보고서 정보
주관연구기관 |
국립축산과학원 National Institute of Animal Science |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2016-02 |
과제시작연도 |
2015 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201600003222 |
과제고유번호 |
1395041400 |
사업명 |
축산시험연구 |
DB 구축일자 |
2016-06-25
|
DOI |
https://doi.org/10.23000/TRKO201600003222 |
초록
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Ⅳ. 연구개발결과
본 연구과제를 수행하여 얻은 결과는 다음과 같다. 제1세부과제는 돼지정액의 동결과정 중에 발생하는 정자손상을 최소화하는 동결전 정액 전처리 방법을 평가하였고, 동결정액 품질개선을 위한 동결보호제 첨가효과를 제시하는 한편, 정자의 내동성을 평가할 수 있는 지표를 제시하였다. 또한, 국내 보유 우수 씨수퇘지 유전자원(정액)을 수집하여 동결보존함으로써 유사시 보존된 유전자원을 공동으로 활용할 수 있는 기반을 마련하였다. 제2세부과제는 액상정액의 정자활력을 개선시킬 수 있는 첨가물에 대한 효과를 제시함과 동시에 액상
Ⅳ. 연구개발결과
본 연구과제를 수행하여 얻은 결과는 다음과 같다. 제1세부과제는 돼지정액의 동결과정 중에 발생하는 정자손상을 최소화하는 동결전 정액 전처리 방법을 평가하였고, 동결정액 품질개선을 위한 동결보호제 첨가효과를 제시하는 한편, 정자의 내동성을 평가할 수 있는 지표를 제시하였다. 또한, 국내 보유 우수 씨수퇘지 유전자원(정액)을 수집하여 동결보존함으로써 유사시 보존된 유전자원을 공동으로 활용할 수 있는 기반을 마련하였다. 제2세부과제는 액상정액의 정자활력을 개선시킬 수 있는 첨가물에 대한 효과를 제시함과 동시에 액상정액 제조와 보관과정의 적정 온도관리 기준을 제시함으로써 액상정액의 보존성을 높일 수 있는 기술을 제시하였다. 제3세부과제는 번식모돈의 성장단계 및 발정주기에 따른 내분비 변화와 단백질 발현 수준을 분석하였으며, 난소에서 발정주기별로 차등발현되는 유전자들을 확인하였다. 제4세부과제는 국내의 돼지 인공수정센터의 분포와 씨수퇘지 보유현황, 정액의 생산과 판매현황, 정액제조 및 품질관리 방법 및 인공수정센터의 기술인력 현황 등에 대한 조사결과를 제시하였다.
Abstract
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1. Study on prediction technology of semen freezability and quality improvement of frozen boar semen
Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT
1. Study on prediction technology of semen freezability and quality improvement of frozen boar semen
Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen Post-thawed in boar. Recently, polymorphisms reported to be significant association with sperm MOT. This study was conducted to evaluate the candidate genes (PLCz, ESR1, CD9) as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.158T>C SNP of PLCz was significantly associated with frozen semen motility and kinematic characteristics. g.158T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p < 0.0001,p = 0.0002, p < 0.0001 and p < 0.0001, respectively). Also, the SNP was low significantly associated with ALH (p = 0.0160).The g.35756T>C SNP of ESR1 was significantly associated with frozen semen motility and kinematic characteristics. The g.35756T>C SNPwas high significantly associated with MOT, VCL, VSL and VAP (p < 0.0001, p = 0.0003, p < 0.0001 and p < 0.0001, respectively). Also, the SNP was low significantly associated with ALH (p = 0.0248).The g.358A>T SNP of the CD9 gene was significantly associated with the traits such as MOT, Curve Linear Velocity, Straight Line Velocity , Average Path Velocity and Amplitude of Lateral Head displacement. Particularly, the g.358A>T SNP significantly has the highest association with MOT and animals with AA genotype (p < 0.001).Therefore, we suggest that the intron region of the porcine PLCz, ESR1 and CD9, may be used as a molecular marker for Duroc boar Post-thawed semen quality, although its functional effect was not defined yet.Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.
On the other hand, ascorbic acid 2-glucoside (AA-2G), a stable ascorbate derivative, is a prominent antioxidant characterized by its stability in an oxidative environment. The objective of this study was to evaluate the effect of AA-2G on sperm motility, viability and acrosome integrity during semen cryopreservation. Semen samples were diluted into a freezing extender (LEY) containing lactose hen-egg yolk with glycerol and Orvus Es Paste (OEP) and loaded in 0.5ml plastic straws. In this experiment, each ejaculated semen sample was split into five aliquots and extended with LEY that supplemented with 0, 100, 200, 300 or 400 μM AA-2G. For freezing the semen samples were cooled from 5℃ to -5℃ at 6℃/min, held at -5℃ for 30s while ice crystal formation was induced, then further cooled from -5 to -80℃ at 40℃/min, and thereafter from -80℃ to -150℃ at 60℃/min using programmable semen freezer. To evaluate the post-thaw sperm quality, semen was thawed at 38℃ for 20sec. Sperm quality in terms of motility, viability and acrosome integrity was improved by addition of AA-2G to freezing extender during semen cryopreservation. In particular, the sperm motility and acrosome integrity at 1 and 2 h of incubation was higher (P<0.05) in the group treated with 300 μM AA-2G during freezing procedure when compared to the control group. The result of this study shows that the addition of AA-2G to the freezing extender (LEY) improves motility and acrosome integrity of frozen-thawed boar sperm.
2. Study on quality improvement of liquid semen for swine artificial insemination
Cysteine is one of the main components of glutathione, which plays an antioxidant role to prevent the lipid peroxidation activity of ROS in the spermzoatozoa. Also, cysteine consists of thiol groups, which individually act as nonenzymatic antioxidant and easily penetrates into the sperm. The objective of this study was to determine the effects of cysteine as an antioxidant on sperm motility parameters in liquid storage of boar semen at 17℃. Boars were kept under uniform feeding and handling conditions in the National Institute of Animal Science, RDA, Korea. In this experiment, whole semen was collected from five mature Duroc boars by a gloved-hand technique. The ejaculates were diluted with BTS extender with cysteine at the doses of 0.5, 1 and 2 mM and an extender containing no cysteine (control). A computer-assisted sperm analysis was used to analyze sperm motion characteristics. The sperm motility parameters such as total motility, curvilinear velocity (VCL), straight linear velocity (VSL), average path velocity (VAP) and linearity (LIN) were determined in the storage of semen at 17℃ for a period of 72 h. Sperm quality in terms of motility was improved by addition of cysteine to BTS extender during liquid semen storage. In particular, the BTS extender supplemented with 2 mM cysteine led to higher (P<0.05) percentages of total motility and VAP in comparison to the control group. The results of this study shows that supplementation of cysteine to the semen extender can improve the sperm motility.
Also, boar liquid semen are sensitive to a fast reduction in temperature because of lipid phase transition and phase separation processes. Temperature management of semen and extender during semen processing may determine the quality of stored semen samples. This study was to evaluate the effect of temperature management during semen processing on boar sperm quality. ejaculates were first diluted (1:1) with BTS(Beltsville Thawing Solution), then secondary diluted with 32℃, 26±3℃ or 21 ℃ BTS, stored at 17 C. And then sperm samples assessed on days 0, 1, 3, 5, 7 and 11. Semen quality based on motility, viability and membrane integrity was lower in group treated with 21℃ compared with other groups(P<0.05). The results of this study shows that chilling injury associated with hypothermic dilution results in lower quality of stored boar semen compared with isothermic dilution.
3. Study on TGF-β and LIF and hormonal changes during estrus cycle in Sow
This study was conducted to investigate the changes of hormone levels of serum progesterone (P4) and estradiol-17 β (E2) in sows of Landrace (L), Yorkshire (Y) and F1 (L × Y) (respectively n=3) with excellent ability, and to provide a baseline data for improving reproductive performance. In this experiment, the sows at the age of 12 months or more were used. The sows were fed by two way methods, one is conventional methods and the other is 3 days-flushing feed before estrus. Each pig’s blood was collected in 3, 6, 9, 12, 15 and 18 days after the estrus for the analyses of P4 and E2. Serum was separated by centrifugation for 15 min. with 3,000 rpm. Progesterone and estradiol-17β were measured by immunochemical assay (ELIZA test). In conventional feeding, serum progesterone levels were significantly (p<0.01) higher in F1 than in L and Y. No significant differences in P4 concentrations were seen between the L and Y of sows. Serum E2 levels were similar the serum progesterone levels. In the case of flushing feed, the tendency of hormonal changes were similar to conventional methods. But almost of hormonal levels were a little higher than that of conventional methods. P4 level of L and Y in flushing feed were significantly different (p<0.01). Serum E2 level of Y in flushing feed was significantly different among the breeds (p<0.01). These results were similar to the tendency of hormonal changes in general sows and moreover, flushing feed is known to develop the swine production, these results proved the fact of the methods. And these results suggested that more studies about hormonal changes in sows according to seasonal and nutritional factors should be needed.
In other study, we conducted to investigate the changes of TGF-β(transforming growth factor-β) and LIF(leukemia inhibitory factor) levels during estrus cycle in sows. Blood serum was collected on days 3, 5, 7 and 9 after the estrus detection. The level of TGF-β in the blood serum was gradually increased during estrus cycle in sows. However, there was no different in the blood serum LIF level. These results suggested that more studies about changes of TGF-β and LIF level in sows according to estrus cycle should be needed.
4. An analysis of survey data on South Korea boar stud practices
The aim of this study is to examine current status of swine AI and boar stud in South Korea using survey and data analysis. This survey included 48 boar studs registered as ‘semen processing business’. The survey data were collected by direct visitation, FAX and/or telephone conversation for 7 months from June through December in 2013. 48 boar studs owned a total of 3,537 boars and the Duroc breed accounted for the highest rate (75.3%) of all boar breeds. In case of ownership, agricultural management corporations was the highest (50.0%) and followed by individual ownership (33.3%). Large-scale boar studs in terms of own over 151 boar were surveyed as 4.2% and most boar studs owned less than 100 boars (77.1%). The amount of liquid semen provided by 48 boar studs were 1,889,000 doses and each boar stud provided average of 39,000 does, which is represented for 90% consumption by sows in South Korea.
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