• Development and Reproduction
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• v.12 no.1
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• pp.19-30
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• 2008

Proper development of fertilized oocyte to blastocyst is a key step in mammalian development to implantation. During development of preimplantation embryos, the mammalian embryo needs supply the energy substrate for keep viability. Usually mammalian oocyte get substrate especially energy substrate from oviduct and uterus, because it does not store much substrate into cytoplasm during oogenesis. Carbohydrates are known as a main energy substrate for preimplantation stage embryos. Glucose, lactate and pyruvate are essential component in preimplantation embryo culture media and there are stage specific preferences to them. Glucose transporter and $H^+$-monocarboxylate cotransporter are a main mediator for carbohydrate transport and those expression levels are primarily under the control of intrinsic or extrinsic factors like insulin and glucose. Other organic substances, amino acids, lipids and nucleotides are used as energy substance and cellular regulation factor. Though since 1960s, successful development of fertilized embryo to blastocyst has been accomplished with chemically defined medium for example BWW and give rise to normal offspring in mammals, the role of metabolites and the regulation of intermediary metabolism are still poorly understood. Glucose may permit expression of metabolic enzymes and transporters in compacting morula, capable of generating the energy required for blastocyst formation. In addition, it has been suggested that the cytokines can modulate the metabolic rate of carbohydrate in embryos and regulate the preimplantation embryonic development through control the metabolic rate. Recently we showed that lactate can be used as a mediator for preimplantation embryonic development. Those observations indicate that metabolites of carbohydrate are required by the early embryo, not only as an energy source, but also as a key substrate for other regulatory and biosynthetic pathways. In addition metabolites of carbohydrate may involve in cellular activity during development of preimplantation embryos. It is suggested that through these regulation and with other regulation mechanisms, embryo and uterus can prepare the embryo implantation and further development, properly.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.161-166
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• 2004

This study was designed to evaluate the effects of nitric oxide scavenger (hemoglobin) and inhibitor (L-nitro-L-arginine methyl ester; L-NAME) with or without cumulus cell on the development of bovine IVM/IVF embryos. When CR/sub 1aa/ medium were supplemented with different dosage (lug/m, 5ug/m and 10ug/ml) of hemoglobin at 48hrs for in vitro culture, the proportion of embryos developing beyond morulae stage in 0, 1ug/ml and 5ug/ml with or without cumulus cell were 23.8%, 33.3 % and 26.8%, and 39.5%, 54.8% and 48.8%, respectively. There was a significantly difference the developmental rate of 1ug/ml hemoglobin intact cumulus cells to any other groups (P<0.05). On the other hand, when added to hemoglobin at 96 hrs, 1 ug/ml hemoglobin with cumulus cell group was significantly increased the percentage of developing into morulae and blastocysts to any other groups (P<0.05), and similar trend that of added at 48hrs. The overall means of the percentage of developing into morulae and blstocysts in 1ug/ml hemoglobin group was significantly increased than those of any other groups (P<0.05) and cumulus co-culture with hemoglobin was increased the in vitro developing rate of IVM/IVF embryos. In CR/sub 1aa/ medium treated with L-NAME 0, 10, 50 and 100mM, the developmental rate of morula plus blastocysts were 55.6%, 64.9%, 58.8% and 66.7%, respectively. The L-NAME did not affect the developmental rate and the cell numbers of blastocysts in all treated groups. These results indicate that hemoglobin and cumulus co-culture can increase the proportion of embryos that developed into morulae and blastocysts, but cell numbers of blastocysts were not affect in all groups.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Journal of Aquaculture
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• v.8 no.3
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• pp.195-207
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• 1995

In order to study the embryonic development and hatching of wild long shanny, Stichaeus grigorjewi, were caught with the gill nets in the East Sea of Korea, and stocked at indoor tanks to induce natural spawning in February 25, 1994 and February 16 to 24, 1995. They were already matured when stocked, and average body length (50.66 cm) and body weight (1,192.74 g) of 57 females and average body length (48.62 cm) and body weight (612.58g) of 43 males were recorded. Before stocking, they were inserted with identification tags(ID tags) in the dorsal muscle, and spawning was traced by the portable reader (Destron/lDl Ltd.) Forty females among 57 spawned successfully in the average of 4 days after stocking. Females spawned almost all eggs contained in the ovaries at one time in the form of an egg mass and averaging 227,200 eggs Per egg mass. The egg mass was oval in shape, translucent milky in color, 20.32cm long axis and 14.57cm short axis in size, and 803.7g in weight. Male parents guarded their egg masses and circulated water with the tail part of the body. Fertilized egg was spherical in shape, and their average diameter was 1.54 mm. Each egg had a containing single oil globule, and it's average diameter was 0.37 mm. The average water temperature was $13.2^{\circ}C$ and incubation times after fertilization were 5 hours 25 minutes up to 2-cell stage, 13 hours up to morula stage, and 66 hours 35 minutes up to embryo formation stage. Hatching rate was approximately 10 percent in 368 hours 50 minutes after fertilization, and approxionateoly 90 percent of eggs were hatched in 425 hours 30 minutes after fertilization.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.3
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• pp.334-342
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• 2015

The present study was designed to evaluate how environmental factors in a dry-summer subtropical climate in Terceira-Azores (situated in the North Atlantic Ocean: $38^{\circ}43^{\prime}N27^{\circ}12^{\prime}W$) can affect dairy cow (Holstein) fertility, as well as seasonal influence on in vitro oocytes maturation and embryos development. Impact of heat shock (HS) effects on in vitro oocyte's maturation and further embryo development after in vitro fertilization (IVF) was also evaluated. For such purpose the result of the first artificial insemination (AI) performed 60 to 90 days after calving of 6,300 cows were recorded for one year. In parallel, climatic data was obtained at different elevation points (n = 5) from 0 to 1,000 m and grazing points from 0 to 500 m, in Terceira island, and the temperature humidity index (THI) was calculated. For in vitro experiments, oocytes (n = 706) were collected weekly during all year, for meiotic maturation and IVF. Further, to evaluate HS effect, 891 oocytes were collected in the cold moths (December, January, February and March) and divided in three groups treated to HS for 24 h during in vitro maturation at: C (Control = $38.5^{\circ}C$), HS1 ($39.5^{\circ}C$) and HS2 ($40.5^{\circ}C$). Oocytes from each group were used for meiotic assessment and IVF. Cleavage, morula and blastocyst development were evaluated respectively on day 2, 6, and 9 after IVF. A negative correlation between cow's conception rate (CR) and THI in grazing points (-91.3%; p<0.001) was observed. Mean THI in warmer months (June, July, August and September) was $71.7{\pm}0.7$ and the CR ($40.2{\pm}1.5%$) while in cold months THI was $62.8{\pm}0.2$ and CR was $63.8{\pm}0.4%$. A similar impact was obtained with in vitro results in which nuclear maturation rate (NMR) ranged from 78.4% (${\pm}8.0$) to 44.3% (${\pm}8.1$), while embryos development ranged from 53.8% (${\pm}5.8$) to 36.3% (${\pm}3.3$) in cold and warmer months respectively. In vitro HS results showed a significant decline (p<0.05) on NMR of oocytes for every $1^{\circ}C$ rising temperature ($78.4{\pm}8.0$, $21.7{\pm}3.1$ and $8.9{\pm}2.2$, respectively for C, HS1, and HS2). Similar results were observed in cleavage rate and embryo development, showing a clear correlation (96.9 p<0.05) between NMR and embryo development with respect to temperatures. Results clearly demonstrated that, up to a THI of 70.6, a decrease in the CR occurs in first AI after calving; this impairment was confirmed with in vitro results.

• Development and Reproduction
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• v.10 no.2
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• pp.105-113
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• 2006

Reactive oxygen species(ROS) generated in cellular metabolism have an effect on cell maturation and development. In human reproductive tract, oxidative injury by ROS may induce female infertility. Also, oxidative injury may be responsible for developmental retardation and arrest of mammalian preimplantation embryos. Activating transcription factor 4(ATF4) is a member of the cyclic-AMP response element-binding(CREB) familiy of basic region- leucine zipper(bZip). ATF4 is known to regulate stress response to protect cell from various stress factors and inducer of apoptisis. The purpose of this study was to investigate whether ATF4 is involved in the defensive mechanism in oxidative stress condition during the development of mouse preimplantation embryos. To verify the expression of ATF4 in oxidative stress condition, 2-cell stage embryos were cultured in HTF media containing 0.1mM, 0.5mM or 1mM hydrogen peroxide($H_2O_2$) for 1hr(2-cell), 8hr(4-cell), 17hr(8-cell), 24hr(morula), 48hr(early blastocyst) or 64hr(late blastocyst). The developmental rate decreased in the 0.1mM $H_2O_2$ treated group compared with control group. In embryos treated with 0.5mM and 1mM $H_2O_2$ showed 2-cell block. As a results of the semi-quantitative RT-PCR analysis of SOD1, ATF4 and Bax gene expression, SOD1, ATF4 and Bax genes were increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. In 2-cell embryos, expression of SOD1, ATF4 and Bax genes were notably increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. Immunofluorescence analysis showed that ATF4 protein was localized at the cytoplasm of preimplantation embryos. The increase in ATF4 immunoreactivety was observed in the 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. It suggests that oxidative stress by $H_2O_2$ induces expression of ATF4 and may be involved in protection mechanism in preimplantation embryos from oxidative injury.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.79-85
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• 2002

We investigated the optimal concentration and exposure time of cycloheximide(CHX) on development of activated porcine oocytes following electrical pulse(EP). After 42~44 h maturation, oocytes were treated with 0.1% hyaluronidase, and denuded cumulus cells by pipetting. Oocytes were stimulated by electric pulse (1.2 kV/cm, 30 $\mu$sec, 1 pulse) or incubated for 3, 5 and 7 h in cycloheximide (1, 5 and 10 $\mu\textrm{g}$/$m\ell$, respectively) following electric pulse, and cultured for 8 days. Cleavage rate of oocytes activated with 10 $\mu\textrm{g}$/$m\ell$ CHX following EP was significantly (P＜0.05) higher than those of 1 $\mu\textrm{g}$/$m\ell$ (86.8% vs. 74.4%). The developmental competence of oocytes incubated to 5 $\mu\textrm{g}$/$m\ell$ of CHX was significantly (P＜0.05) higher development to blastocysts (13.3%), compared with 10 $\mu\textrm{g}$/$m\ell$ of CHX (5.6%). When the oocytes were activated with 5$\mu\textrm{g}$/$m\ell$ CHX for 3, 5, and 7 h following EP, the cleavage rate of oocytes in 5 h group(86.6%) was significantly (P＜0.05) higher than that in 3 h group(73.2%). The developmental rate of oocytes to morula in 5 and 7 h groups(26.7% and 16.4%) were significantly (P＜0.05) high than that in 3 h group(14.5%). Matured oocytes were activated with electric pulse (EP) or electric pulse combined with cycloheximide (EP + CHX) and cultured for 8 days. The rate of cleavage and development to blastocyst (80.1% and 11.6%) of activated with EP group were similar to EP combined with CHX group. When activated with EP or EP combined with CHX, the mean cell number of blastocysts were less in the activated with EP (18.67$\pm$5.53) than in the activated EP combined CHX (20.71$\pm$6.16), but not significantly different. This results suggest that, when the porcine oocytes were activated with CHX following EP, the developmental rate of activated oocytes can be improved by treated with a concentration of 5 $\mu\textrm{g}$/$m\ell$ CHX for 5 h exposure time.

• 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.21 no.2
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• pp.101-107
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• 2006

The objective of this study was to investigative the effects of retinol supplement to IVM and/or IVC medium on maturation, fertilization and development of pig oocytes. North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF) was used as base medium. Each 1 uM, 5 uM and 10 uM concentration of retinol was supplemented to IVM and /or IVC medium. When the retinol was supplemented to maturation medium, the maturation rates were not different (p>0.05) among treatment groups ($66.7{\pm}6.0{\sim}69.2{\pm}5.3%$), but the developmental rate to blastocyst stage was higher (p<0.05) in $5{\mu}M$ group ($20.4{\pm}2.6%$) than in 0 uM ($13.6{\pm}2.1%$) and 10 uM groups ($9.7{\pm}1.7%$). Moreover, total cell number was significantly greater (p<0.05) in the 5 uM group ($37.0{\pm}1.6$) than in the other groups ($29.8{\pm}1.0{\sim}33.2{\pm}1.0$). Retinol supplement to maturation medium did not significantly affect the rates of fertilization and polyspermy (p>0.05). When the retinol was supplemented to culture medium or both maturation and culture medium, the rates of cleavage, and develop to morula and blastocyst stage were not affected, while those of 10 uM group were significantly decreased (p<0.05). These results indicate that 5 uM retinol supplement in maturation medium significantly stimulates embryo development, also improves the total cell number of blastocyst stage in pig.