초록 ▼

Ⅳ. 연구개발결과
1) Anti-prolactin 제재와 천연 화합물 복합투여를 통한 발정동기화
2) 발정유도견의 혈액채취 및 분석으로 일반발정견과의 호르몬 변화 비교분석
3) 발정유도견의 인공수정유도에 의한 임신 및 분만유도
4) 식혜에서 Anti-prolactin 성분함량의 분석
5) GnRH/GnRH agonist 주사에 의한 과배란유도 기술 확립
6) 과배란 유도견의 외과적 수술에 의한 난자 회수율 조사
7) 발정유도견의 호르몬 변화 비교분석
8) 과배란유도견의 난자회수율 및 배란점

Ⅳ. 연구개발결과
1) Anti-prolactin 제재와 천연 화합물 복합투여를 통한 발정동기화
2) 발정유도견의 혈액채취 및 분석으로 일반발정견과의 호르몬 변화 비교분석
3) 발정유도견의 인공수정유도에 의한 임신 및 분만유도
4) 식혜에서 Anti-prolactin 성분함량의 분석
5) GnRH/GnRH agonist 주사에 의한 과배란유도 기술 확립
6) 과배란 유도견의 외과적 수술에 의한 난자 회수율 조사
7) 발정유도견의 호르몬 변화 비교분석
8) 과배란유도견의 난자회수율 및 배란점 등의 비교분석
9) MII 단계 난자를 이용한 복제 수정란 생산
10) 배란된 미성숙난자의 체외성숙 유도기술 개발
11) 발정 전ㆍ후 단계 (비발정기, 발정전기, 배란 후 1일 및 채란 후 1일)암캐의 혈액 채취 및 프로테옴 분석에 최적화 된 혈장 단백질 분획 법 확립
12) 프로테옴 (1-DE 및 2-DE)분석을 통한 발정 전ㆍ후 단계 (비발정기, 발정전기, 배란 후 1일 및 채란 후 1일)암캐의 혈장 내 차등 발현 단백질의 확인
13) 프로테옴 (LC-MS/MS)분석을 통한 발정 전ㆍ후 단계(비발정기, 발정전기, 배란 후 1일 및 채란 후 1일)암캐의 혈장 내 차등발현 단백질의 선별
14) 발정 전ㆍ후 단계(비발정기, 발정전기, 배란 후 1일 및 채란 후 1일)암캐의 혈장 내 차등 발현 단백질의 메커니즘 분석
15) LC/MS분석을 통한 발정 전ㆍ후 단계별 유의성이 높은 단백질 선별
16) Fetuin-beta에 대한 발정주기 관련 메커니즘 분석을 위한 모식도
17) 개의 estrogen 및 progesterone 특이적 단클론 항체 제작과 이를 이용한 진단 키트 개발
18) 개 estrogen 및 progesterone 발정 주기 진단 키트의 유효성 평가
19) LH, Estrogen, Progesteron 특이적 단클론 항체 단백질 제작
20) 항체 단백질을 이용한 배란 적기 판별 진단 키트 제작 및 효능 검증
21) 자연발정 난자 공여견에서 계절 및 경산/미경산에 따른 혈청내 P4변화 및 난자 성숙단계 확인
22) 자연발정 난자 공여견의 Cotisol 수치 따른 혈액(혈청)내 호르몬변화 및 배란 난자수 및 성숙단계 확인
23) 자연발정 난자 공여견에서 멜라토닌 투여에 따른 황체수, 난자수, 난자성숙정도 비교
24) 자연발정 난자 공여견에서 멜라토닌 투여에 따른 호르몬 변화 확인
25) Estrogen 수치에 의한 배란시점 탐색연구
26) 혈청 내 Progesterone 분석시, ECLI 시스템과 RIA 시스템간의 분석값 비교분석
27) RIA시스템과 ECLI시스템의 배란추정 정확도 비교분석
28) ECLI시스템에서의 P4농도와 E2농도에 의한 배란추정 정확도 비교분석

Abstract ▼

The purpose of this study have to establish of effectively production and utility method of oocyte by estrus synchronization and superovulation technology, and contribute of consistently and safely supply and produce of MII oocyte for effectively production and distribution of special aim’s dog. To

The purpose of this study have to establish of effectively production and utility method of oocyte by estrus synchronization and superovulation technology, and contribute of consistently and safely supply and produce of MII oocyte for effectively production and distribution of special aim’s dog. To do this study, my research group need to evaluate of estrus and control mechanism of estrous timing for effective production of higher quality and mass number oocyte, discover of specific factors in related with ovulation and oocyte maturation, produce and utilize of diagnosis kit for optimal estimating of estrus and ovulation, and also establish of reproductive characteristic confirm of natural estrus donor dog and oocyte collection conditions for appling of cloning.
The contents of main research are as follow,
1. to develop the superovulation technology after confirmation of estrus induction by estrous induced materials and effective oocyte production technology by induction of synchronization
2. to develop the exactly ovulation control and oocyte matured related marker technology for high quality oocyte’s mass production by proteomic methods through estrous female dog’s blood
3. to develop the monoclonal antibody of estrogen, progesteron and LH for effective estrous diagnosis and diagnosis kit for optimal ovulation timing
4. to develop the optimal timing of surgical collection of natural estrous female dog derived matured oocytes through progesteron and LH concentration by confirmation of hormone change in the blood.
The results of this research was summarized by order by year as follow. In the 1^st subject, primary year, six of 10 (60.0%) and seven of 10 (70.0%) bitches showed pro-oestrual bleeding in Fermented rice punch (1 liter, Group 1) and fermented rice punch + bromocriptine (1 liter + 100 ug/kg/day, Group 2), respectively. The mean and median values (min–max) to estrus induction was not significantly different between Groups 1 and 2 (9.7±7.3, 6.5 (3–22) and 11.3±6.6, 7.9 (5–21) days) after treatment commencement (P>0.05). The pregnancy rate was very similar between Groups 1, 2 (66.0%) and control (66.0, 57.0 and 50.0%). Secondary year, the concentration of dopamine in fermented rice punch was 47.2 mg/kg (parts per million) after component analysis of fermented rice punch. The artificial estrus induced dogs were superovulated by treatment of hCG group. The mean number of corpus luteum was higher compared with control (12.0 vs. 4.5±4.9) and also total recovered oocyte number was higher in hCG group than in control group (10.5±3.5 vs. 5.5±4.9). Third year, the mean number of recovered total oocytes were significantly higher in GnRH + hCG, PMSG + hCG groups rather than in control group. The recovery rate of MII oocytes was higher in the GnRH + hCG, PMSG + hCG groups than in control group (52.6% and 54.6% vs. 37.5%), but immatured oocytes recovered were 25.0%, 26.3% and 21.2% in control, GnRH + hCG, PMSG + hCG groups. So if we can develop in vitro maturation technology, we can produce effectively MII stage oocytes.
In the 2^nd subject, the primary year, we prepared blood samples to analyze proteome (protein body) expression quantity before and after the ovulation for acquire information of differentially expressed proteins from female dog's blood before and after it's ovulation, established blood post-treatment method to perform proteomics. Secondary year, we confirmed differentially expressed proteins (hormones or growth factors) from before and after the ovulation blood samples to find proteins which can be used as a marker for investigating estrus-related molecular mechanism, real ovulation and ovum maturity by using differentially expressed proteins(hormones or growth factors) and investigated ovulation regulatory- and ovum maturity mechanism after purifying proteins that expressed differentially with the presence of estrus. Third year, we suggested ovulation- and ovum maturity- regulatory target proteins which can be used as a marker to analyze target protein's effect on ovulation and ovum maturity, confirmed marker candidate protein's physiological effect and then we performed research to find accurate ovulation-regulatory and ovum maturity related markers by utilizing proteomic methods for produce superior ovum.
In the 3^rd subjects, the objective of this study is to make novel canine ovulation diagnostic kit using novel biomarker proteins in canine. We identify the novel canine biomaker candidates, APOB and GSP by the proteomics analysis using canine serum protein. Monoclonal antibody against APOB and GSP, including estrogen, progesterone and LH hormone is established for ovulation diagnostic kit. We obtained several different monoclonal antibody candidates through the immunization of each antigen peptides into mouse and rat animal model and confirmed the specific binding efficiency by ELISA test. To confirm the actual diagnostic ability of antibodies, we tested the established ELISA kit with the canine serum protein by indirect or sandwich ELISA test. The kit against estrogen and progesterone, as well as LH could be useful to determine canine ovulation time. In addition, the diagnostic kit of APOB and GSP is tested with canine serum protein and confirm that GSP is the best diagnostic marker protein which is detected the change of expression following ovulation time.
In the 4^th subject, we attempt to establish the method to predict accurate ovulation day using hormonal analysis of blood progesterone and LH. Firstly, we examined that various environmental factors which may affect to ovulation and hormone values such as season and parity. In addition, we examined the other hormones which may be related to progesterone and LH values such as cortisol, melatonin, and estradiol. In result, it was known that season and parity in canine rarely affect to ovulation. However, melatonin and estradiol affected to ovulation and extruded oocytes number as well as progesterone values. We introduce the effect of melatonin to ovulation.
Expectation of this results are the establishment of new technology to research work for whole year by estrous induction technology during anestrous season and of safely oocyte collection and supplication by superovulation of estrous female dogs, mass production and utilize maximization of special aim dog of somatic cell nuclear transfer by consistently egg supply system and increase of research efficiency by possibility of induced estrous and superovulation technology. And also we can obtained benefits that are research grant’s efficiency, contribution of animal welfare, research output’s maximization by superovulation of induced estrous female dog and increase the research efficiency by recovered egg number per dog by application of new development technology.
In conclusion, we have established an artificial estrous induction technology of anestrus dog by treatment of fermented rice punch and bromatocriptine, and then can be superovulated of artificial estrus induced dogs by hCG + PMSG injection, of which could be applied safely for production of special aim dog by consistently supply of enough number of in vivo ovulated MII stage oocytes. The finding related factors that can diagnose estrus and ovulation period thorough proteomic analysis and molecular mechanism analysis itself means the possibility of procuring and utilizing mass superior ovum. We produced the five independent canine ovulation kit, estrogen, and progesterone, LH, APOB and GSP in this study. From the test of ELISA kits, we show that the novel biomarker protein, GSP may be used for making novel canine ovulation diagnostic kit in canine. And also we offer de novo method which is more accurate to detect ovulation by simultaneous analyzing progesterone and estradiol value.