it is the first time with it has become possible, artificial insemination also "with Schweinijiin to be carried out economically.

The applicant has "in her experiments of every method Stage samples are taken and examined microscopically for viability of the sperm. In the final phase it showed the sperm solution has a motility of 50 to 70 ^ while with the previous method sperm solutions with a maximum mobility of $ 30 have been obtained. The sperm solutions prepared according to the invention fertilized well and resulted in litters of average 9-10 piglets,

It is believed that the instant invention Inactivation, i.e. the reduction in the metabolism of the sperm immediately after the sperm has been removed makes it more resistant to freezing treatment. The type of thawing is also essential to the invention. The tablets are not at body temperature, but instead given a 60 to 70 ö warm solution; that goes through Introducing the approx. - 190 ° C cold tablets into the solution, the latter cools down, results in the invention. if the method were to produce a solution of about body temperature,

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while in the "known Yerfihren the resultant according to the thawing solution has a temperature of about 20 ⁰ G. YoIlig is also new to both the production of a Yorverdünnang during thawing, which only slightly diluted sperm (about 1 ml to 1 pellet) and perform the thawing process with a metabolism inactivating solution, and to make the final concentration with the mobilizing solution only in the uterus in this ^T;. / EIEE sperm remain until the time ,, au where they are used in a mobile less j

Condition, which means that they are considerably spared.

The three solutions used in the method according to the invention can of course differ in their composition from that given above. It is essential that the inactivation solution contains a suitable buffer system and D-gluose; With the τ ¹ iff freezer thinner it is important that the amount of egg yolk is not significantly exceeded. "The mobilizing effect of the third solution is caused by the increased * M _en - ^ 3ps Natriuasai ^ e ??. The solution must also be a Tu ff he sy s tem included and appropriately sets r = ir. ulfonamid her "too, bor to extensive IC ^ inifreiheit ^Ί Αη Tjntollen.

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Claims (9)

- 1ο - 2Ι353Ί8 claims 1. Method for preserving boar sperm by freezing, thawing and breezes in the sow's uterus, "in which the ejaculate is centrifuged after removal, the sediment is diluted, ^{frozen to a temperature of - 180 to - 200 0} C, in due course For thawing, add 40 to 50 warm thinner to such an amount that a sperm dilution suitable for rinsing the uterus is created and this thinning is introduced into the uterus, characterized in that the ejaculate is inactivated before it is removed and before centrifugation a sperm mobilizing solution is added to the sperm solution after thawing 2. The method according to claim 1, characterized in that the inactivation by adding an aqueous Solution containing glucose and a buffer system is effected. 3. The method according to claim 1, characterized in that thawing is carried out with an insufficient amount of thinner to achieve the desired final dilution, and the final concentration is carried out by adding the solution; au der Χ ^Γ οχ "νβχ * άίίηη.ιΐΓ. v ; o stf-. · ill, L, ■ j. 209885/1275 4. Procedure according to. Claim 3 »dadurcii characterized in that thawing is carried out with thinner up to 70 ⁰ G warm. 5. The method according to claim 3 »characterized in that the thawing with a 60" to 70 ° warm inactivation solution are made. b. A method according to claim 1 and any one of the preceding claims, characterized in that a wäeerige g, H sodium salts as the sperm mobilizing solution ,, a ICaliumsalz, G-Lukose, a Puff he syst em and a sulfonamide-containing solution is used. 7. Method according to claim 1 and any of the preceding Claims, characterized in that the final thinning is only produced in the uterus by first the thawed, prediluted sperm was introduced and then the solution that mobilized the sperm was put in ac is nodding. 8. The method of claim 1 and any of the preceding Claims, characterized in that the centrifuging sperm is thinned prior to freezing is made with a freeze thinner, the one watery one, fructose, egg yolk, citric acid and solution containing glycerine and a puifer system 209885 / 127B - Λ 2 - 9. The method according to claim 1 and any one of the preceding claims, characterized in that the
Freezing is done gradually by cooling the sperm to which your deep freeze thinner has been added for only 1 1/4 "to 1 1/2 hours at 5 ° C and then dripping it onto CC ^ dry ice (carbonated snow). 209885/1276 ORIGINAL INSPECTED