• 한국수정란이식학회지
• /
• 제14권3호
• /
• pp.145-153
• /
• 1999

This study was conducted to investigate the effects of vitrification solution and developmental stage on the survival rate of vitrified-thawed human blastocyst embryos. Human blastocyst embryos were cryopreserved by vitrification using EFS and GE solution, and their survival rates were examined after thawing and further culture. EFS solution was consisted of 40% ethylene glycol, 18% Ficoll 70 and 0.3M sucrose. GE solution was consisted of 25% glycerol and 25% ethylene glycol. Embryos were exposed to EFS and GE solution by 2 steps and 3 steps, respectively, and plunged into liquid nitrogen after loading into 0.25ml plastic straws. Blastocysts were classified into 4 groups in accordance with their developmental stage: into 1) EEB, 2) MEB and 3) EdB, of blastocysts developed on day 5, and 4) 6d-Bla(the blastocysts which formed on day 6). The blastocysts at each stage were vitrified by GE solution and cryopreserved in LN2. After thawing them, we examined their survival rates, respectively. The resulted of this study were as follows: 1. The survival rate of blastocysts vitrified by GE solution was 64.4%, significantly higher than that (5.7%) vitrified by EFS solution (P<0.001). 2. When the blastocysts were vitrified by GE solution according to each developmental stage, the survival rates of EEB, MEB, EdB and 6d-Bla were 65.9%, 65.9%, 73.2% and 58.1%, respectively. In conclusion, the cryopreservation of human blastocysts by vitrification is likely to have a marked advantage in terms of cost, work and time as compared to the conventional slow freezing in IVF-ET programs.

• 한국수정란이식학회지
• /
• 제17권3호
• /
• pp.227-234
• /
• 2002

본 연구는 한우미경산우에 인공수정 7일째와 복제수정란 이식시 hCG를 투여에 의해 수태율 향상을 위하여 실시하였다 인공수정 후 임신 60일째에 임신감정을 실시한 결과 대조구, CIDR 그리고 hCG를 투여한 결과 53%, 46%, 71%였으며, 처리간에 유의적인 차이를 보였다(P<0.05). 복제수정 란의 이식시 hCG를 투여한 결과 대조구, hCG투여구는 각각 5.8%, 10.4%를 나타냈으며, 유의차는 인정되지 않았다. 인공수정 7일째에 hCG를 투여한 한우 미경산우의 발정일부터 발정 28일까지의 혈중 P4농도는 발정초기에는 처리구 모두 약간의 상승을 나타냈으며, 임신을 위한 가장 중요한 시기인 발정 13~16 일에는 무처리구에 비하여 hCG투여구가 3배 이상 의 수준을 나타냈으며, CIDR처리 임신우에 비교 하여도 약 B배 이상의 P4농도를 나타내었다. 그 이 후에도 P4의 농도는 2~3배의 수준을 지속적으로 유지하여 임신을 위하여 안정된 P4의 농도를 나타내었다. 인공수정 7일째 hCG를 투여한 한우 미경산우의 IGF-I 및 IGF-II의 혈중호르몬 농도는 IGF-I은 임신.비임신 그리고 hOG 및 CIDBt의 처리에 관계없이 큰 차이를 보이지 않았다. IGF-II는 CIDR(E2 capsule 제거) 처리구에서 임신이 비임신에 비하여 높은 경향을 나타냈으나, hCG 투여구에서는 임신과 비임신간에 큰 차이를 보이지 않았다. 인공수정 7일째에 hCG를 투여한 한우 미경산우의 발정일로부터 28일째까지의 혈중 cortisol 농도 는 발정당일과 발정 16일째의 임신한 미경산우에 서는 다른 발정주기일보다는 약간 높은 경향을 보였으며, 임신CIDR의 경우가 비임신보다는 전반적으로 높게 나타났고, hCG를 투여한 것은 큰 변화를 보이지 않았다. 이상의 결과에서 인공수정 및 복제수정란의 이식시 hCG 1,5001u를 투여한 한우 미경산우는 임신유지를 위한 혈중 P4의 농도를 상승시키며 수태율도 향상시키는 것으로 나타났다. IGF와 cortisol은 hCG 투여가 초기 임신기에는 큰 영향을 미치지 않는 것으로 나타났다.

• 한국수정란이식학회지
• /
• 제22권1호
• /
• pp.53-61
• /
• 2007

본 연구는 PI한 한국흑염소 수정란의 이식 결과를 통해 수란 흑염소의 수태율에 영향을 미칠 것으로 생각되는 여러 가지 요인을 분석함으로써, 높은 수태율을 얻을 수 있는 수란 흑염소의 최적 조건을 찾아낼 목적으로 수행하였다. 분석 결과, 수태율에 유의적인 영향을 주는 요인들은 발정형태, 수술 빈도, 이식 부위, 황체의 발육 단계, 수정란의 발육단계, 이식된 수정란의 수 등이었다. 자연 발정이 관찰되어 이식된 흑염소(59.1%, 13/22)들이 $CIDR^(R)$로 발정이 유도된 후 이식된 흑염소(36.8%, 118/321)에서보다 높은 수태율을 나타내었으며(P<0.05), 두 번째 수술 받은 흑염소의 수태율(56.5%, 13/23)이 처음 이식 받은 흑염소(36.5%, 116/318)에 비해 수태율이 높았다(P<0.05). 이식한 부위에 따른 수태율은 좌측 난관에 이식한 흑염소(49.0%, 50/102)가 오른쪽 난관에 이식한 흑염소(35.9%, 46/128)에 비해 수태율이 더 높게 나타났으며(P<0.05), 황체의 발육 단계에 따른 수태율에서는 $CH_1$단계(47.5%, 57/120) 출혈체를 가진 수란 흑염소에서 $CH_3(17.9%,\;7/39)$의 출혈체를 가진 수란 흑염소보다 높은 수태율을 얻었다(P<0.01). 수정란의 발육단계에 따른 차이는 난관 이식의 경우에 1세포기 배가 이식된 수란 흑염소의 수태율(51.6%, 49/95)이 4세포기배를 이식한 경우(24.5%, 12/49)보다 높았다(P<0.01). 수정란의 수는 2개를 이식했을 때(27.%, 37/137)보다 3개를 이식했을 때(47.6%, 50/105) 더 높은 수태율을 얻었다(P<0.01). 수태율에 유의적인 영향이 없는 요인들은 난관 유착이나 자궁 유착, 난소 유착, 자궁각의 크기, 황체의 수, 대형 난포의 존재 유무, 이식의 난이도 등이었다 그러나, 중간 크기의 자궁을 가진 수란 흑염소(38.9%, 122/314)에서 직경 5mm 이하의 작은 자궁을 가진 수란 흑염소(20%, 1/5)나 20mm 이상의 큰 자궁을 가진 수란 흑염소(18.2%, 2/11)보다 수태율이 높은 경향을 보였고, 배란 황체가 있는 같은 쪽 난소에 대형 난포가 존재할 경우(53.3%, 16/30)에 존재하지 않는 경우(37.1%, 104/280)보다 수태율이 높아지는 경향을 보였으며, 이식이 쉽게 이루어진 경우(39.2%, 125/319)에 이식이 어렵거나(27.8%, 5/18) 곤란한 경우(0%, 0/3)에서보다 높은 수태율을 얻을 수 있었다. 따라서, 자궁각의 크기나 대형 난포의 존재 유무, 이식의 난이도 등도 수정란 이식 후의 수태율에 영향을 줄 수 있을 것으로 생각된다. 이상의 결과로 볼 때 PI한 한국 흑염소 수정란의 이식 시 높은 수태율을 얻기 위해서는 수란 흑염소는 자연 발정 온 개체를 이용하고, 난관에 이식하고자하는 경우에는 난소에 $CH_1$ 단계의 출혈체가 존재하는 쪽 난관에 1세포기의 수정란을, 그리고 자궁에 이식하는 경우에는 난소에 발육 단계가 $CL_3$인 황체가 존재하는 쪽 자궁각에 중기 배반포나 후기 배반포를 이식하는 것이 가장 바람직하다는 결론을 얻었다.

• 한국수정란이식학회지
• /
• 제29권2호
• /
• pp.157-161
• /
• 2014

The study aimed to determine the physical characteristics of estrus mucus and conception rates in dairy cattle. Samples of estrus mucus from the cervix were collected from 108 dairy cattle during heat and were examined for color and consistency. Samples were taken from bred animals at starting from day of breeding to the completion of one estrus cycle. The color of the cervical mucus was studied based on its transparency while the consistency was based on the thinness and thickness of the cervical mucus. The dairy cattle were bred and the pregnancy diagnosis was performed at the $60^{th}$ day post breeding. Findings showed that the estrus mucus of the dairy cattle was transparent in 58.3%, turbid in 31.5% and dirty in 10.2%. It was further observed that the mucus consistency of the dairy cattle was thin in 74.1% and thick in 25.9%. In the pregnant group, 67.3% mucus samples were found transparent, turbid in 23.6% and dirty in 9.1%. However, the corresponding figures for the non-pregnant group had 49.1%, 39.6% and 11.3%. The consistency of cervical mucus was found to be thin in 74.1% and thick in 25.9% of dairy cattle. The conception rates of dairy cattle with thin and thick consistency of cervical mucus were 81.8% and 18.2%, respectively. Pregnant was associated with consistency of cervical mucus (p<0.10). Findings indicated that dairy cattle with thin consistency of cervical mucus and had clear discharge were pregnant cows.

• Clinical and Experimental Reproductive Medicine
• /
• 제47권4호
• /
• pp.284-292
• /
• 2020

Objective: This study investigated whether adding outer-well medium to inhibit osmotic changes in culture media in a dry-type incubator improved the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) cycles. Methods: In culture dishes, the osmotic changes in media (20 µL)-covered oil with or without outer-well medium (humid or dry culture conditions, respectively) were compared after 3 days of incubation in a dry-type incubator. One-step (Origio) and G1/G2 (Vitrolife) media were used. Results: The osmotic changes in the dry culture condition (308 mOsm) were higher than in the humid culture conditions (285-290 mOsm) after 3 days of incubation. In day 3 IVF-ET cycles, although the pregnancy rate did not significantly differ between the dry (46.2%) and humid culture (51.0%) groups, the rates of abortion and ongoing pregnancy were significantly better in the humid culture group (1.5% and 49.5%, respectively) than in the dry culture group (8.3% and 37.8%, respectively, p<0.05). In day 5 IVF-ET cycles, the abortion rate was significantly lower in the humid culture group (2.2%) than in the dry culture group (25.0%, p<0.01), but no statistically significant difference was observed in the rates of clinical and ongoing pregnancy between the dry (50.0% and 25.0%, respectively) and humid culture groups (59.5% and 57.3%, respectively) because of the small number of cycles. Conclusion: Hyperosmotic changes in media occurred in a dry-type incubator by evaporation, although the medium was covered with oil. These osmotic changes were efficiently inhibited by supplementation of outer-well medium, which resulted in improved pregnancy outcomes.

• 한국수정란이식학회지
• /
• 제15권2호
• /
• pp.191-196
• /
• 2000

the present study was carried out to develop a completely defined culture system and determine if high NaCl concentrations in defined (PVA added) or semi-defined (BSA added) medium is toxic to bovine embryos. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro. After 30 h of insemination, only 2-cell stage embryos were selected and cultured for this experiment. The culture media used were as follows : TLP(114 mM of NaCl) + BSA (3 mg/ml), TLP + PVA (1 mg/ml), mTLP(96 mM of NaCl) + BSA, mTLP + PVA. Six to ten embryos were placed into a 30$\mu$1 drop of each medium and the embryos were examined at 10 day post-insemination without medium renewal. The experiment was replicated 4 times. All data were analyzed by chi-square. There were no significant differences among TLP-BSA, mTLP-BSA and mTLP-PVA in blastocyst development (21.6, 17.2 and 20.2%), respectively. Also, no differences were obtained in hatching rates (11.7, 9.9 and 12.2%), respecitively. However, there were significant differences between TLP-PVA (1.7% and 0.6%) and other group in blastocyst formation and hatching rates, respectively (p<0.01). Development of in vitro produced embryos cultured in BSA containing medium was not affected by high NaCl concentration, but in the completely defined medium, embryonic development was highly affected by NaCl. This study shows that reduced NaCl concentration in completely defined medium is beneficial for development of bovine pre-implantation embryos in vitro.

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권2호
• /
• pp.194-199
• /
• 2007

Effects of recombinant bovine somatotropin (bST) on plasma hormonal concentration, embryo quality, and pregnancy rate were examined during the superovulation and synchronization treatment in donor and recipient cows. Hanwoos (Korean native beef cattle) were treated with controlled internal drug release (CIDR) combined with bST (CIDR+bST) or without bST (CIDR) as donor cows. The embryos recovered from donors were transferred into Holstein recipient heifers treated with bST (CIDR+bST) or without bST (CIDR) for synchronization. The correlation between IGF-I and P4 showed a positive pattern in the CIDR+bST group (r=0.44, p<0.01), but a negative pattern was shown in the CIDR group (r = -0.59, p<0.02) at day 7 of estrous cycles. Although the number of recovered, transferable, and degenerated embryos was not different, quantities of grade 1 (excellent) embryos in CIDR+bST group were significantly higher than those of the CIDR group (p<0.01). The pregnancy rate was higher in the CIDR+bST recipient group compared to CIDR group (p<0.05), when the embryos were recovered from the donors treated with CIDR. However, the pregnancy was maintained highly in both recipient groups, when the embryos were produced by CIDR+bST treated donors. It can be concluded that bST administration combined with CIDR is an effective method for superovulation and synchronization treatment to stabilize plasma hormonal levels, to obtain excellent quality of embryos, and to get higher pregnancy rate.

• 한국가축번식학회지
• /
• 제26권4호
• /
• pp.385-394
• /
• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• Clinical and Experimental Reproductive Medicine
• /
• 제45권3호
• /
• pp.122-128
• /
• 2018

Objective: To determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal. Methods: This study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n = 87) and the no fragment removal group (n = 104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in $Ca^{2+}$- and $Mg^{2+}$-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of $30{\mu}m$. Results: There were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres ($7.1{\pm}1.7$ vs. $6.9{\pm}1.6$) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group ($1.9{\pm}0.7$) was significantly higher than that of the control group ($3.1{\pm}0.5$, p< 0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p< 0.05). Conclusion: Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.

• 한국수정란이식학회지
• /
• 제27권3호
• /
• pp.133-139
• /
• 2012

This study was carried out to develop the useful inducing method of estrus for Korean native cows. Under the condition of estrus induction by administering $PGF_{2{\alpha}}$ for the cows in which corpus luteum (CL) in ovaries was detected by ultrasonography, ovarian responses and the changes of progesterone ($P_4$) concentration against $PGF_{2{\alpha}}$ compared with conception rate were observed in cows and heifers. In inducing estrus administering $PGF_{2{\alpha}}$. to the cows which has corpus luteum in ovaries, ovarian reponses, the changes of progesterone concentration, and conception rate were identified and compared. The results attained from the studies were as follows. Significant decreases of CL in size over time after $PGF_{2{\alpha}}$ administration were detected in both cow and heifer groups (p<0.001), but not different between groups in the CL regression rate (p>0.05). In addition, the percentage changes relative to the plasma $P_4$ concentration on day 1 after $PGF_{2{\alpha}}$ treatment were decreased to below 1ng/ml. The growth rate of follicle was observed as 31% on day 1 and 42% on day 2 in cows, and 34% on day 1 and 97% on day 2 in heifers, resulting that growth of heifers are faster than that of cows (p<0.05). The conception rate after $PGF_{2{\alpha}}$ treatment were 60.5% and 64.2% in cows and heifers, respectively. It also indicated that the conception rate after estrus observation with $PGF_{2{\alpha}}$ injection was as high as 66.6% while that with timed-artificial insemination (TAI) regardless of the estrus observation was 56%, which means the pregnancy rate of artificial insemination after estrus observation was higher than that of TAI (p<0.05). In the result of all above, there were significant decreases in CL size and the plasma $P_4$ concentration by days but rapid growth in follicles, which has no differences in cows and heifers. The conception rate was commonly high after estrus observation and more than 50% under TAI.