• 한국수정란이식학회지
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• 제33권3호
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• 한국수정란이식학회지
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• 제27권2호
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• pp.93-100
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• 2012

To investigate the biochemical nature of changes in vaginal physiology during estrus and pregnancy, we examined the cytology and viscosity, and monitored the protein expression profile in vaginal mucus during estrus and pregnancy. The viscosity progressively decreased from estrus to pregnancy. Cell type analysis revealed that white blood cells progressively increased from estrus to pregnancy, while red blood cells progressively decreased during pregnancy. The cornification index (CI) was higher in estrus than in pregnancy. Protein mass spectrumetry identified the presence of ribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domain of unknown function729) domain-containing protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrix metalloprotease)-9 in vaginal mucus. MMP-2 and MMP-9 proteins in the vaginal mucus were active throughout estrus and gestation, as measured by a gelatinase assay, but most abundant in the vaginal mucus on day 0 of estrus. Results from ELISA of MMP-2 and MMP-9 were in accordance with the gelatinase assay. In light of the crucial role of metalloproteinases in extracellular matrix remodeling, the level of MMP-9 in vaginal mucus might be useful as an indicator of estrus and pregnancy to increase the efficiency of reproduction.

• 한국수정란이식학회지
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• 제25권1호
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• pp.21-27
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• 2010

The increase in the total number of cows on farms, which breed Korean Native Cattle (KNC), is associated with many problems. In particular, the services per conception and calving interval have increased. In this study, we examined the effect of dietary supplementation with vitamin and mineral complex on the estrus rate, pregnancy rate, gestation length, and birth weight of KNC calves. Multiparous or primiparous KNC were divided into 3 groups with 40 heads per group. Experimental group 1 was administered a consisting of 35,000 IU vitamin A, 100 IU vitamin E, 200 mg $\beta$-carotene, 200 mg Zn methionine, and 1.5 mg Se. Experimental group 2 was administered a vitamin complex (100 g/day) consisting of 100 IU vitamin E and 200 mg $\beta$-carotene. In the case of multiparous KNC, the estrus detection rate in the control group was 90.0% and those in the experimental groups were 75.0% to 95.0%. However, the first-service pregnancy rate after parturition in the control group was 41.2%, which was significantly lower than that in the experimental groups (71.0% to 76.7%; p<0.05). The average duration of pregnancy in the group supplemented with the vitamin complex was days, which was similar to that in the case of the control group. The birth weight of calves from cows fed with vitamin complex was 25.3 to 27.0 kg, which was similar to that in the case of the control group (25.2 to 26.0 kg). In the case of primiparous KNC, no differences in the estrus rate, pregnancy rate, gestation length, or the birth weight of calves were noted between the groups. Thus, dietary supplementation vitamin and mineral complex have no effect on the reproductive efficiency in primiparous cows, but the pregnancy rate was observed to have increased in multiparous KNC with these supplements.

• 한국수정란이식학회지
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• 제9권2호
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• pp.153-160
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• 1994

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 $\mu$sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

• 한국수정란이식학회지
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• 제29권4호
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• pp.321-325
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• 2014

The present study was performed on farm animals to test the effectiveness of progesterone-releasing intravaginal device (Cue-Mate$^{(R)}$ 1.56 g) and injection of prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$) for synchronization estrus in Hanwoo cattle. The cattle were at random stage of the estrus cycle. The cows were artificially inseminated at day 7 after Cue-Mate withdrawal, using commercial semen from Korean native bulls. There was a season effect on the estrus synchronization rate. It was higher in spring (94.3%) followed by winter (93.3%), autumn (90.4%) and summer (67.2%). In summary, The results of this study revealed that season has influences on estrus behavior of cattle with no significant effect on pregnancy rate. In summary, we suggest summer reproductive management to alleviate the effects of heat stress. It should be based on intensive cooling combined with hormonal treatment. Given that different subgroups of cows benefit differently from the treatments, selective hormonal administration should be considered.

• 한국수정란이식학회지
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• 제18권3호
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• pp.249-255
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• 2003

종빈돈의 번식효율 증진을 위해 후보종빈돈의 성성숙일령, 첫교배일령 및 등지방 두께 등이 번식능력에 영향을 미치는 요인을 조사$.$분석하여 우수한 후보종빈돈의 조기선발에 활용코자 수행한 결과는 다음과 같다. 1. 출생계절별 첫발정일령 및 첫수정일령은 봄에 출생한 후보종빈돈은 194.14일 및 222.05일, 여름에 출생한 후보종빈돈은 163.25일 및 193.00일, 가을에 출생한 후보종빈돈은 160.25일 및 199.20일, 겨울에 출생한 후보종빈돈은 159.72일 및 190.11일로 봄에 출생한 후보종빈돈의 첫발정일령이 겨울, 가을, 여름에 출생한 후보종빈돈보다 유의적으로 늦게 나타났다(P< 0.01). 2. 등지방두께가 13∼16mm인 후보종빈돈의 첫발정일은 180.32일, 17∼20mm인 후보종빈돈은 171.24일, 21∼23mm인 후보종빈돈은 162.20일로 등지방두께가 얇을수록 첫발정일령이 지연되었으나 유의적인 차이는 인정되지 않았다. 3. 등지방두께가 13∼16mm인 후보종빈돈의 첫수정일령은 211.12일, 17∼20mm인 후보종빈돈은 202.43일, 21∼23mm인 후보종빈돈은 195.43일로 등지방 두께가 얇을수록 첫수정일령이 지연되었으나 유의적인 차이는 없었다. 4. 첫발정일령이 160일령 이하일 때에 산자수는 9.64두였으며, 161∼180일렬인 경우 10.14두, 181∼200일령인 경우 9.56두, 201일령이상인 경우 9.13두로 첫발정일령이 161∼180일이었을 때가 산자수가 가장 많았으나 유의적인 차이는 없었다. 5. 첫수정일령이 180일령 이하일 때에 산자수는 9.13두였으며, 181∼200일령인 경우 9.75두, 201∼220일령인 경우 10.13두, 221일령 이상인 경우 9.45두로 첫수정일령이 201∼220일령이었을때가 산자수가 가장 많았으나 유의적인 차이는 없었다. 6. 첫수정시 등지방두께가 13∼16mm일 때에 산자수는 9.33두였으며, 17∼20mm인 경우 9.81두, 21∼20mm인 경우 10.17두로 첫수정시 등 지방두께가 21∼23mm이었을 때가 산자수가 가장 많았으나 유의적인 차이는 없었다.

• 한국수정란이식학회지
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• 제27권4호
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• pp.223-228
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• 2012

This study was designed to adopt two estrus synchronization protocols in zebu and crossbred heifers and their effects on pregnancy rate after timed artificial insemination (TAI). A number of 120 cyclic heifers were allotted for two different treatment groups and one control group. Heifers under protocol A were injected with GnRH at first day followed by a single dose of $PGF_{2{\alpha}}$ at Day 11 and injection of GnRH at the day of AI; and heifers belonged to protocol B were treated with GnRH, two $PGF_{2{\alpha}}$ injections at 11 days apart and injection of GnRH at AI. AI was done at fixed time (within 72~96 hours after $PGF_{2{\alpha}}$ injection) in both protocols and pregnancy was confirmed by rectal palpation on 80~120 days of post AI. In control group; local heifers were conceived higher (30%) proportion than that of crossbred heifers (25%; p<0.05). In protocol A, the local breed were conceived higher (38.9%) proportion compared with crossbred (25%; p<0.05). In protocol B, local breed heifers were conceived higher (38.9%) proportion compared with crossbred heifers (33.3%; p<0.05). The overall pregnancy rate in protocol A and protocol B was 33.3% and 36.6%, respectively. The proportion of pregnancy rate of local heifers (38.9%; Protocol A) was significant (p<0.05) in comparison with local heifers (30%) in control group (p<0.05). The overall pregnancy rate between pooled control group (28.3%) and treatment group (35%) was significantly (p<0.05) differ from each other's. Results of present study concluded that estrus synchronization followed by fixed time AI could be applied for higher pregnancy rate in zebu and crossbred heifers.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• 한국수정란이식학회지
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• 제33권3호
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• pp.149-157
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• 2018

The objective of this study was to evaluate the effect of treating dairy cattle with exogenous human chorionic gonadotrophin (hCG), five (5) days post artificial insemination (AI) on serum progesterone (P4) concentration and pregnancy rate. In this experiment, five days after AI, cows were assigned randomly to two groups namely: a) treated group (67) which were administrered with 1500 IU hCG (Chorulon) and b) control group (61), which received no treatment. On day 5, 10, 15 and 20 after the artificial insemination, blood samples from a total of 8 cows (4 from each group) were collected and were analyzed for serum P4 concentration. Cows were detected for estrus according to standing heat by visual observation. Cows that were detected still in estrus after days 18-24 were re-inseminated and recorded as not pregnant (open). Pregnancy diagnosis was conducted by ultrasonographic examination and transrectal palpation of the uterus on approximately 60 days in cows that observed to be not in estrus. The conception rate in hCG treated and control groups were 52.5 and 36.1%, respectively. The results proved that there were no significant differences in conception rate between two groups (p=0.0568). However, pregnancy rates were reduced by hCG treatment. Average serum P4 concentrations did not differ between Hcg-treated and control groups on day 5 (0.377 versus 0.375 ng/ml). On day 20 serum P4 concentrations were greater in the treated group compared with the control group (3.085 versus 2.010 ng/ml). The treatment with hCG seemed to increase P4 level compared with the control. In conclusion, the results of this study showed that 1500 IU of hCG administered on 5 day post AI increased conception rate in dairy cows. This was supported by the results on serum P4 concentration which was greater in hCG treated group.

• 한국수정란이식학회지
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• 제30권2호
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• pp.115-119
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• 2015

The study was carried out to assess the production performances of Japanese quail parent stock raised under open housing system and the study was conducted in a local farm with 2000 Japanese parent stock quail birds (Coturnix coturnix japonica). Observed body weight gain was recorded in each weekend up to 7 weeks of their age; birds gained weight 15.73, 31.45, 47.20, 90.80, 125.27, 153.14 and 175.20 grams, respectively. There have been observed sharp decline of fertility and hatchability of birds with increase of their age. Optimum fertility was recorded at third week and optimum hatchability at fifth week of their age. Average flock uniformity was found 83.68%. The hen day egg production of the flock was recorded 81, 83, 85, 86, 89, 89, 89, 89 and 88% in each weekend, respectively. It may therefore be concluded that Japanese quail parent stock performs well under open housing system in Bangladesh and future study can be taken to better understand their production performances.