• Parasites, Hosts and Diseases
• /
• v.27 no.4
• /
• pp.231-248
• /
• 1989

The growth and development of the metacercariae of F. seoulensis cultivated in vitro or on the chick chorioallantois were assessed by comparison with the optimum process of maturation in albino rats and new born chickens. The process of maturation was divided for convenience into six stages: Stage 1 ; cell multiplication, Stage 2; body shaping, Stage 3; separation of genital anlagen, Stage :1 organogeny, Stage 5; gametogony, and Stage 6: oviposition. In Hank's and Tyrode's .solutions, the metacercariae were alive up to 200 days or more at $4^{\circ}C$ without any development. The in vivo maturation process in rats or chicks was as follows: stage 1 from 6 hours; stage 2 from 24 hours; stage 3 from 48 to 72 hours; stage 4 from 3 to 4 days; stage 5 from 4 to 5 days; and stage 6 from 5 to 8 days. Despite unsuccessful infection of the metacercariae to 12 day old chicks, fully mature worms of stage 5 or 6 were recovered from new born chicks (1 to 2 days old), The metacercariae of F. seoulensis grown in vitro were up to stage 3 and no further maturation was observed. Of various media employed, the medium NCTC 109 (Gibco) or NCTC 135(Gibco) supplemented with 20% egg yolk or 20% whole egg macerate or 0.5% yeast was basically required for the earlier development of the fluke. It took 16.1 days(in average) to reach the stage 3 after cultivation. The metacercariae cultivated on the chorioallantoic membranes of 6∼13 day old chick embryo at 37∼38℃ showed their full development up to stage 5 or 6. However, the worms were in general remarkably retarded, compared with those grown in rats or chickens. In the experiments of worm transplant, although the transfer was failed from in vitro culture to in vivo of rats(Per os), the transplants from in vitro culture to the chorioallantois and from the choriollantois to in vivo of rat host were successful with or without development of the transferred worms. In the present study, it was observed that the metacercariae of F, seoulensis can be maintained in vitro media with poor development as well as fully matured in 1 to 2 day-old chicks or on the chorioallantois at a very low rate.

• Korean Journal of Animal Reproduction
• /
• v.10 no.2
• /
• pp.204-210
• /
• 1986

As a part of in vitro fertilization(IVF) for farm animals, IVF experiment was conducted using New Zealand white rabbits with their sperm capacitated in vivo. The effect of uterine conditions on sperm capacitation and effect of sperm concentration and fertilization media on IVF rate and implantation of in vitro fertilized ova were studied. The results obtained are summarized as follows; 1. Acrosomal reaction, noted after staining, of sperm recovered from ligated and intact uterus of capacitators was 83.0% and 65.7%, respectively. 2. IVF rate of ova inseminated with sperm from ligated uterus tended to be higher in DM or with higher concentration of sperm than in the modified F12 medium or with lower sperm concentration. Cleavage rate of fertilized ova for 48hr in DM was 31.5% for 106/ml and 30.0% for 104/ml of sperm and that in modified F12 medium was 26.0% for 106/ml and 22.3% for 104/ml of sperm. 3. Using the sperm from intact uterus, cleavage rate of fertilized ova showed same tendency as those shown with ligated uterus. The rate was 82.0% for 106/ml and 66.5% for 104/ml of sperm in DM and was 69.0% for 106/ml and 56.5% for 104/ml of sperm in the medium. 4. When normal ova up to 48hr after IVF were cultured for 4 days in either DM or modified F12 medium, ova developed to blastocyst stage showed higher rate in the groups of higher sperm concentration in the both media. The rate was 80.9% and 60.0% for 106/ml and 104/ml of sperm in DM and 91.7% and 71.4% for 106/ml and 104/ml of sperm in the modified F12 medium, respectively. 5. Rate of implantation after transfer of 4- or 8-cell embryos was 36.8%.

• Animal Bioscience
• /
• v.35 no.9
• /
• pp.1360-1366
• /
• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.73-73
• /
• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.3
• /
• pp.345-353
• /
• 1999

In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those 01 in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.8
• /
• pp.1193-1203
• /
• 2019

We investigated the protective effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) from Polysiphonia morrowii Harvey against hydrogen peroxide ($H_2O_2$)-induced apoptosis in Vero cells. BDB exhibited scavenging activity for DPPH, hydroxyl, and alkyl radicals. BDB also inhibited $H_2O_2$-induced lipid peroxidation, cell death, and apoptosis in Vero cells by inhibiting the production of ROS. To evaluate the molecular mechanisms of apoptosis inhibition, the expression of Bax/Bcl-xL and $NF-{\kappa}B$ was assessed by western blot assay. BDB significantly suppressed the cleavage of caspase-9 and PARP and reduced Bax levels in $H_2O_2$-induced Vero cells. Besides, BDB suppressed the phosphorylation of $NF-{\kappa}$B and the translocation of p65 in $H_2O_2$-induced cells. Furthermore, we evaluated the effect of BDB on ROS production, cell death, and lipid peroxidation in an $H_2O_2$-stimulated zebrafish embryo model. Taken together, these results indicated that ROS generation and cell death were significantly inhibited by BDB in zebrafish embryos, thereby proving that BDB exerts excellent antioxidant activity in vitro and in vivo.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.128-128
• /
• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.7
• /
• pp.855-862
• /
• 2010

Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful pathways. The Mx protein has direct antiviral activity and inhibits a wide range of viruses by blocking an early stage of the viral replication cycle. Cloning, characterization, and expression of Mx in vivo and in vitro have been conducted. The chicken Mx gene spans 21 kb and is made up of 14 exons and 13 introns, of which the promoter region was analyzed. The real-time PCR results showed that Mx expression was increased in chicken embryo fibroblasts (CEF) after 12- and 24-h induction with polyI: C. Induction of Mx expression by poly I: C in vivo revealed tissue-specific patterns among the chicken tissues tested. A trace expression of Mx was detected in healthy chicken liver tissues from adult chickens without inducement; the expression levels in the liver, heart, and gizzard were higher than in the muscle and kidney. This is the first report to demonstrate the expression of a glutathione-S-transferase-tagged-Mx fusion protein of 75 KDa, as well as the biological activity tested by SDS-PAGE and western blotting.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.1
• /
• pp.1-8
• /
• 1999

In order to study the implantation mechanism various methods for culture of endometrial cells in vitro have been attempted. However, a disadvantage is that primary cultures of stromal and epithelial cells do not have the ability to differentiate, and therefore cannot be reproduced in the same manner as in vivo endometrium. The object of this study is to establish a three dimensional culture of endometrial cells which are both morphologically and functionally identical to in vivo endometrium. Endometrial tissues obtained after hysterectomies were cut into thin slices and treated with collagenase and trypsin-EDTA. The stromal cells and the epithelial cells were separated by centrifugation and cultured for 24 hours in DMEM media containing 10% FCS, 100 nM progesterone, and 1 nM estradiol. The cultured stromal cells were mixed with collagen gel and solidified, after which it was covered with matrigel. Epithelial cells were inoculated on the top and then cultured for 3 days. The three dimensionally cultured endometrial cells were stained for integrin ${\alpha}1,\;{\alpha}4,\;{\beta}3$, and cyclooxygenase-l, -2 by immunohistochemistry, which all showed strong expression. The cultured epithelial cells showed the formation of microvilli, tight junctions and pinopodes by electron microscopy. Studies are currently under way utilizing this three dimensional culture model to ascertain the interaction between the embryo and human endometrial cells at the time of implantation, and it is thought that further studies into a new culture environment which would allow longer periods of culture will be necessary.

• BMB Reports
• /
• v.34 no.3
• /
• pp.206-211
• /
• 2001

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and the growth of several primary tumors. However, the opinions on the activity of endostatin derivatives deleted N- or C- terminal are still controversial. In this regard, we produced mouse endostatin and its derivatives in the prokaryotic system, and studied their anti-tumor activity. The [$^3H$]-thymidine incorporation assay demonstrated that N-terminal deleted mouse endostatin, and a C- and N-terminal deleted mutant, effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVECs). The biological activity of endostatin was also shown by its in vivo anti-angiogenic ability on the chorioallantoic membrane (CAM) of a chick embryo. Treatment of $200\;{\mu}g$ of mouse endostatin, or N-terminal deleted mouse endostatin, inhibited capillary formation of CAM 45 to 71%, which is comparative to a 80% effect of positive control, $1\;{\mu}g$ of retinoic acid. An in vivo mouse tumor growth assay showed that N-terminal deleted mouse endostatin, and the N-/C-terminal deleted mutant, significantly repressed the growth of B16F10 melanoma cells in mice as did the full-length mouse endostatin. According to these results, N-and N-/C-terminal deleted mouse endostatins are the potent inhibitors of tumor growth and angiogenesis.