• Korean Journal of Animal Reproduction
• /
• v.22 no.4
• /
• pp.307-317
• /
• 1998

This study was to investigate the vitrification method for cryopreservation technique of bovine in vitro fertilization(IVF) embryos. The morphological appearance and viability following vitrification of IVF bovine blastocysts and expanded blastocysts were examined. Embryos obtained 6, 7, 8 or 9 days after IVF were vitrified in both 40% ethylene glycoI(EG) plus 0.3M trehalose and 20% polyvinylpyrrolidone(PVP) in DPBS(ETP, Exp. 1) and ETP solution added 0.375M dextrose (ETPD, Exp. 2). The viability of Days 6, 7, 8 and 9 vitrified /thawed embryos at 24∼48 h culture after thawing was 11.9%, 19.8%, 23.4% and 15.3% in ETP(Exp. 1), and 34.6%, 54.5%, 37.9% and 13.0% in ETPD(Exp. 2), respectively. The viability of vitrified embryos produced from the culture days after IVF did not differ in Exp. 1, but significantly differ in Exp. 2(P＜0.05). The viability of blastocysts and expanded blastocysts significantly differed(P＜0.05) in 15.2% and 23.3%(Exp. 1), and 25.0% and 45.8%(Exp. 2). The result of Exp. 1 was similar to that of Exp. 2 on the viability of embryo according to developmental stages, but ETP solution plus sugar(dextrose) was increased the viability of vitrified embryos. In Experiment 3, The viability of vitrified embryos was not different between 12% and 20% PVP concentrations in ETP solution according to culture days or developmental stages. To investigate the effect of addition of sugar, two type of carbohydrates and a mixture of cryoprotectants for vitrification on the survival of bovine IVF embryos, bovine Days 7 to 9 blastocysts and expanded blastocysts were cryopreserved in either 20% glycerol plus 20% EG, 0.375M sucrose and 0.375M dextrose (GESD, Exp. 4) or 20% glycerol plus 20% EG, 0.3M trehalose and 20% PVP(GETP, Exp. 5) in DPBS. Survival rates of Day 7, 8 and 9 embryos at 24∼48h culture after thawing were 71.4%, 94.6% and 40.5% in GESD, and 59.5%, 81.5% and 62.5% in GETP, respectively. Hatching rates of Day 7, 8 and 9 embryos after thawing were 28.6%, 35.1% and 16.2% in GESD, and 27.0%, 33.3% and 18.8% in GETP, respectively. These results indicates that a mixture of cryoprotectants(glycerol and EG) and addition of sugar can improve the survival rates of the bovine IVF embryos(Day 7 or 8) vitrified, and the expanded blastocyst embryos are more suitable for vitrification than early blastocysts stage.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.1
• /
• pp.9-20
• /
• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• Development and Reproduction
• /
• v.16 no.2
• /
• pp.137-143
• /
• 2012

The main objective of this study is to investigate the effects of ovulation induction by treating HCG, LHRHa, GnRHa, ovaprim and pimozidein in long snout bullhead, L. longriostris. All hormons were injected into the muscles of back. Concentration of LHRHa to injection were 20, 50, 100, 150 and 200 ${\mu}g/kg$ and same concentration of LHRHa was injected after 24 hour. The ovulation induction rate was 100% and fertilization and hatching rates were 68.4, 45.2, 58.4 and 33.6% in 50 and 100 ${\mu}g/kg$. The times to ovulation were between 28 and 44 h. HCG was injected in long snout bullhead at 5,000, 10,000, 15,000, 20,000 and 25,000 IU/kg. The ovulation induction rate was 50% in 15,000 and 20,000 IU/kg. Fertlilzation and hatching rates were 55.2, 45.6, 52.8 and 44.8%. Ovulation time was between 72~80 h. HCG concentration of 500, 1,000, 2,000 and 4,000 IU/kg were injected with $50{\mu}g$ of LHRHa. Ovulation and hatching rates in 2,000 IU/kg were 75 and 35%. Ovulation time was 28~48 h. Ovaprim of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL/kg were injected to the abdominal cavity. The ovulation induction rate was highest at 2.5 mL/kg to 50% and ovulation time was between 66~86 h. LHRHa concentration of 50, 100, 200, 300 and 400 ${\mu}g/kg$ was injected with pimozide (1,000 ${\mu}g/kg$). Ovulation induction rate was 87% at 100 and 200 IU/kg with pimozide. Ovulation time was between 66~86 h.

• Korean Journal of Ichthyology
• /
• v.1 no.1_2
• /
• pp.9-18
• /
• 1989

Blenniid fish, Istiblennius stellifer(jordan et Snyder) is distributed in the coastal waters of south-eastern Korea and Japan. Matured adults of blenniid fish were collected from the rocky shore of Namchun-dong, Nam-gu, Pusan, Korea on May 15, 1988. The fertilized eggs were incubated and the larvae were reared in laboratory. The eggs of this species were demersal and adhesive, and their diameters varied from 0.84 to 0.88 mm(mean 0.86 mm, n=30). They have a number of small oil globules. The water temperature throughout incubation ranged from 18.5 to $23.3^{\circ}C$ and salinity was maintained at $28.2-29.5\;^{\circ}/_{\circ\circ}$. The hatching took place in 130 hours after fertilization. The newly hatched larvae were 2.70 mm in total length with 11 (abdominal)+22~25 (caudal)=33-36 myomeres. The larvae absorbed the yolk material and oil globule completely in 10 days after hatching and became postlarvae. Total lengths of the larvae reached 4.65 and 5.75 mm in 10 and 13 days after the hatching, respectively.

• Development and Reproduction
• /
• v.12 no.3
• /
• pp.283-288
• /
• 2008

The Pseudogobio esocinus were caught at Wyuleo-ri, Gyeombaek-myeon, Boseong-gun, Jeollanamdo from April to May 2003. The fishes were incubated in transparent aquarium located at the laboratory of Chonnam National University, and their embryonic and larval development were observed. The fertilized eggs were spherical, semitransparent, and adhesive, and were $1.98{\pm}0.19mm$ (n=50) in diameter. The embryo, including 31$\sim$32 myotomes, hatched through egg membrane at 164 hrs after fertilization. The newly-hatched larvae were $4.61{\pm}0.83mm$ (n=10) in total length (TL). At that moment, yolk was not absorbed, and mouth and anus were not open. Star and spot shaped melanophores were distributed on the lens, and dorsal, ventral, and caudal parts. At 42 days after hatching, larva was $16.22{\pm}0.65mm$ (n=10) in TL. Melanophores were scaterred at head, back, and side parts. Morphological features of the embryo were transferred to juvenile stage showing similar features with those of the adult fish.

• Journal of Embryo Transfer
• /
• v.29 no.2
• /
• pp.101-109
• /
• 2014

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups ($41.46{\pm}10.66$ vs. $37.73{\pm}8.92$ vs. $45.11{\pm}11.41%$ vs. $41.59{\pm}11.88$, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups ($79.84{\pm}12.63$ vs. $83.3{\pm}17.46$ vs. $78.55{\pm}14.48%$ vs. $72.02{\pm}14.09$). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.293-300
• /
• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.12
• /
• pp.1820-1826
• /
• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• The Korean Journal of Malacology
• /
• v.30 no.2
• /
• pp.107-116
• /
• 2014

Gonadal development and maturation mechanism were studied on the freshwater marsh clam Corbicula papyracea (Heude), which is the endangered species in Korea. The specimens were collected in the rearing ponds and waterway of NFRDI in Gapyeong-gun, Gyeonggi-do from January to December 2004, and then investigated by condition factor, relative growth, gonadal development phases and gonad histological characters based on 30-50 individuals every month. Comparing with the freshwater marsh clam, C. papyracea is small, light olive brown shell and violet interior. The hermaphrodite individuals of C. papyracea take an internal fertilization and fertilized eggs are stored in the foster-sack in the gills, then the hatched juveniles are released outside after an ovoviviparous process. The average water temperature of inhabit area was in range of $1.8-27.0^{\circ}C$ and usually took great effects on the gonad maturation of C. papyracea. The condition factor ranged from 0.14 to 0.21 throughout the year, which was the lowest during winter season (December-February), and gradually increased to the highest value of 0.21 in May. The ratio of meat weight to total weight was 25.9-38.7%, indicating the similar trend with condition factor. The highest values of condition factor and the ratio of meat weight appeared 1-2 months later than gonadosomatic index reached the peak value, it was probably because that the ovoviviparous eggs would spend a long period before hatching from the foster-sack in the gills. To synthesize the characters of meat weight, condition factor and gonad development by histological study, reproductive cycle of C. papyracea could be divided into five successive stages: multiplicative stage (December to February), growing stage (February to May), mature stage (June to August), spawning stage (August to November), recovery stage (November to December). The smallest shell length of matured C. papyracea was 12.6 mm, and individuals, larger than 16 mm, was formed the nursery in the gills.

• The Korean Journal of Malacology
• /
• v.26 no.2
• /
• pp.151-156
• /
• 2010

For industrialization of the hard clams, Meretrix petechiails (Lamarck), spawning was induced per spawning induction technique in the artificial maturation group administered of parent maturation control and the natural maturation group of which parents were transported for artificial spawning per time period. Then, fertilization rates, hatching rates and D-shaped larva development rates were investigated. In addition, growth and survival rates of larvae were investigated per larva breeding technique. The results of spawning induction by exposure in the artificial maturation group indicated that response rates were relatively higher at 23% and 32% respectively at the 4th hour and the 8th hour of exposure. In terms of water temperature increase, responses began only when the temperature reached $28^{\circ}C$ or higher. In the experiment group administered with both exposure and water temperature increase techniques, response rate was found to be 45% or higher at the 4th hour of exposure and the temperature of $28^{\circ}C$. At the temperatures of 29, 30 and $31^{\circ}C$, significant differences were not observed. Therefore, it was indicated that the response rates of parent hard clams were higher toward water temperature increase than exposure time. As for spawning induction per time period of the transported parent group, response rate and D-shaped larva development rate were the highest at 67.6% and 96% respectively on August 6, 2009. In terms of water temperatures during larva breeding experiment, growth was faster as water temperature was higher. In addition, growth and survival rates were relatively higher at the salinity of 25. In terms of stocking density, growth and survival rates were relatively higher at 5 inds./mL.