보고서 정보
주관연구기관 |
경남과학기술대학교 GYEONGNAM NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLOGY |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2014-02 |
과제시작연도 |
2011 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201400011454 |
과제고유번호 |
1395021406 |
사업명 |
FTA대응 경쟁력향상기술개발 |
DB 구축일자 |
2014-07-05
|
초록
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Ⅳ. 연구개발결과
○ 정액 보존일수에 따른 세균 오염도 변화 구명
- 보존 3일째 세균농도 증가
○ 정액 보존일수별 세균농도 증가가 정자의 성상에 미치는 영향
- 보존 3일째 정자활력 감소, 7일째 생존율 감소
○ 액상정액 오염정도에 따른 체외수정란 생산효율 검정
- 체외수정란 배 발달율은 보존 3일째 감소
- 돼지 인공수정용 액상정액의 항생제 무처리시 적정 사용기간은 3일
○ 정액에 대한 E. coli 오염농도가 5000 cfu/ml 이상일 경우 정액성상 저하 및 체외수정란 생산효율 감소
Ⅳ. 연구개발결과
○ 정액 보존일수에 따른 세균 오염도 변화 구명
- 보존 3일째 세균농도 증가
○ 정액 보존일수별 세균농도 증가가 정자의 성상에 미치는 영향
- 보존 3일째 정자활력 감소, 7일째 생존율 감소
○ 액상정액 오염정도에 따른 체외수정란 생산효율 검정
- 체외수정란 배 발달율은 보존 3일째 감소
- 돼지 인공수정용 액상정액의 항생제 무처리시 적정 사용기간은 3일
○ 정액에 대한 E. coli 오염농도가 5000 cfu/ml 이상일 경우 정액성상 저하 및 체외수정란 생산효율 감소
○ 인공수정용 돼지 액상정액 제품 제조공정 중 세균 오염원 확인
- 정액 희석제 교반용 항온수조의 관리부실로 인한 공기 중 부유세균 유입 확인
- 이러한 세균오염 희석액을 사용한 제품정액에서 지속적인 세균오염 발생
○ 정액제조 과정 중 세균오염원 제거 및 위생관리 방안 제시
- 항온수조는 매번 깨끗이 세척하고 증류수로 세척하여 관리
- 교반기에서 희석제를 용해할 때는 공기 중 부유세균이 항온수조로 유입되지 않도록 교반기의 덮개를 닫아놓은 상태로 작업 실시
- 희석액 채취 및 분주용 용기관리 철저 : 한번 사용한 용기는 자외선 소독기에 보관하면서 멸균처리를 하도록 하고 필요시 마다 꺼내서 사용
○ 세균오염을 최소화하는 위생적인 돼지정액 제조방법
- 세균제어가 가능하고 정자운동성에 영향을 주지않는 적정 항생제 농도 평가 : gentamicin sulfate 50~150㎎/ℓ
- 우수 씨수퇘지 유전자원(정액) 위생적 동결보존 : 1,772 스트로 (0.5㎖ 용량)
- 돼지 인공수정센터 정액 내 세균검사비용 지원(정책제안)
○ 돼지 인공수정 기술제공 프로그램 개발 및 보급
- 돼지가족계획 : 기기종속적인 앱(App)에서 모바일 웹(Web)방식으로 전환(2013.2.7.)
○ 돼지 인공수정센터 통합관리시스템(AI PigMoS)의 맞춤형 기능개발
- 인터넷 홈페이지 : http://www.aipig.or.kr
- 모바일 홈페이지 : http://www.aipig.or.kr/m
Abstract
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The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome int
The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome integrity and hypo-osmotic swelling test were performed during 7 days of liquid preservation. Semen samples were prepared using antibiotic free BTS extender and 4 different levels of E. coli were treated to semen with following concentrations; 3,000, 5,000, 7,000, 10,000 CFU/ml ofsperms. Semen samples were preserved at 17℃ for 7 days in semen storage until analyzed. Aliquots were subjected to measure the sperm viability, motility and agglutination using computer assisted sperm analysis (CASA) system, acrosome integrity was performed using chlortetracycline (CTC) staining method and hypo-osmotic swelling test was performed using hypotonic solution from day 1(day of semen collection) to 7. Detrimental effects on sperm motility and viability were observed 3 days after preservation at the level of 5,000 CFU/ml (P< 0.05). Percentage of sperm abnormalitywas higher (P< 0.05) in over 5,000 CFU/ml groups. Sperm agglutination rate was also significantly higher (P< 0.05) in groups of 5,000 and 7,000 CFU/ml. The rate of acrosome reacted sperm was higher as preservation time goes in all the samples but the pattern was clearly higher among E. coli contaminated groups(P< 0.05). The sperm membrane integrity in terms of hypo-osmotic test, E. coli affects little compared to other sperm parameters. The deleterious effects observed due to the bacterial contamination in semen suggest that importance of hygiene protocol to minimize the bacterial contamination during semen collection and processing.
Artificial insemination (AI) has globally been used in swine industry. Bacterial contamination is an unavoidable finding of the semen collection process in boar and can lead to deleterious effects on semen quality and longevity if proper control absent. The purpose of this study was to investigate the prevalence of bacterial contaminations in boar semen, and select the effective antimicrobials to control of the contaminated bacteria. Three hundred twenty four of fresh unextended semen and 329 of extended semen samples were collected from 20 AI centers during 2011 to 2012. The semen samples were inoculated onto blood agar and MacConkey agar plate for bacterial isolation. After aerobic incubation for 24h at 37°C, pure cultures of colony were identified by VITEK 2 Compact (bioMérieux, France). The number of bacteria contaminated in semen samples was counted by standard count method. The antimicrobial susceptibility test was also performed by Kirby-Bauer disk diffusion method using 12 antimicrobials. Among the semen samples, a total of 320 isolates were identified; 245 (76.6%) were gram negative (GN) bacteria, and 75 (23.4%) were gram positive (GP) bacteria, respectively. The most prevalent GN bacteria was P seudomonas aeruginosa (n=59), following Escherichia coli (n=43), and Sphingomonas paucimobilis (n=23), respectively. The top three GP bacteria were as follows: Kocuria
varians (n=26), Staphylococcus spp. (n=15) and K ocuria kristinae (n=10). Although most of the fresh unextended semen samples in high grade AI centers were contaminated with bacteria (245/258; 95.0%), only 78 (29.8%) in 262 extended semen samples were contaminated with bacteria showing median average 0 CFU/mL. The effective top five antimicrobials for GN bacteria were gentamicin (68.3%), enrofloxacin (59.4%), colistin (58.3%), ceftiofur (57.5%), and florfenicol (52.4%), whereas gentamicin (68.0%), ceftiofur (58.7%), florfenicol (56.9%), enrofloxacin (53.7%), and bacitracin (50.1%) for GP bacteria.
GN bacteria were more frequently isolated from the boar semen than GP bacteria. The average number of the bacteria was remarkably decreased in extended semen compared to that of fresh unextended semen. We speculated that the level of contaminated bacteria could be controlled by dilution step in semen processing and use of effective antimicrobials in extender. Although florfenicol, ceftiofur and gentamicin were selected for the control of contaminated bacteria in this study, these antimicrobials were not effective all the contaminants. Therefore, we suggested that the mixture of two or three antimicrobials with high susceptibility would be better than using single antimicrobial to control the bacteria in boar semen.
Also, we determined the effects of E . coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro(Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E . coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E . coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Sperm motility and viability were significantly decreased(P< 0.05) on day 0 (4hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased(P< 0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90~6.86) and T4 (6.86~6.65) from day 3 to day 5 (P< 0.05). Sample pH was maintained 7.0~7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E . coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. To evaluate the field fertility outcomes in bacteria contamination semen, we performed AI with 56 herds of weaned Duroc sows. Each semen sample for AI was artificially contaminated with E. coli at different concentrations of 0, 500, 5000, 50000 cfu/ml, respectively. The result of AI, the rate of pregnancy and farrowing tend to decrease by increasing the concentration of E. coli of semen. These results suggest that E . coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality. On the other hand, we determined the effect of different concentration of E. coli and gentamicin sulfate on sperm motility in boar extended semen. In experiment 1, semen were diluted Beltsville Thawing Solution(BTS) without antibiotic to yield a final sperm concentration of 3×10(7) cells/ml. Each sample was artificially contaminated with E. coli at different concentrations of 0, 2×10(4), 2×10(5), 2×10(6) and 2×10(7) cfu/ml, respectively. There was a reduction in the percentage of sperm motility for groups contaminated above 2×10(5)cfu/ml E. coli(72.2±2.4%) compared control group(80.7±6.9%) on day 3. In experiment 2, five aliquots were diluted with manufactured BTS containing different concentration of gentamicin sulfate concentration of 0, 50, 100, 150, 200, 250 and 300 mg/l, to yield a final sperm concentration of 3×10(7) cells/ml and infected 2×10(5)cfu/ml E. coli. Addition of gentamicin sulfate in liquid semen was effective at eliminating bacterial growth. After 1 day of incubation, no bacterial colonies were yielded more than a level of 300mg/l of gentamicin sulfate. At concentration above 250mg/l, gentamicin sulfate has shown inhibitory effect on sperm motility from day 3 after incubation. These results suggest that E. coli contamination has a concentration-dependent detrimental effect and E. coli growth in extended boar semen may be controlled by gentamicin sulfate at concentration of 50∼200mg/l.
Artificial Insemination(AI) technique in the hog industry became a basis of the industrial development by having been distributed to domestic hog farmers in earnest after 1994. Currently AI supply system consists of 3 steps such as suppliers who produce and manage boars, centers that produce AI from boars and its distribution, and producers who bring up hogs after obtaining excellent AI in our country. AI PigMoS system proposed by the study can manage nationwide AI centers comprehensively based on the web, and also designed and developed Bar-Code generating functionality so that the record tracing on boars, semen production and sales management etc can be possible. Therefore it is expected that effective hog improvement system would be set up because this could manage AI centers comprehensively and will be able to be utilized to integrated analysis of AI centers and record tracing of various uses as well. The artificial insemination(AI) centers in the country’s hog-raising industry play a key role in the development and dissemination of artificial insemination technology. But the artificial insemination information management systems in AI centers are operated and managed based on independent systems, so there are many limitations in performing integrated information management analysis on nationwide AI centers and performing cause analysis and traceability of infectious diseases. In this thesis, an improved AI PigMoS that can be operated in the fixed mobile convergence(FMC) environment was designed and embodied. The embodied FMC-based AI PigMoS can be operated in the web and mobile environment simultaneously and is expected to contribute to establishing the artificial insemination information management system of nationwide AI centers and enhancing competitiveness of individual AI centers.
목차 Contents
- 표 지 ... 1
- 제출문 ... 2
- 요 약 문 ... 3
- SUMMARY ... 6
- 목 차 ... 9
- 제 1 장 서 론 ... 10
- 제 2 장 국내외 기술개발 현황 ... 12
- 1절: 국내 연구 현황 ... 12
- 2절: 국외 연구 현황 ... 12
- 제 3 장 연구개발수행 내용 및 결과 ... 13
- 제1세부과제 : 돼지정액의 세균오염정도가 번식성적에 미치는 영향 ... 13
- 제2세부과제 : 정액 내 세균허용기준 및 적정 항생제 사용 기준 설정 ... 23
- 제3세부과제 : 위생적인 돼지정액 제조기술 개발을 통한 번식성적 향상 ... 56
- 제 4 장 연구개발 목표 달성도 및 대외 기여도 ... 93
- 1절 목표대비 달성도 ... 93
- 2절 : 정량적 성과(논문게재, 특허출원, 기타) ... 94
- 제 5 장 연구개발결과의 활용계획 ... 99
- 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 100
- 제 7 장 기타 중요 변동사항 ... 101
- 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 101
- 제 9 장 참고문헌 ... 102
- 끝페이지 ... 106
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