• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.293-300
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• 1998

The objective of this study was to determine effects of $\beta$-mercaptoethanol ($\beta$-ME) and cyst-eine (CYS) on the development of bovine em-bryos obtained from in vitro matured and fertil-ized oocytes. Cumulus-oocyte-complexes (COC-s) were matured in micro-drop of TCM-199 medium containing 10% FBS, 17$\beta$-Estradiol and FSH-p under paraffin oil at 39$^{\circ}C$ for 24 hrs. The fertilization of COC were induced in Fert-TALP medium supplemented with PHE, heparin, BSA and then the fertilized oocytes were cultured in CR1aa medium for 24 hrs. To investigate the effects of the agents on the development of the embryos, the embryos developed to the late 2-cell stage were cultured in the media with and without $\beta$-ME, CYS for 9 days. In experiment 1, to select appropriate concentration of $\beta$-ME and CYS during whole culture period (9 days), various concentrations of $\beta$-ME and CYS were add ded to the CR1aa medium. Addition of 25TEX>$\mu$M of $\beta$-ME and O.1mM of CYS to the culture medium 1 increase the incidence of embryos developed to the blastocyst. In experiment 2, we evaluated the effects of 25$\mu$M of $\beta$-ME and O.1mM of CYS addition on the blastocyst formation when emb bryos at different stages were exposed to 25$\mu$M $\beta$-ME and O.1mM of CYS. $\beta$-ME and CYS enhanced in vitro development of embryos to the blastocyst stage. The effect was greater in 8-ceII to morula embryos than in embryos fewer than 2-cells at the initiation of treatment. These results suggested that the addition of 25$\mu$M B-ME and O.1mM cysteine enhanced development to the blastocyst and hatching stage of in vitro derived bovine embryos, also addition of $\beta$-ME and cysteine were effective later stage embryo than early embryo development.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.149-159
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• 2015

This study was designed to know the possibility in repeat uses of elite donor cows for getting mass production of OPU-derived embryo production (OPU-IVP). Ultrasound transvaginal ovum pick-up (OPU) performed in 6 Korean native cows was aged 4 to 10 years old. The aspiration of immature oocytes for OPU derived embryo was carried out 2 times per week, and OPU-IVP of $1^{st}$ period was carried out 22~48 sessions from each donors. And the break time for OPU-IVP of $2^{nd}$ period after $1^{st}$ OPU from each donors were 2~25 months. The OPU-IVP of $2^{nd}$ period each donors conducted total 15~65 times for 2~8 months by an ultrasonographic, was guided follicular aspiration system. The average numbers of collected oocytes, grade 1 + grade 2(G1+G2) oocytes and cleavage embryo from $1^{st}$ period OPU-IVP were significantly differences between donors (p<0.05). Total collected oocytes of donor D were significantly higher compared with donors of A, B, C, E and F (average 17.0 per session vs. 11.2, 10.1, 8.5, 10.2 and 9.6; p<0.05) and also oocytes of G1+G2 were significantly higher compared with r A and D and subsequently to donors of B, C, E and F (average 7.9 and 8.5 per session vs. 5.0, 2.7, 6.0 and 1.6; p<0.05). Cleavage rate of donor D was significantly higher compared with donors of A, B, C, E and F (average 13.1 per session vs. 10.1, 9.1, 6.9, 8.9 and 6.7; p<0.05). The average numbers of OPU-IVP for $1^{st}$ period was significantly higher from donors of B, D and E than those from donors of A, C and F (average 6.5, 7.1 and 6.5 per session vs. 3.5, 4.2 and 2.8; p<0.05). The possibility investigation of $2^{nd}$ OPU-IVP was carried out after 2~25 months rest periods from $1^{st}$ period OPU session. Total average numbers of collected oocytes, cleavages and blastocyst development rates were significantly higher from $1^{st}$ period OPU compared with $2^{nd}$ period one (p<0.05). The OPU-IVP efficiency by break for more embryo production from elite cow was analysis comparing without rest of donor A, under 6 months rest period as B and over 6 months rest period as C and then the average numbers of collected oocytes, cleavages and blastocysts were significantly higher from A group (11.8, 9.5 and 5.2 per session) than those from B and C groups (7.9, 6.2 and 2.6 vs. 9.2, 7.5 and 3.9, p<0.05), and also C group was significantly higher than B group. In conclusion, $1^{st}$ period OPU-IVP was more efficient compared with $2^{nd}$ period repeated uses of donor, and the break times for additional production of embryo on donor were needed more than over 6 months after $1^{st}$ period OPU-IVP. This repeating uses of elite donor cows given more emphasis for getting the opportunity on mass production of elite cow OPU-IVP embryo should be increased G1+G2 possibility of genetic improvement of livestock within short period.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.407-417
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• 2000

The present study was to examine effects of various electrical stimulus treatments used for electro-fusion on the preimplantation development of bovine nuclear transfer (NT) embryos with fetal fibroblast cells. Fetal fibroblast cells were isolated from one fetus at day 45 of gestation in Holstein cow, and passaged 3 to 4 times before being transferred into enucleated oocytes. Single fibroblast cells were individually placed into the perivitelline space of enucleated oocytes by using a micromanipulator. At first, the fusion and developmental rates of reconstructed oocytes were compared between different electric stimulation conditions. When fusion of the reconstructed oocyte was induced by different electric pulse periods (15, 30 and 45 $\mu$sec) at a DC pulse of 1.8 kV/cm, 15 (45.5%, 120/264) or 30 $\mu$ sec group (43.9%, 106/241) showed a higher fusion rate than 45 $\mu$sec group (23.2%, 58/250, P＜0.05). However, no difference was detected in the development rate of the fused oocytes to blastocysts between groups. Next experiment was to examine the effects of different electrical field strengths (1.5, 1.8 and 2.1 kV/cm) for 15 $\mu$sec at electrofusion on in vitro development of the NT embryos. As results, there was no difference in the fusion and developmental rates of the NT embryos between electrical strength (P＞0.05). Finally, developmental competence of bovine NT embryos with somatic cells was compared with IVF-derived embryos. Of enucleated oocytes fused with fibroblast cells, 27.4% (75/274) developed to the blastocyst stage, which is similar to that (24.5%, 58/237) of IVF-derived embryos. However, mean nuclei number of NT blastocysts was smaller than that of IVF-derived blastocysts. Thus, we have established an optimal condition (1.8 kV/cm, 15 $\mu$sec) for electric fusion of bovine NT oocytes with somatic cells. The present study indicates that bovine reconstructed embryos with somatic cells normally develop to blastocyst stage in vitro, although having smaller nuclei numbers of blastocysts as compared to IVF-derived embryos.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.73-81
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• 2014

The in vitro production of porcine embryos was essential to increase of blastocyst development rate and select of high quality blastocyst in early stage. There were a lot of reports about in vitro porcine embryo development, but there was no report about the selection of high quality embryos. Therefore, in this study, we investigated the effect of vitamin $K_1$ (vit $K_1$) on the development and survival rate of porcine in vitro fertilized embryos. When vit $K_1$ was treated for 24 hr at day 1 in vitro culture, blastocyst development rate in the control group ($35.5{\pm}3.2%$) was significantly lower compared to $1.0{\mu}M$, $3.0{\mu}M$, or $6.0{\mu}M$ groups ($14.5{\pm}4.3$, 0.0, or 0.0%; p<0.05). The survival rates of blastocysts at day 8 in $1.0{\mu}M$, $3.0{\mu}M$ or $6.0{\mu}M$ of vit $K_1$ treated groups ($22.2{\pm}2.9$, 0.0 or 0.0%) were significantly lower than that of the control group ($31.8{\pm}2.6%$; p<0.05). We were added at $1.0{\mu}M$, $3.0{\mu}M$ or $6.0{\mu}M$ vit $K_1$ for different durations of time at day 1 in vitro culture. The development rate and survival rate in the group of $1.0{\mu}M$ vit $K_1$ for 6 hr was $26.5{\pm}2.9%$ and $47.2{\pm}2.8%$, respectively, which were differed significantly in the group of 12 hr (p<0.05). In the group of $3.0{\mu}M$ vit $K_1$, the blastocyst development in control group was $36.4{\pm}3.1%$ but, the survival rate $41.7{\pm}3.2%$ in the group of 3.0 hr was significantly higher than that of the control group (p<0.05). In the group of $6.0{\mu}M$ vit $K_1$, the control group's the blastocyst development was $32.0{\pm}2.8%$ and the 0.5 hr supplement group's survival rates was $42.9{\pm}1.8%$ higher than other groups. We added vit $K_1$ at day 1, day 2, day 4 and day 6 of in vitro culture, on the based the results of supplemented concentration and duration. In the group of $1.0{\mu}M$ 6.0 hr addition, the blastocyst development rate of day 4 and the survival rate of day 2 were the highest in each group. In the groups of $3.0{\mu}M$ 3.0 hr addition or $6.0{\mu}M$ 0.5 hr addition, the blastocyst development ($59.5{\pm}4.1%$ and $50.0{\pm}3.6%$) and survival rates ($72.7{\pm}5.4%$ and $79.2{\pm}4.0%$) on day 4 were significantly higher than that of control and other experiment groups (p<0.05). Meanwhile, the number of cells in blastocysts that produced by vit $K_1$ supplementation was $53.4{\pm}5.8$, $49.4{\pm}3.8$ and $51.5{\pm}4.5$ respectively, which were significantly higher than that of $40.2{\pm}2.3$ in the control group (p<0.05). There was no difference of the number of apoptotic cells between control and experiment groups. In addition, gene expression of survival blastocyst, the Bax mRNA expression was similar between the control and the experiment groups. However, Bcl-xL mRNA expression's in the group of $6.0{\mu}M$ 0.5 hr on day 4 was highest among control and experiment groups (p<0.05). In this study suggested that the control of concentration, duration and time was effective on the survival and cell number of porcine blastocyst derived from in vitro. We are not know what the exact reasons of the effect of vit $K_1$ on embryo development and need to fur ther study. However, vit $K_1$ might be using the selection of high quality porcine blastocyst.

• Journal of Aquaculture
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• v.21 no.4
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• pp.234-238
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• 2008

We studied the effects of temperature and salinity on the egg development and hatching rate of chub mackerel Scomber japonicus under laboratory culturing condition. The fertilized eggs were transparent, spherical, separate in shape and turned out to be separately and floated, and they contained one oil globule. Fertilized eggs are $0.91{\sim}1.33\;mm$ in diameter. The time of egg development was positively proportional to water temperature with 70 hrs, 48 hrs, 42 hrs, 34 hrs, after fertilization in $16^{\circ}C$, $20^{\circ}C$, $24^{\circ}C$, $28^{\circ}C$, respectively. Hatching rate was highest with the range of $20{\sim}24^{\circ}C$ and $33{\sim}35\;psu$. The relation between the time of egg development (t: hour) and water temperature (T:$^{\circ}C$) was represented by the mathematical formulae. The mean biological minimum temperature was $6.9^{\circ}C$.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.61-61
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• 2003

자바리, Epinephelus bruneus는 제주에서 다금바리로 불리우고, 제주도 남부 연안에서 주로 어획되며, 식용어로서 기호도가 높은 어종이다. 자바리의 자원량 격감과 가격상승으로 양식 산업화를 위한 종묘생산에 기술개발이 요구되는 실정이다. 이 실험에 이용된 자바리 어미는 전장 81.5$\pm$3.5 cm, 체중 7,38$\pm$1.06 kg에서 인공 채란된 난과 정자를 인공 수정방법을 이용하여 얻었다. 알의 직경은 900.11$\pm$2.52 $\mu$m이었고, 유구경은 233.98$\pm$2.48 $\mu$m이었다. 수정란은 수온 $25^{\circ}C$에서 32시간 30분에 부화(50%)되었고, 부화율을 96.76$\pm$0,49%였다. 부화자어는 90일 동안 로티퍼, Artemia nauplii, 인공사료를 공급하였다. 부화 직후의 자어는 전장이 2.02$\pm$0.02 mm이였다. 부화 후 3일째(2,76$\pm$0.08 mm) 난황이 대부분 흡수되고, 입이 열렸다. 개구시 입의 크기는 219$\pm$10 $\mu$m 이었다. 부화 후 3일째 막지느러미 후부에 색소포 침적이 일어났다. 부화 후 11일째(4.12$\pm$0.09 mm) 등지느러미 제2극조와 배지느러미 극이 돌출하였다. 부화 후 17일째(6.10$\pm$0.14 mm) 자어에 있어서 꼬리지느러미, 등지느러미의 기조 부위가 발생하기 시작하였다. 부화 후 54일째(41.12$\pm$l.20 mm) 모든 지느러미의 기조는 대부분 분화되어 성어와 비슷한 체색과 체형을 갖는 치어로 발달하였다. 부화 후 78일째 치어는 전장 55.86$\pm$1.26 mm, 체중 3,64$\pm$0.25 g으로 성장하였다.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.413-421
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• 1997

In vitro culture has provided new information on the mechanisms involved in fertilization how sperm and oocyte fuse together. At the same time, results obtained in vitro have led to new questions. Techniques for In vitro maturation of porcine oocytes have progressed such that the problem of the low rate of pronucleus formation with in vitro matured oocytes after in vitro fertilization has been nearly improved. On the other hand, porcine spermatozoa have been shown to be capacitated if the fertilization medium contains caffeine and Ca$^2+$, but the incidence of polyspermy in IVM-IVF oocytes is still high. To prevent polyspermy, co-culture with oviductal cells, sperm preincubation with porcine follicular fluid or control of sperm concentration, have been examined with significant effects but still remarkably high rates of polyspermy. The under standing of these influences is a prerequisite to enhancing in vitro production of porcine em bryos.

• Journal of Aquaculture
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• v.18 no.1
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• pp.1-6
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• 2005

This study focused on spawning season, induce spawning, spawning and larval development of the Jicon scallop in Daehuksan Island of southwestern waters in Korea. The condition index and gonadosomatic index were used to investigate the reproductive pattern of the Jicon scallop. The major spawning season was from July to August, showing an unimodal gametogenic cycle per year. Several different tests were carried out to induce spawning of the mature male and female C. farreri. For females, the injection of serotonin, temperature induction technique and the combination of the both treatments produced significantly faster gamete release. Unlike females, males spawned only in response to the UV rays irradiation stimulation. Mean size of fertilized eggs was 69.5 $\mu$m in diameter. After fertilization, the zygote could be divided into 2 cells as early as 2 hours. It took about 8 hours to develop the 8-cell stage, about 20 hours to hatch trochophore larvae, and about 40 hours to be D-shaped larvae.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.35-44
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• 2003

The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

• Journal of Veterinary Clinics
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• v.17 no.1
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• pp.234-237
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• 2000

The dornor, 2 years old, 20kg and mixed breed, was bred naturally on day 1 and day 3 of estrus and eight gastrulae were collected by flushing the uterus of the donor after laparatomy on day 13 after the second mating. The embryos were frozen by programmable freezer and preserved for about 3 months in liquid nitrogen. Another bitch in natural estrus, 2 years old, 30kg, mixed breed, was selected as the recipient and the frozen embryos(8 gastrulae) were thawed and each 4 embryos were transferred into upper partr of left and right uterine horn, respectively, on day 13 after the proper mating day determined by vaginal smear. The ecipient delivered 6 offspring 48 days after embryo transfer. Of 6 puppies, one was still birth and two puppies died one month after birth. Parentage test was performed by DNA analysis using microsatellite sequences for 3 puppiers, the recipient, the donor, the male dog bred with the donor, and the male dog raised near to the recipient. The markers selected for the test were CXX 873(133-157 base pair) and CXX 894(141-165 base pair). Using primers manufactured according to the markers, the blood samples were processed for polymerase chain reaction and the PCR products were treated for electrophoresis. The three puppies showed identical band to that of recipient, consequently, it was concluded that the puppies were offspring of the recipient mated naturally by the male dog, not the offspring by embryo transfer.