KR20010036569A - Method for cryopreservation of sperm cells from Korean native goats - Google Patents

Method for cryopreservation of sperm cells from Korean native goats Download PDF

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KR20010036569A
KR20010036569A KR1019990043640A KR19990043640A KR20010036569A KR 20010036569 A KR20010036569 A KR 20010036569A KR 1019990043640 A KR1019990043640 A KR 1019990043640A KR 19990043640 A KR19990043640 A KR 19990043640A KR 20010036569 A KR20010036569 A KR 20010036569A
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sperm
semen
diluent
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korean native
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KR100328511B1 (en
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장규태
한동운
장희성
강문일
김태윤
류재웅
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장규태
강문일
한동운
장희성
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation
    • A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
    • A61D19/021—Apparatus for collecting seminal fluids; Artificial vaginas

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Abstract

PURPOSE: A freeze-drying method of sperm collected from Korean sheep harboring the gene of mWAP/hGH is provided, thereby the sperm can be safely stored. CONSTITUTION: The freeze-drying method of sperm collected from Korean sheep comprises the steps of: collecting sperm from Korean sheep harboring the gene of mWAP/hGH; diluting the sperm into a diluting solution(Citrate-Egg york-Tris Extender) in a volume ratio of 1:2 and cooling at 4 deg. C for 2 to 3 hours; secondly diluting the sperm solution into a diluting solution(Citrate-Egg york-Tris Extender) which contains glycerol at 3 to 5 deg. C; and inserting the sperm solution into a vessel, sealing and dipping the vessel into liquid nitrogen having -79 or -196 deg. C, in which the diluting solution contains sodium citrate, sodium phosphate diabasic, glycerin, glucose, fructose, Tris, EDTA, sodium bicarbonate, BSA, Streptomycin-penicillin-G, and Egg york.

Description

한국 재래산양 정자의 동결보존 방법{Method for cryopreservation of sperm cells from Korean native goats}Method for cryopreservation of sperm cells from Korean native goats}

본 발명은 한국 재래산양 정자의 동결보존 방법에 관한 것이다. 보다 상세하게는, 본 발명은 한국 재래산양으로부터 정자를 채취하여 희석액으로 1차로 희석하고 4℃의 냉장실에서 2-3시간 동안 냉각(0.3-0.5℃/min)을 실시한 다음, 상기 냉동 희석 정액을 동결보호제인 글리세롤을 첨가한 희석액으로 2차로 희석하여 액체질소에서 보존하는 방법에 관한 것이다.The present invention relates to a cryopreservation method of Korean native goat sperm. More specifically, the present invention is obtained by collecting sperm from Korean native goats and diluting it first with diluent and then cooling (0.3-0.5 ° C / min) for 2-3 hours in a refrigerator at 4 ° C. It relates to a method of preliminary dilution with a diluent containing glycerol, a cryoprotectant, and preservation in liquid nitrogen.

포유동물 정자세포 보존은, 예컨대 멸종에 처한 종의 보존, 동물종의 육종, 연구 개발 등의 여러가지 목적을 위해 수행되고 있으나 오염 방지, 사멸방지 및 활성 유지 등의 문제점 때문에 많은 방법이 강구되어지고 있다.Mammalian sperm cell preservation is performed for various purposes, such as preservation of extinct species, breeding of animal species, research and development, etc. However, many methods have been devised due to problems such as pollution prevention, prevention of death and maintenance of activity. .

특히, 한국 재래산양의 경우 일반 포유동물의 정액에 비해 정자가 고밀도 (10-30억 마리/ ml)이기 때문에 정액 희석은 물론 인공수정이 불가능한 것으로 알려져 있다. 한편, 최근에는 한국 재래산양을 이용한 생활성물질 생산을 위한 연구가 급속히 진전되고 있기 때문에 우수한 형질을 가진 개체의 대량생산을 목적으로 한국 재래산양의 보존 필요성 또한 절실히 요구되고 있는 실정이다.In particular, Korean native goats are known to be inseparable from artificial semen as well as semen dilution because the sperm has a higher density (10-30 billion / ml) than semen in normal mammals. On the other hand, in recent years, research for the production of bioactive materials using Korean native goats is rapidly progressing, and the necessity of preserving Korean native goats is also urgently required for the purpose of mass production of individuals with excellent traits.

본 발명자들은 우수한 유전형질을 가진 개체의 대량생산을 목적으로 한국 재래산양 정액을 동결보존한 다음 인공수정 방법을 적용한 결과, 기존의 자연적 교배법에 비하여 약 30배 정도 효율적인 것으로 확인되어 본 발명을 완성하게 되었다. 본 발명은 이와같이 한국 재래산양에 있어서 채취한 정액을 동결보존한후 융해한 정액중의 정자 생존율이 인공수정에 충분히 이용되어질 수 있는 생존율을 달성하였고, 본 발명 방법에 의한 정자로 인공수정을 실시한 결과 재래산양의 산자를 생산할 수 있었기 때문에 본 발명 정자 보존방법을 통해 아주 체계적이고 유전형질이 높은 한국 재래산양의 대량 생산에 크게 이바지할 것으로 기대된다.The present inventors cryopreserved Korean native goat semen for the purpose of mass production of individuals with excellent genetic traits, and then applied artificial insemination, which was confirmed to be about 30 times more efficient than conventional natural breeding methods, thus completing the present invention. Was done. The present invention thus achieved a survival rate in which the sperm survival rate in the frozen semen after freezing preservation of the semen collected in Korean native goats can be fully used for artificial insemination, the result of artificial insemination with sperm by the method of the present invention Since it is possible to produce goats of goats, it is expected that the method of sperm preservation of the present invention will greatly contribute to the mass production of Korean native goats having a very systematic and high genetic trait.

따라서, 본 발명의 목적은 한국 재래산양 정자의 동결보존 방법을 제공하는 것이다.Therefore, it is an object of the present invention to provide a cryopreservation method of Korean native goat sperm.

이하, 본 발명의 구성 및 효과를 상세히 설명한다.Hereinafter, the configuration and effects of the present invention will be described in detail.

본 발명에 따른 정자는 본 발명자들이 구성한 구연산-난황(Egg york)-트리스 (Tris) 희석액 (extender)으로 희석되어져 보존 되어진다. 바람직하게는 본 발명의 정액(정자)은 동결보호제 글리세롤이 첨가된 희석액으로 최종적으로 부피비 15-30 배 범위로 희석되어 액체질소에서 보존되어진다. 본 발명에 따른 희석액은 구연산 나트륨, 인산 나트륨, 글리세린, 포도당, 과당, 트리스, EDTA, 중탄산나트륨, BSA, 스트렙토마이신-페니실린 G, 난황 등의 조성분을 포함한다. 동결보호제 글리세롤의 최종 농도는 7%가 가장 바람직하였다. 동결보호제가 첨가된 희석액으로 희석된 정액은 액체질소 조건하에서 인공수정에 충분히 이용되어질 수 있는 생존율 60-70%를 유지하며 보존되어진다.The sperm according to the present invention is diluted and preserved with the citric acid-egg yolk-tris extender configured by the present inventors. Preferably, the semen (sperm) of the present invention is a diluent to which the cryoprotectant glycerol is added and finally diluted to a range of 15-30 times by volume to be preserved in liquid nitrogen. Diluents according to the present invention include the composition of sodium citrate, sodium phosphate, glycerin, glucose, fructose, tris, EDTA, sodium bicarbonate, BSA, streptomycin-penicillin G, egg yolk and the like. The final concentration of cryoprotectant glycerol was most preferably 7%. Semen diluted with a cryoprotectant-diluent is preserved with 60-70% survival under liquid nitrogen conditions, which can be fully used for artificial insemination.

동결보존한 정액은 급속 동결융해방법으로 37℃의 온수조에서 2분간 정액이 보존된 봉입 스트로우를 서서히 저어가면서 융해시킨 후 수정이나 기타 목적에 이용되어질 수 있다. 상기 정액을 담을 수 있는 스트로우와 같은 보존매체는 얼마든지 대체가 가능하다는 것은 당업자에게 명백할 것이며, 편의상 다른 보존매체로 얼마든지 대체하여 사용할 수도 있다.The cryopreserved semen can be used for fertilization or other purposes after melting by slowly stirring the enclosed straw, which was preserved for 2 minutes in a hot water bath at 37 ° C. by rapid freezing thawing. It will be apparent to those skilled in the art that a storage medium such as a straw that can contain the semen can be replaced by any number, and can be replaced by any other storage medium for convenience.

이하 실시예를 통하여 본 발명을 보다 상세히 설명한다. 그러나 하기 실시예는 본 발명을 예시하기 위한 목적일 뿐이며 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely for the purpose of illustrating the invention and do not limit the scope of the invention.

실시예 1 : 1차 희석액의 제조Example 1 Preparation of Primary Diluent

본 발명에서 정액을 희석하기 위해 사용한 1차 희석액의 조성분은 다음과 같으며 각 조성분의 함량은 1리터에 함유된 양이다 : 구연산 나트륨 1,920mg, 인산 나트륨(diabasic) 80mg, 글리세린 170mg, 포도당 83mg, 과당 170mg, 트리스(Tris) 500mg, EDTA 25mg, 중탄산나트륨 166mg, BSA 3mg/ml, 스트렙토마이신-페니실린-G 10,000 IU/100ml, 난황(Egg york) 20%(v/v). 상기 조성분을 가진 1차 희석액을 제조하여 Citrate-Egg York-Tris Extender라 명명하였으며, 정액 채취전 제조하여 정액채취후 희석전까지 37℃에 보존하였다.The composition of the primary diluent used to dilute semen in the present invention is as follows, the content of each component is the amount contained in 1 liter: sodium citrate 1920 mg, sodium phosphate (diabasic) 80 mg, glycerin 170 mg, glucose 83 mg, Fructose 170 mg, Tris 500 mg, EDTA 25 mg, Sodium bicarbonate 166 mg, BSA 3 mg / ml, Streptomycin-penicillin-G 10,000 IU / 100 ml, Egg york 20% (v / v). The first dilution with the composition was prepared and named Citrate-Egg York-Tris Extender, prepared before semen collection, and stored at 37 ° C. until dilution after semen collection.

실시예 2 : 정액채취 및 희석Example 2 Semen Collection and Dilution

정액을 채취하기 위하여 약 15-20개월령의 한국 재래산양으로부터 인공질법 (FHK, Japan)을 실시하였으며, 인공질의 온도는 약 40℃로 유지하였고 음압에 의한 자극법으로 인공 의빈대로 유인하여 정액을 채취하였다. 체내정자가 외부로 사출되어 온도의 차이가 10℃ 이상일 때 나타나는 온도 쇼크를 방지하기 위하여 미리 준비해둔 37℃의 1차 희석액으로 채취된 정액의 희석(8X107마리/ml)을 실시하였으며, 정액 일부(20-50μl)는 정자의 사용가능성 여부를 알아보기 위하여 정자의 기형, 생존율 여부 및 수정능력을 가지고 있는 지를 판단하는 MRT(methylene-blue reaction test) 검사를 실시하였다.In order to collect semen, artificial method (FHK, Japan) was performed from Korean native goats of about 15-20 months of age, and the temperature of artificial material was maintained at about 40 ° C. . In order to prevent temperature shock when the internal sperm were injected to the outside and the temperature difference was more than 10 ℃, dilution of semen (8X10 7 grains / ml) was performed with the primary dilution of 37 ℃ prepared in advance. (20-50μl) was carried out a methylene-blue reaction test (MRT) test to determine whether sperm malformation, survival rate and fertilization ability to determine whether sperm availability.

실시예 3 : 2차 희석액의 제조Example 3 Preparation of Secondary Dilutions

채취한 정액은 1차 희석액과 1:2로 희석하여 4℃의 냉장실에서 2-3시간 동안 냉각(0.3-0.5℃/min)을 실시한 다음, 충분히 냉각되었을 때 2차 희석을 실시하였다. 2차 희석액은 1차 희석액을 기준으로 하였고, 정자의 액체질소 조건하(-70 또는 -196℃)의 동결보존시 흔히 발생하는 정자의 첨체막 탈락, 꼬리 절단, 세포내 탈수 등을 방지하기 위하여 동결보호제(cryoprotective agent)로 독성이 거의 없다고 알려진 글리세롤을 이용하였다. 이때, 동결보호제의 농도는 세포막 내의 탈수에 물리적 손상을 줄일 수 있고, 가장 안정적으로 탈수시킬 수 있는 농도인 7%를 최종농도로 설정하여 3단계 평형방법(즉, 글리세롤 농도가 1.5%, 3% 및 7% 함유/1차 희석액중)을 적용하였다.The collected semen was diluted 1: 2 with the primary diluent and cooled (0.3-0.5 ° C./min) for 2-3 hours in a 4 ° C. refrigerating chamber, followed by secondary dilution when sufficiently cooled. Secondary diluents were based on the first diluents and were used to prevent sperm dropping of the sperm of the sperm, tail cleavage, and intracellular dehydration, which often occur during cryopreservation under liquid nitrogen conditions (-70 or -196 ° C). Glycerol, which is known to have little toxicity as a cryoprotective agent, was used. At this time, the concentration of the cryoprotectant can reduce physical damage to dehydration in the cell membrane and set the final concentration of 7%, which is the most stable dehydration concentration, to a three-step equilibrium method (ie, glycerol concentration of 1.5% and 3%). And 7% containing / first dilution).

실시예 4 : 2차 희석 실시Example 4 Second Dilution

상기 실시예3의 희석된 정액이 충분히 냉각된후 4℃에서 글리세롤이 첨가된 2차 희석액과 혼합한 후 2시간동안 평형을 실시(최종농도가 7%가 되도록 글리세롤 첨가)하여 약 15-30배로 희석되게 하였다.After the diluted semen of Example 3 was sufficiently cooled, the mixture was mixed with the secondary diluent to which glycerol was added at 4 ° C., followed by equilibration for 2 hours (adding glycerol such that the final concentration was 7%) to about 15-30 times. Allowed to be diluted.

실시예 5 : 액체질소 보존Example 5 Liquid Nitrogen Preservation

상기 실시예 3의 평형이 끝나면 혼합정액을 0.5ml 스트로우에 봉입후 -79℃ 또는 -196℃(LN2)에서 보존하였다.When the equilibrium of Example 3 was completed, the mixed sperm was stored in -79 ° C or -196 ° C (LN 2 ) after being enclosed in 0.5ml straw.

실시예 6 : 동결보존된 정액의 융해Example 6 Thawing of Cryopreserved Semen

동결보존한 정액의 융해방법은 급속 동결융해방법으로 37℃의 온수조에서 2분간 스트로우를 서서히 저어가면서 융해하였다.The freeze-preserved semen thawing method was a rapid freeze thawing method, which was thawed while stirring the straw for 2 minutes in a hot water bath at 37 ° C.

실시예 7 : 동결보존된 정자의 생존율 검사Example 7 Survival Test of Cryopreserved Sperm

상기 실시예 6과 같은 방법으로 동결보존한 다음 융해한 한국 재래산양 정자의 생존율을 MRT 검사법에 의하여 생존율을 판정하였는 바, 60-70%의 정자 생존율이 판정되어 일반적으로 소에서의 인공수정시 이용되어지고 있는 동결보존후의 생존율과 거의 비슷한 결과를 나타내었다.Survival was determined by the MRT test for the survival rate of frozen Korean native goat sperm after cryopreservation in the same manner as in Example 6, 60-70% of sperm survival rate was determined and is generally used for fertilization in cattle. The survival rate after cryopreservation was similar.

실시예 8 : 산자 시험(progeny test)Example 8 progeny test

한국 재래산양에 PGF2α를 투여하여 1차 투여후 4일째 66%의 재래산양 발정유기율을 보였고, 2차 투여시는 대부분의 산양에서 발정(약 96%)이 유기되었다.PGF2α was administered to Korean native goats and showed a 66% native goat estrus yield on the 4th day after the first dose, and estrus (about 96%) was induced in most goats during the second dose.

PGF2α 투여후 1차 시기에 발정이 유도된 개체에 약 40일간 동결보존된 정액으로 16두의 산양에 인공수정을 실시하여 4두를 임신시켰고 임신되어 총 7두(암컷 3, 수컷 4)의 자양을 생산하였다.Four pigs were conceived by artificial insemination on 16 goats with sperm that had been cryopreserved for 40 days in the estrus-induced individuals at the first time after PGF2α administration, and a total of 7 (females 3 and 4) were pregnant. Produced.

본 발명은 정액 희석은 물론 인공수정이 불가능한 것으로 알려진 한국 재래산양의 정액을 동결보존하는 벙법에 관한 것으로, 본 발명에 따르면 동결보존된 정액의 정자 생존율이 60-70%로 나타나 본 발명은 인공수정시 인공수정에 충분히 이용될 수 있는 정자의 보존이 가능하여 농축산업상 매우 유용한 발명인 것이다.The present invention relates to a method of cryopreservation of semen from Korean native goats known to be impossible to artificially inseminate as well as semen dilution. According to the present invention, the sperm survival rate of cryopreserved semen is 60-70%. Preservation of sperm that can be fully used for artificial insemination in the city is a very useful invention in the enrichment industry.

Claims (4)

한국 재래산양의 동결보존 방법에 있어서,In the cryopreservation method of Korean native goats, (a)한국 재래산양으로부터 채취한 정액을 희석액(Citrate-Egg york-Tris Extender)으로 1차 희석(정액:희석액(v/v)=1:2)하고 4℃에서 2∼3시간 냉각시키는 단계;(a) First dilution of semen collected from Korean native goats with diluent (Citrate-Egg york-Tris Extender) (fixed solution: diluent (v / v) = 1: 2) and cooling at 4 ° C for 2-3 hours. ; (b)상기 (a)단계의 희석 냉동된 정액을 상기 희석액에 글리세롤을 첨가시킨 희석액으로 3-5℃에서 2차 희석하고 평형을 실시하는 단계;(b) diluting the frozen frozen semen of step (a) with a diluent to which glycerol is added to the diluent at a secondary dilution at 3-5 ° C. and performing equilibration; (c)정액을 담을 수 있는 보존매체에 상기 (b)단계의 평형을 이룬 정액을 넣은 후 봉입하여 -79℃ 또는 -196℃의 액체질소에 보존하는 단계를 포함함을 특징으로 하는 한국 재래산양 정자의 동결보존 방법.(c) a conventional Korean goat comprising the steps of putting the equilibrium semen of step (b) into a storage medium capable of containing the sperm and then encapsulating it in liquid nitrogen at -79 ° C or -196 ° C Cryopreservation method of sperm. 제1항에 있어서, 상기 희석액이 구연산 나트륨, 인산 나트륨(diabasic), 글리세린, 포도당, 과당, 트리스(Tris), EDTA, 중탄산나트륨, BSA, 스트렙토마이신-페니실린-G, 난황(Egg york)을 포함함을 특징으로 하는 한국 재래산양 정자의 동결보존 방법.The method of claim 1, wherein the diluent comprises sodium citrate, sodium phosphate (diabasic), glycerin, glucose, fructose, tris, EDTA, sodium bicarbonate, BSA, streptomycin-penicillin-G, egg york Cryopreservation method of Korean native goat sperm, characterized in that. 제1항에 있어서, 상기 (b)단계의 희석액에 글리세롤을 7% 첨가시킴을 특징으로 하는 한국 재래산양 정자의 동결보존 방법.[Claim 2] The cryopreservation method of Korean native goat sperm according to claim 1, wherein 7% of glycerol is added to the diluent of step (b). 상기 제1항의 방법에 의해 제조된 정액 보존매체를 37℃의 온도에서 2-3분간 급속융해시켜 정자를 활성화시키는 방법.Method to activate sperm by rapidly melting the semen preservation medium prepared by the method of claim 1 at a temperature of 37 ℃ 2-3 minutes.
KR1019990043640A 1999-10-09 1999-10-09 Method for cryopreservation of sperm cells from mWAP/hGH in Transgenic Korean native goats KR100328511B1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030014891A (en) * 2001-08-13 2003-02-20 (주)베티즌 Compositions for cooling and cryopreservation of canine sperm, Methods for cooling and cryopreservation using the compositions, and Canine Articificial Insemination
KR100427850B1 (en) * 2001-05-04 2004-04-28 충남대학교산학협력단 Freezing method of semen or embryo derived from small-species dogs
CN102246744A (en) * 2011-05-03 2011-11-23 陕西省动物研究所 Giant panda semen freezing and preserving fluid and preparation method thereof
WO2012003737A1 (en) * 2010-07-06 2012-01-12 陕西精健新星生物医药有限公司 Method for preparing animal semen freeze-dried powder

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100427850B1 (en) * 2001-05-04 2004-04-28 충남대학교산학협력단 Freezing method of semen or embryo derived from small-species dogs
KR20030014891A (en) * 2001-08-13 2003-02-20 (주)베티즌 Compositions for cooling and cryopreservation of canine sperm, Methods for cooling and cryopreservation using the compositions, and Canine Articificial Insemination
WO2012003737A1 (en) * 2010-07-06 2012-01-12 陕西精健新星生物医药有限公司 Method for preparing animal semen freeze-dried powder
CN102246744A (en) * 2011-05-03 2011-11-23 陕西省动物研究所 Giant panda semen freezing and preserving fluid and preparation method thereof

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