• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• 한국수정란이식학회지
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• 제15권2호
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• pp.167-173
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• 2000

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

• 한국양식학회지
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• 제4권1호
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• pp.13-18
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• 1991

참돔 종묘생산을 위한 생물학적 기초자료를 얻고자, 난발생에 미치는 환경요인으로서 수온의 영향에 대하여 실험한 결과는 다음과 같다. 1. 난발생의 각 단계에 이르기까지의 수온($T\;:\;^{\circ}C$)에 따른 발생속도(t : hour)는 수온이 높을수록 빨랐으며, 그 관계식은 다음과 같다. 8 세포기 : 1/t=0.0618T-0.5877(r=0.9899) 상실배기 : 1/t=0.0284T-0.2556(r=0.9948) Kupffer씨포 출현기 : 1/t=0.0076T-0.0829(r=0.9902) 부화자어기 : 1/t=0.0031T-0.0350(r=0.9985) 2. 참돔의 초기발생에 있어서 난발생이 진전되지 않는 생물학적 영도는 평균 $10.2^{\circ}C$로 나타났다. 3. 설정수온별로 수정에서 부화에 이르기까지의 소요시간은 $15^{\circ}C$에서는 87시간, $18^{\circ}C$에서는 48시간, $21^{\circ}C$에서는 32시간, $24^{\circ}C$에서는 27시간이었다.

• 한국양식학회지
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• 제10권3호
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• pp.357-362
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• 1997

참가자미의 종묘생산을 위한 생물학적 기초자료를 얻기 위하여 난발생에 미치는 수온과 염분의 영향을 조사하였다. 수정란의 정상적인 부화는 9-$15^\circC$에서 가능하였으며 생존율은 $12^\circC$에서 가장 높게 나타났다. 난발생의 각 단계에 이르기까지의 수온 (T : \circC$)에 따른 발생속도 (t : hour)는 수온이 높을수록 빨랐으며, 그 관계식은 다음과 같았다. 8세포기 1/t=0.0284T-0.0554 (r=0.9999) 상실기 : 1/t=0.0137T-0.0527 (r=0.9998) Kupffer씨포 출현기 : 1/t=0.0035T-0.0133 (r=0.9762) 부화자어기 : 1/t=0.0012T-0.0007 (r=0.9981) 참가자미의 난발생이 개시되는 생물학적 영도는 평균 $2.6^\circC$로 나타났다. 설정 수온별로 수정에서 부화에 이르기까지의 평균 소요시간은 $9^\circC$에서 95.5시간, 12$12^\circC$에서 72.5시간 및 $15^\circC$에서 56.0시간이었다. 염분별 부화까지의 생존율은 35-$38\textperthousand$에서 높게 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권4호
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• pp.455-461
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• 2001

The effect of exogenous gonadotrophins on superovulation in rabbits was examined. One hundred and sixteen sexually mature California, Chinchilla and New Zealand White rabbits were randomly allocated to control (100 IU hCG), PMSG-treated (100 IU HCG following 150 IU PMSG) and FSH-treated groups (0.3 mg/head /12 h for 3 days followed by 100 IU hCG). All does were mated after hCG injection and were sacrificed or laparotomized within 1 to 4 days postcoitus for counting the number of ovulation points. The number of ovulations was higher in FSH-treated animals than in the control and PMSG-treated groups (37.2 vs. 10.4 and 14.5, p<0.05). Follicle haemorrhagicum was observed in many cases in the PMSG-treated group. No significant difference in ovulation number was observed between left and right ovaries regardless of gonadotropin treatment. In another experiment, 2-cell stage embryos were collected at 26 h postmating and blastomeres were separated by mechanical pipetting or gentle pressure with a fine glass needle. Aggregated or chimeric embryos were produced from two single blastomeres from two breeds, New Zealand White and Chinchlla, with different coat colors. All the embryos were cultured in Ham's F-10 medium supplemented with 1.5% BSA (bovine serum albumin fraction V) and 10% PRS (pregnant rabbit serum), and incubated in a humidified atmosphere with 5% $CO_2$ at $38^{\circ}C$. After development to morula or early blastocyst, the embryos were transferred into the oviducts of recipient does. Results showed that 7 out of 10 does (70%) receiving intact embryos (control) became pregnant and 41 kits were delivered. However, no pregnancy was obtained from the recipient of either denuded demi- or aggregated embryos. It is suggested that embryos without zona pellucida could not develop to term in rabbits.

• Asian-Australasian Journal of Animal Sciences
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• 제26권4호
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• pp.501-508
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• 2013

The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode's albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2-4 cell, 8-16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.

• Asian-Australasian Journal of Animal Sciences
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• 제14권10호
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• pp.1360-1366
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• 2001

The aims of this study were first to evaluate the effects of different levels (20, 40 and 100%) and sources (follicular size: large, >7 mm; medium, >5-7 mm; small, 3-5 mm) of porcine follicular fluid (pFF) on the in vitro maturation (IVM) of porcine oocytes, and the effects of fertilization treatments and different culture conditions on development of fertilized oocytes were also investigated. No differences in the maturation (63.6-76.6%) and cleavage (24.8-34.3%) rates were observed among the 20,40 and 100% pFF groups (p>0.05). The cleavage rates of oocytes cultured and fertilized in 40% and 100% pFF maturation media were significantly higher than those fertilized in m199-NBCS (51.0-61.2% vs. 12.8-31.8%. p<0.05), regardless of sources of the pFF. When oocytes were fertilized in m199-NBCS followed by culture in rabbit oviducts for 4 days, the cleavage rate in 40% pFF group was better than that in 100% pFF group (46.9% vs. 32.5%, p<0.05). Two oocytes recovered from the oviducts in the 40% pFF group developed to blastocysts after IVC. However, none developed to blastocysts when fertilized in the IVM medium after being transferred to rabbit oviducts. In conclusion, addition of pFF accompanied with gonadotropins (FSH, LH) in IVM medium enhanced maturation and cleavage rates of porcine oocytes. Direct addition of sperm suspension to IVM medium may be an alternative to simplify the fertilization procedures and to reduce the mechanical lesion during manipulation. Furthermore, rabbit oviducts provide a better environment for the in vitro fertilized oocyte developing to the morula and blastocyst stages.

• 한국수산과학회지
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• 제36권2호
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• pp.136-143
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• 2003

The eggs of Favonigobius gymnauchen attached on the under side of a small stone were collected off Seongsan-eup Cheju-do in August, 2000 to investigate their development of egg, larvae and juveniles. The fertilized eggs were elliptical in shape (mean long axis: 1.50 mm; mean short axis: 0.57 mm) and transparent. There were filaments on one side of the egg membrane. Larvae hatched at 48 hrs 50 mins after morula stage with 25-26 myotomes in $22.8-28.5^{\circ}C\;(mean\;24.7^{\circ}C).$ The newly hatched larvae were 2.31-2.49 mm (mean 2.37 mm n=10) in total length (TL) and their mouth and anus were already opened. Their melanophores were appeared on the over gas globule, around anus and the part of caudal peduncle with 24-25 myotomes. At 4-5 days after hatching. larvae attained 3.81-4.07 mm (mean 3.96 mm, n=10) in TL and their yolk sac was completely absorbed. They began to eat rotifer and transformed to postlarvae stage. At 14 days after hatching, postlarvae attained 6.17-6.31 mm (mean 6.21 mm, n=10) in TL and their caudal notocord was flexed $45^{\circ}$ upward. At 24 days after hatching, postlarvae attained 8.69-9.10 mm (mean 8.87 mm, n=10) in TL had reached the juvenile stage. All fins were formed with the complete set of fin rays with the following counts: dorsal fin rays IV-I, 9-10; anal fin rays I, 9; pectoral fin rays 17; ventral fin rays: I, 5; caudal fin rays: 9+8= 17.

• 한국발생생물학회지:발생과생식
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• 제15권1호
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• pp.17-24
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• 2011

Lipid metabolites involved in cellular regulation as signaling mediators. Prostaglandins (PGs), metabolites of lipid are involved to pregnancy at the time of implantation but the functional roles of PGs on embryo development are still controversy and largely unknown. In previous report, the levels of $PGE_2$ and $PGF_{2a}$ at embryos of morula stage and blastocyst stage were explored (Cheon et al., 1998). In this study, the previous suggestion was confirmed and the possible downstream mediator of prostaglandin $E_2$ and prostaglandin $F_{2a}$ on the expansion and hatching of mouse embryo was examined. As expected, developmental rate of the blastocyst to expanded stage was a concentration-response curve that showed the highest expansion rate at 10 ${\mu}M$ $PGE_2$, but at 100 ${\mu}M$ $PGE_2$, the rate was decreased. In contrast to the $PGE_2$, $PGF_{2a}$ stimulated expansion without toxicity at highest concentration. Cotreatment of PGs with indomethacin overcame the inhibitory effects of indomethacin in expansion. Exogenous PGs also improved the development of expanded embryos to the hatching stage. Besides, PGs receptors' transcripts detected at blastocyst. $PGE_2$ was caused of calcium fluctuation in the blastocyst but $PGF_{2a}$ did not. The changes of intracellular calcium concentration were different between indomethacin pretreated embryos and non-treated embryos. Based on these results it is suggested that PGs work as paracrine and/or autocrine factors through calcium and the others which were not identified in this study.

• 한국수정란이식학회지
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• 제4권1호
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• pp.1-13
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• 1989

This paper reviews the most important steps that have generated consistent progress in principles and developmental progress of embryo cryopreservation, and also study on freezing procedure and its application by conventional method and current improved method for freezing procedure and its appilcation of embryo cryopreservation in farm animals. Four were of particular interest: 1.The transport of water across the ccli membrane (zona pellucida) during freezing and thawing accordinglyplays a role in determing whether the celi survives. This movement of water is controlied mainly by extracellular phase changes and by the nature and concentration of any cryoprotective agent present. Therates of cooling, freezing and warming, and the intervals over which they are applied are further decisi've factors in determining whether a cryopreservation procedure allows survival after thawing. 2.The first successful deep freezing experiments with sheep morula and blastocysts during the seventies were based on the early procedures used for mouse embryos.Current research during the eighties is developed with the aim of simplifying and improving current procedures such as one-step dilution and rapid or ultra-rapid cooling by using the model of laboratory animals. 3.The conventional method for the embryo cryopreservation is described. An alternative to this method which may result in high survival and also in reducing of the freezing and thawing time is done by combing a permeable cryoprotectant such as glycerol, DMSO or propanediol and a non-permeable compound such as sucrose, trehalose, raffinose or lactose. 4.Finally a different approach to the preservation of embryos, named vitrification, is introduced. This procedure depends upon the ability of concentrated solutions of cryoprotective agents such as glycerol and propanediol to supercool to very low temperature (-196$^{\circ}C$) during rapid cooling before solidifying without formation of ice. However, more complete data are necessary for successful vitrification of blastocysts.