초록 ▼

○ 연구결과
자궁적출술로 conventional 토끼에서 Pasteurella multocida, Bodetella bronchiseptica, Salmonella spp. Eimeria spp. Ear mange, body mange, Sendaivirus(HVJ), mycoplasma이 부재한 토끼를 인공유제조를 위한 모토착유법 개발과 인공포유기술 확립으로 국내에서 최초로 작출을 성공하였고, 이 동물을 이용하여 정액의 동결보존과 인공수정, 수정란의 동결보존과 이식 기술을 확립하였고, 각 부분별운영에 관련된 SOP를 제작하고

○ 연구결과
자궁적출술로 conventional 토끼에서 Pasteurella multocida, Bodetella bronchiseptica, Salmonella spp. Eimeria spp. Ear mange, body mange, Sendaivirus(HVJ), mycoplasma이 부재한 토끼를 인공유제조를 위한 모토착유법 개발과 인공포유기술 확립으로 국내에서 최초로 작출을 성공하였고, 이 동물을 이용하여 정액의 동결보존과 인공수정, 수정란의 동결보존과 이식 기술을 확립하였고, 각 부분별운영에 관련된 SOP를 제작하고 이에 준하여 Barrier를 운영한 결과 국내에서 최초로 SPF 토끼를 공급할 수 있는 기반을 구축하게 되었다.

Abstract ▼

Part Ⅰ
A. Production of germ free rabbit.
In this study, we have attempted to establish of specific pathogene free(SPF) rabbit producted by hysterectomy and artificial nursing as Pasteurella multocida, Bordetella bronchiseptica, Salmonella spp, Eimeria spp, Ear/Body mange.
The fetal was tak

Part Ⅰ
A. Production of germ free rabbit.
In this study, we have attempted to establish of specific pathogene free(SPF) rabbit producted by hysterectomy and artificial nursing as Pasteurella multocida, Bordetella bronchiseptica, Salmonella spp, Eimeria spp, Ear/Body mange.
The fetal was take out from uterus at 30 day pregnancy of New Zealand White rabbit in the operating isolator and than nursed with rabbit milk according to body weight(10%) by artificial nipple(∅1.52㎜) for 20 days after operated. From 36 days to weaning, autoclaved cabbage and assorted feed was feed to newborn rabbits. Fixing of microorganism in a digestive organ, dung of rabbit growth in the barrier system (temperature:22±2℃, moisture: 50±D5%) was feed by mixed with saline at firstly nursing.
On the microbiological test, pathogens free as Pasteurella multocida (bacteriology), Bordetella bronchiseptica(bacteriology), Salmonella spp (bacteriology), Eimeria spp(parasitology), Ear/Body mange(parasitology) of rabbit producted by hysterectomy and artificial nursing. In the current report, we introduce SPF rabbit by hysterectomy and artificial nursing in the operating vinyl isolator.
B. Invention of milking device and milking method in rabbit.
Milk from laboratory animals is used in studies of mammary tumor virus, commercial feed production, and neonatal nutrition. Several investigators have reported devices for collecting milk from mice, rats, hamsters and guinea pig.
However, the use of these devices for collection milk is impractical because these devices are too small, they collect very small volumes of milk, and they are difficult for one person to use.
We have designed a device and method for milking a rabbit without pain, which can be adjusted to fit the nipple of a rabbit, and milk a large volume of milk during a short period. In addition, this devices is constructed easily, requires little physical exertion, and can be used by one person or by two people for simultaneous collection from both milk lines of one rabbit. To milk a rabbit(New Zealand White), the mother was separated from her litters immediately after delivery and oxytocin(6.0IU/㎏) was injected to stimulate the milk letdown. The rabbit is placed on a restraining board and its nipples are cleaned with 70% ethyl alcohol. The milk was suctioned into the collection bottle by 400㎜HG of continuous vacuum. Milking continued for approximately 30 minutes at which time milk flow decreased.
We recorded the volume of milk collected from each rabbit during each milking period, successful or unsuccessful, on collection period. On two occasions before perfecting the milking technique, milk was contaminated by blood, these contaminated samples were not used in the study.
Using this milking device, we collected a volume of milk ranging from 15 to 140㎖with a mean of 44.3㎖during 30 minutes of the milking period per rabbit. Successful milking continued for an average of 20 days among the 45 rabbits, with a rage of 14 to 33 days.
The device and milking procedures are tolerated well by rabbits, as evidenced by the good quality of milk obtained. The device is also easy to construct and requires little physical effort to operate.
C. Environmental and microbiological monitoring technique with the relating SOPs for SPF rabbits
For the cleanness of the breeding facilities and the health of SPF rabbits, our continuous effort, and the construction of environmental and microbiological monitoring are essential parts. For these, we must construct good laboratories and make the SOPs of environment and microbiology to the facilities. To make
SOPs means to accomplish the mile stone of good monitoring system.
PartⅡ
A. Cryopreservation of SPF rabbit sperm.
Glycerol is not an effective cryprotectant for rabbit spematozoar; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. The objectives of this research were to compare extenders for freezing rabbit spermatozoa, to study the effect of freezing on the acrosome, viability on cooling rates at which the temperature fell down to 25℃ from 5℃ and to test the possibility that an effect on the acrosome could alter the optimal time for insemination relative to expected ovulation time.
In experiment 1, viability of the post-thawed sperm increased at the rapid cooling rates, with cell membrane penetrating in egg-yolk cryoprotective (p<0.05): on the hand, sperm viability was shown high at slow cooling rates, with cell membrane non-penetrating in egg-yolk cryprotective(p<0.05).
Rates of normal acrosome of post-thawed sperm were higher when methyl cellulose among cell membrane non-penetrating cryoprotectants was added to the extender than when cell membrane penetrating cryoprotectives was added to egg-yolk extender.
In experiment 2, viability of sperm was higher when both cell membrane penetrating and non-penetrating cryoprotectives were added to the extender containing egg-yolk than when cell membrane penetrating or non-penetrating cryoprotctants alone(p<0.05) were added to extender.
In experiment 3, 30 does were inseminated with frozen-thawed semen 0 or 10h after being given an ovulating dose of a hCG. The percentage of does pregnancy with frozen-thawed semen inseminated at 0 and 10 h after hCG injection was 93.3 and 40. Litter size and the number of young born was greater(P<0.05) at 0h than at 10h, which was the expected time of ovulation.
The results of this experiment showed that cooling rates should be modulated according to physiological characteristics of cryoprotectives; and a combination of cell membrane penetrating and non-penetrating cryoprotectives had more synergistic effects in preserving rabbit spermatozoa than penetrating of non-penetrating cryoprotectives alone was used. And if freezing did reduce the time required for capacitation, it was not detectable by these experiments.
B. Effects of age, litter size and sex on bodyweight of newborn rabbits in SPF New Zealand White rabbits(Yac:NZW).
This studies have investigated changes of the bodyweight according to week, litter size and sex after birth of the newborn SPF New zealand White rabbit breeding in the barrier system.
In 26 liters containing 183 young(average liter size:7) there were 91(49.82%) male and 92(50.18) female. The eyes do not open until day 10 after birth. The young being to emerge from the nest at about 3 weeks of age and take some solid food.
The change of bodyweight according to weeks, at 6(1,138.8±ﾃ28.0g) weeks old were 10 times compared with bodyweight at first week old(112.10±)4.10) and every weeks grown about 200∼00g (P<0.05).
The bodyweight were significantly higher in below 7 litter size than above 8 litter size on 4 to 11 weeks old after birth(P<0.05). The bodyweight on the sex were higher in the female than male(P<0.05). These data provide select weight and weeks old for use in toxicological research with SPF New Zealand White rabbit.
C. Environmental control system and disinfection programs
For the environmental control of clean rooms and isolators we made the SOPs of the environmental control. High quality filters (hepa filters) are equipped in every rooms to maintain the cleanness of facilities. Physical cleaning of airs and effective control programs of disinfection are also another important essentials for good breeding.
Air ventilation must be constructed within the consideration of the meaning of proper temperature, humidity, flow, cleanness. And blocking system from the pollution of deteriorated air do work smartly with regular change of air filters and consumptions and operating of related SOPs.
Part Ⅲ
A. Cryopreservation of SPF rabbit embryos
As an innovative method for embryo cryopreservation, vitrification not only reduced the cooling stage duration to a minimum, but also eliminated any injuries cased by extra cellular ice, which is a major cause of cell injury. As a component of a vitrification solution, a permeating cryprotective agent is essential, and additional inclusion of a macromolecular and a small saccharide makes the solution more effective.
This study was carried out to investigate the effect of vitrification(40% ethylene glycol, 18% Ficoll, 0.3M sucrose; EFS-40), non-step(10% ethylene glycol, EG) and step wise(10% glycerol, G) freezing methods on the post-thaw developmental ability of SPF rabbit morulae and blastocyst embryos. In vitrification, equilibrate all the solutions and instruments at 20℃. Connect a 1㎖% syringe and the straw with a silicone tube. By aspirating the syringe carefully, load 60㎜ 0.5M sucrose solution, 15㎜ air, 3.0㎜ EFS-40, 4㎜ air, and 13㎜ EFS-40, successively into the 0.25㎖straw. Exposure of embryos to EFS-40 and loading into the straw: Pour approx 0.2㎖of EFS-40 into a watch glass.
Prepare two pipets: one aspirated with PBS solution, and the other with EFS-40. Under a dissecting microscope, pick up about 10 embryos at the tip of the pipet with PBS solution, suspended the embryos in the EFS-40 in the watch glass with a minimal volume of PBS solution for 2.0min. Aspirate the embryos into the other pipet containing EFS-40 and transfer the embryos to the part of the EFS-40 in the watch glass to wash glass in the solution. Pick up the embryos in the pipet and transfer them into the 13㎜ EFS-40 column in the straw. pour liquid nitrogen into a dew flask and float a styrofoam on the liquid nitrogen. Two minutes after exposure of he embryos to EFS-40, place the straw on the styrofoam to allow it to cool slowly in the liquid nitrogen vapor. Leave it for at least 3 min and then store the straw in the liquid nitrogen tank.
The post-thaw development rates to expended blastocyst of rabbit morulae was not different among the vitrification(86.5%), non-step(86.9%) and step-wise(89.2%). From this result, it was indicated that this methods using EFS-40(vitrification), 10% ethylene glycol and 10% glycerol solutions will be of practical use for reduced breeding cost and conservation of strains in SPF rabbit.
B. Physiological values of SPF rabbit of the ages
At the ages of 3, 9, 16 and 27 weeks we sacrificed the SPF rabbits and checked and calculated the values of organ weights ,serum biochemistry, microbiology and hematology. These values are the first data of SPF rabbits producted in Korea and will be a meaningful reference for the area of safety test and pharmacological toxicology test.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 4
• SUMMARY ... 14
• CONTENTS ... 20
• 목차 ... 21
• 제1장 연구개발과제의 개요 ... 22
• 제1절 연구개발의 필요성 ... 22
• 1. 기술적 측면 ... 23
• 2. 경제ㆍ산업적 측면 ... 24
• 3. 사회ㆍ문화적 측면 ... 25
• 제2장 국내외 기술개발현황 ... 25
• 1. 국내 ... 25
• 2. 국외 ... 26
• 제3장 연구개발수행 내용 및 결과 ... 26
• 제1절 무균자토의 생산기술, 사육시설의 청정화 ... 26
• 1. 토끼의 자궁적출기술 확립과 인공포유기술 개발 ... 27
• 2. 모토유 착유기술 확립 ... 43
• 3. 환경모니터링 및 미생물 모니터링 기술 확립과 관련 SOP 제작 ... 47
• 제2절 SPF 토끼 정액동결 보존과 각종 모니터링법 확립 ... 61
• 1. SPF 토끼 정액동결 보존 기술 확립 ... 61
• 2. SPF 토끼의 증체속도 측정에 의한 유전monitoring ... 71
• 3. SPF토끼의 사육시설 과 SPF토끼의 미생물 모니터링 ... 79
• 제3절 SPF 토끼의 수정란 생산과 동결보존 기술의 확립, 월 350두 생산사육시설의 운영에 따른 지침서 작성 및 SPF 토끼의 생리적 특성에 대한 검토 ... 170
• 1. SPF 토끼 수정란의 동결보존 ... 170
• 2. 월 350두 생산사육시설의 운영에 따른 지침서 작성 ... 181
• 3. Conventional 토끼와 SPF 토끼의 생리적 차이분석 ... 183
• 제4장 목표달성도 및 관련분야에의 기여도 ... 194
• 제5장 연구개발결과의 활용계획 ... 199
• 1. 현재의 진행상황 ... 199
• 2. 향후 계획 ... 199
• 제6장 참고문헌 ... 200
• 끝페이지 ... 205