• 한국가축번식학회지
• /
• 제13권2호
• /
• pp.113-120
• /
• 1989

In order to determine the sex of preimplantation embryos prior to transfer in cattle, a series of experiments were carried out using 45 Holstein donor cows to examine the ovarian response on the gonadotropin and PGF2${\alpha}$, and the morphology of fresh embryos or frozen/thawed embryos after deep freezing at -196$^{\circ}C$. The sexing of embryos treated with the medium containing H-Y antiserum(10%, v/v) and FITC anti-mouse IgG(10%, v/v) were analysed by chromosomal analysis, and the sex of the embryos which survived were ascertain after delivering the pups. The results obtained were summarized as follows ; 1. The average number of developed follicle and corpus luteum per cow were 13.5 and 8.1, and the ovalation rate was 60.1%. 2. Of 220-ova recovered, 75(34.1%) were morula and 91(41.4%) were blastocyst, and the morphological normal and abnormal rate of ova recovered were 75.5% and 24.5%, respectively. 3. Of 39 frozen/thawed embryos, the scores of normal morula and blastocyst, after thawing were 79.2%(19/24) and 73.3%(11/15). The average rate of frozen/thawed embryos which appeared morphologically normal post thawing was 76.9%(30/39). 4. The sex ratio was measured using the embryos treated with immunofluorescence assay to examine the relationship between embryo developmental stage, sex ratio of morula stage embryo was 42.2%(19/45) fluorescing and 57.8%(26/45) non-fluorescing, on the other hand, the ratio switched to 46.8%(29/62) fluorescing and 53.2%(33/62) non-fluorescing embryo in blastocyst stage. The sex ratio was also measured between fresh and frozen/thawed embryos, fresh and frozen/thawed treated embryos were indicated 45.8%(38/83) fluorescing, 54.2%(45/83) non-fluorescing and 41.7%(10/24) fluorescing, 58.3%(14/24) non-fluorescing. This trend indicated the approximal sex ratio was 1 : 1. 5. The result of karyotype test showed the successful rate of sexing embryo is fluorescing and non-fluorescing was 21.2%(7/33) and 29.6%(8/27). The female to male ratio within 33 fluorescing was 28.6 : 71.4, and the ratio of 27 non-fluorescing embryos was 87.7 : 12.5. 6. Of the embryo transferred after assignment of H-Y phenotype, five of the fluorescing embryos survived to term, all was males. Whereas six non-fluorescing embryos also survived to term and the sexes of the calves were 1 male 5 female.

• 대한수의학회지
• /
• 제38권2호
• /
• pp.394-400
• /
• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• 한국가금학회지
• /
• 제41권4호
• /
• pp.249-259
• /
• 2014

동결 닭 원시 생식 세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 원시 생식 세포의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시 생식 세포는 배양 5.5일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시 생식 세포를 분리했다. 15% 각각의 EG를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 동결 보호제로 10% EG를 이용한 유리화 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 10% EG를 이용한 완만 동결 처리군에서 66.14%로 동일한 농도의 PG 처리군(50.11%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였고, 이는 한국재래닭(오계)의 원시 생식 세포의 동결 보존의 실용화가 보다 더 향상될 수 있는 또 하나의 방법이 될 수 있음을 시사한다.

• Restorative Dentistry and Endodontics
• /
• 제35권4호
• /
• pp.285-294
• /
• 2010

본 연구의 목적은 흰 쥐의 상악 대구치를 발거한 후 치주인대세포를 $0^{\circ}C$/2 MPa고압-저온하에 1주간 보관시켜 MTT, WST-1 검색법을 이용하여 측정한 치주인대세포의 활성도를 저속 냉동법(No Additional Pressure, 2, 3 MPa), 급속 냉동법(No Additional Pressure, 2 MPa), $-5^{\circ}C$/90 MPa초고압 저온보존법과 비교하여 평가하는 것이다. 생후 4주된 암컷 Sprague-Dawley계 흰쥐의 상악 좌우 제1, 2대구치를 발거하여 각 군 당 12개의 쥐 치아를 MTT, WST-1 검색에 이용하였다. 실험군은 9개군으로 대조군은 즉시 발치군이며, 각각 3 MPa, 2 MPa, No Additional Pressure (NAP)의 압력을 가한 후 $4^{\circ}C$에서 $-35^{\circ}C$까지 $-0.5^{\circ}C$/min 속도로 서서히 냉동시킨 뒤 $-196^{\circ}C$에 냉동한 저속 냉동군, 발치 후 동해방지제 처리과정을 거쳐 각각 2 MPa, NP의 압력을 가한 후, $-196^{\circ}C$의 액화질소에 넣어 냉동한 급속 냉동군, 발치 후 각각 2MPa,NP의 압력을 가한 후, $0^{\circ}C$에 보관한 저온 보존군, $-5^{\circ}C$/90 MPa의 초고압 저온 보존군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethylsulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기 위해 MTT, WST-1 측정값을 Eosin 염색 후 530 nm에서 측정한 흡광도 값으로 나누었다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후 검정으로는 Tukey HSD 방법을 사용하였고 결과는 다음과 같다. 1. MTT 검색법 및 WST-1 검색법 결과 $0^{\circ}C$/2 MPa 고압 저온 보존군이 즉시 발치군보다 세포 활성도가 낮았으나 통계적 유의차는 없었으며, 저속 압력 냉동군(NP, 2 MPa, 3 MPa)과, 급속 압력 냉동군(NP, 2 MPa), 저온보존군($0^{\circ}C$/NP), 초고압 저온 보존군($-5^{\circ}C$/90 MPa)보다 통계적으로 유의차있게 높은 세포 활성도를 나타내었다(p < 0.05). 2. MTT검색법 및 WST-1 검색법 결과 $-5^{\circ}C$/90 MPa 초고압 저온 보존군이 가장 낮은 세포 활성도를 나타내었으며, MTT 검사 결과에서는 모든 군에 대해 통계적으로 유의성 있는 결과를 보였다(p < 0.05). 위의 결과를 통해, $0^{\circ}C$/2 MPa (20기압)의 고압-저온 보존법이 다른 급속 냉동 보관법(2 MPa, NAP)이나 저속냉동보관법(3, 2 MPa, NAP), $-5^{\circ}C$/90 MPa 초고압 저온 보존법에 비해 우수한 쥐 치아의 치주인대세포의 활성도를 보여 차후 치아의 재이식시 치아보관을 위한 방법으로의 가능성을 제시하였다.

• 터널과지하공간
• /
• 제21권2호
• /
• pp.145-150
• /
• 2011

사암과 안산암에 대한 풍화특성을 살펴보기 위하여 동결융해 반복시험을 실시한 후, 두 암석에 대한 중량 감소율, 탄성파속도, 일축압축강도 등의 물성 변화를 측정하였다. 중량 변화의 경우 두 암석 모두에서 동결 융해 반복 횟수가 증가함에 중량이 감소하는 경향을 보였다. 특히 안산암에서는 중량 감소 경향이 시험편에 따라 매우 불규칙하게 나타났다. 탄성파속도 변화에 있어서 사암은 5%이상의 감소 경향을 보였으며, 안산암은 동결융해 반복시험 500 사이클까지 탄성파속도 변화가 거의 없다가 1000 사이클부터 5%이상의 감소 현상을 보였다. 이것은 안산암이 비교적 풍화에 강한 암석인 반면, 사암은 풍화받기 쉬운 암석인 것을 의미한다. 또한 안산암의 탄성파속도 변화 양상은 안산암의 중량 변화와 일치한다. 사암의 일축압축시험 결과에서 일축압축강도는 동결융해 반복시험 초기 사이클 구간에서 미미한 강도저하를 보이다 64 사이클부터는 불규칙한 경향을 나타냈다. 결과적으로, 중량 감소가 적은 암석 시험편일수록 강도저하율도 작게 나타났다.

• 한국콘크리트학회:학술대회논문집
• /
• 한국콘크리트학회 2008년도 추계 학술발표회 제20권2호
• /
• pp.397-400
• /
• 2008

$850^{\circ}C{\sim}1000^{\circ}C$ 정도의 저온에서 소성된 MgO 분말을 사용한 콘크리트는 장기적인 팽창성을 가지게 되며, 이러한 팽창의 특성은 지연팽창을 통하여 콘크리트의 수축을 보상하는 특성을 지니게 된다. 이를 통하여 매스 콘크리트의 균열저항성능의 개선 효과를 기대할 수 있다. 현재 사용되는 팽창성 콘크리트 혼화재료는 에트린자이트형(CSA), 산화칼슘(CaO)형, 산화마그네슘형(MgO)가 있다. 본 연구에서는 5%의 MgO를 사용한 콘크리트의 장기재령에서의 내구특성을 평가하기 위하여 56일 양생한 후 탄산화, 염화물 확산계수, 동결융해 저항성, 황산염 저항성 실험을 실시하여, MgO를 혼합 하지 않은 콘크리트와 비교하였다. 또한 10%의 MgO를 넣은 시멘트 페이스트를 대상으로 1일, 3일, 7일, 28일, 56일에 수화정도를 SEM, XRD, DSC 등을 통하여 분석하였다.

• Molecules and Cells
• /
• 제26권6호
• /
• pp.558-565
• /
• 2008

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.

• Clinical and Experimental Reproductive Medicine
• /
• 제18권1호
• /
• pp.81-87
• /
• 1991

There studies were carried for evaluation of the efficiency of freezing of hamster oocytes for use in a human sperm penetration assay. The hamster oocytes fully equilibrated in various cryoprotectant agents and inseminated with human sperm. After insemination with hamster oocytes, there was no difference in penetrated rates. Cumulus free oocytes equilibrated in 1.5M various cryoprotective agents and slowely cooled to temperature $-30^{\circ}C$ before rapid cooling and storage in nitrozen tank. After rapid thawing, survival rates of frozen oocytes according to cryo-protective agents were examined and the human sperm penetration assay with zona free hamster oocytes was conducted. 1. Survival rates of oocytes after cryoprotectants exposure have no significant difference (range 88-91%) and peneration rate was 51.1%. 2. Recovery and survival rate of frozen-thawed oocytes were 85.1 and 66.8%. There was no significant difference on cryoprotective agents. 3. Penetration rates of the frozen-thawed and intact oocytes were 69.0 and 77.0%, respectively. 4. Hamster oocytes cryopreservation provides a convenient way of supplying and trans-porting hamster oocytes for the assessment of the fertilizing potential of human spermatozoa.

• Clinical and Experimental Reproductive Medicine
• /
• 제29권1호
• /
• pp.37-44
• /
• 2002

Objective : Heat shock protein family is related to protective mechanism of cells by environmental changes. This study was performed to evaluate the effect of cryopreservation on the heat shock protein 90 (Hsp90) expression in mouse ovarian tissue. Methods : Cryopreservation of mouse ovarian tissue was carried out by slow freezing method. The mRNA level of Hsp90 expression in both fresh and cryopreserved mouse ovarian tissue was analyzed by RT-PCR. The protein expression of Hsp90 was evaluated by Western blot analysis and immunohistochemistry. Results: The mRNA and protein of Hsp90 were expressed in both fresh and cryopreserved mouse ovarian tissue. The amount of Hsp90 mRNA was increased in cryopreserved ovarian tissue after 60 and 90 minutes after thawing and incubation. The amount of Hsp90 protein was increased in the cryopreserved ovarian tissue after 6 hours of the incubation in Western blot analysis. In immunohistochemical study, Hsp90 protein was localized in cytoplasm of oocytes and granulosa cells. Significant level of immunoreactive Hsp90 protein was detected in theca cells contrast to the weak expression in ovarian epithelial cells. Conclusion: This results showed the increase of Hsp90 expression in both mRNA and protein level in the cryopreserved mouse ovarian tissue. It can be suggested that Hsp90 may play a role in the protective or recovery mechanism against the cell damage during cryopreservaion.

• Reproductive and Developmental Biology
• /
• 제28권4호
• /
• pp.261-265
• /
• 2004

The present study was conducted to assess sperm characteristics in miniature-pig. The semen samples were transported to the laboratory at 17℃ within 3 hours after collection. The extended semen was stored at 17℃, and sperm quality was evaluated at 0, 1, 3, 5 and 7 days after storage. The semen volume of miniature-pig (62±22㎖) was significantly (p<0.05) lower than that of Duroc (155±25㎖) and Yorkshire (154±23㎖). Significant differences were also observed in sperm concentrations. During 3 days of storage, sperm viability did not differ among miniature-pig, Duroc and Yorkshire. However, the viability was significantly (p<0.05) lower in miniature-pig than in Duroc and Yorkshire semen after Day 3 of storage. In abnormality, acrosome intactness and intensity, there were no differences among miniature-pig, Duroc and Yorkshire semen. On the other hand, the viability of frozen-thawed sperm in miniature-pig was significantly (p<0.05) lower than in that of Duroc and Yorkshire. This study also examined CTC patterns in frozen-thawed spermatozoa. The rates of AR pattern were higher in miniature-pig than in Duroc and Yorkshire. However, no difference was found in F, B and AR patterns. The results of present study suggest that further research is necessary to develop of semen extender and freezing methods to improve sperm quality in miniature-pig.