• Journal of Embryo Transfer
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• v.9 no.3
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• pp.269-277
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• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes These experiments were conducted to examine the effect of sperm factor on the IVF and IVD, and the effect of coculture with somatic cells on the IVD of embryos. Although the concentration of epididymal sperm for IVF did not affect on cleavage rate, but 5 x 105 sperm/mi showed the highest cleavage rate(48.7%) and the developmental potential of IVF oocytes from this concentration was also greatly higher (P$^{\circ}C$-stored sperm for l2hrs and the cleavage rate from fresh sperm was significantly higher (P<0.05) than that from frozen sperm, but the developmental potential after IVF was slightly high from the frozen sperm. The cleavage rate of IVF oocytes cocultured with oviductal epithelial cells and cumulus cells was 76.3% and 72.9%, respectively. There was no difference between two coculture systems but this rate was significantly higher(P<0.05) than that of medium alone(42.0%).

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.125-132
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• 1991

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24~48hrs in an incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows : 1. The in vitro fertilizatin and cleavage rate offollicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin method were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7%, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solutin by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin＋BSA, heparin＋caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoa in BO solution with heparin concentrations of 2, 5, 10, 20, 40$\mu\textrm{g}$/ml were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10$\mu\textrm{g}$/ml of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentraton of 10, 20, 30, 40$\mu\textrm{g}$/ml were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3~74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30$\mu\textrm{g}$/ml were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3~66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).

• Development and Reproduction
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• v.20 no.2
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.293-299
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• 2020

Objective: The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. Methods: Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. Results: Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). Conclusion: The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

• Bulletin of the Korean Chemical Society
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• v.32 no.3
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• pp.964-972
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• 2011

This work investigated the difference between $Fe^{2+}$ autoxidation-induced and Fenton-type cleavage of pBR322 plasmid DNA. $^{\cdot}OH$ generation reactions in the absence and presence of $H_2O_2$ under various conditions were also investigated. Although both the $Fe^{2+}$ autoxidation and Fenton-type reactions showed DNA cleavage and $^{\cdot}OH$ generation, there were significant differences in their efficiencies and reaction rates. The rate and efficiency of the cleavage reaction were higher in the absence of 1.0 mM of $H_2O_2$ than in its presence in 20 mM phosphate buffer. In contrast, the $^{\cdot}OH$ generation reaction was more prominent in the presence of $H_2O_2$ and showed a pH-independent, fast initial reaction rate, but the rate was decreased in the absence of $H_2O_2$ at across the entire tested pH range. Studies using radical scavengers on DNA cleavage and $^{\cdot}OH$ generation reactions in both the absence and presence of $H_2O_2$ confirmed that both reactions spontaneously involved the active oxygen species $^{\cdot}OH$, ${O_2}^{\cdot-}$, $^1O_2$ and $H_2O_2$, indicating that a similar process may participate in both reactions. Based on the above observations, a new mechanism for the $Fe^{2+}$ autoxidation-induced DNA cleavage reaction is proposed.

• Journal of Animal Science and Technology
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• v.57 no.8
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• pp.27.1-27.6
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• 2015

This study was designed to investigate the effects of ${\alpha}$-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of ${\alpha}$-tocopherol (0, 5, 10, $15{\mu}g/ml$), oocytes were incubated at $39^{\circ}C$ with 5 % $CO_2$ for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that ${\alpha}$-tocopherol had no effect on GVBD and MII as compared to control group, but when ${\alpha}$-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.73-83
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• 1990

These studies were carried out to find the proper conditions for in vitro maturation and fertilization of bovine follicular oocytes and culture methods capable of further developing early embryos. For these objectives, the cleavage rate of oocytes matured and fertilized in vitro was investigated under medium supplemented with hormones and estrous cow serum and season of oocytes collection as well as different cumulus cell stage before insemination. Finally, 2~8 cell embryos were cultured in in vitro and in vitro culture system to investigate developmental capacity into morula. 1. Cleavage rate of oocytes matured in vitro was 27%(20/73) for A(LH＋FSH＋estradil-17$\beta$＋10% FBS), 38%(27/71) for B(LH＋10% ECS) and 27%(15/56) for C(10% ECS), respectively. Supplement B showed more higher rate and 4~8 cell embryos were also obtained much more in this group(67%, 18/27). In vitro maturation rate of follicular oocytes cultured in TCM 199 supplemented withLH and 10% ECS was 88%(75/85). 2. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes with partially removed cumulus cell before insemination was more higher than that(44%, 27/62) of oocytes with intact cumulus cell. 4. The frequency of development from early cleaved embryos into morula was 6%(4/65), 12%(4/33) for co-culture of cumulus cell monolayer and bovine oviduct epithelial cell monolayer, respectively and 25%(6/25) in ligated rabbit oviduct.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.63-69
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• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• Applied Biological Chemistry
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• v.37 no.6
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• pp.447-450
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• 1994

Rate of hydrolysis ethylene phosphate, dimethylphosphate and hydroxyethylmethylphosphate in neutral water have been measured. Hydrolysis of ethylene phosphate proceeds with P-0 bond cleavage $(k_{obs}=3{\times}10^{-7}s^{-1}\;at\;100^{\circ}C,\;{\Delta}H{\neq}=24\;kcal,\;{\Delta}S{\neq}=25.5\;eu)$. In constrast, hydrolysis of dimethylphosphate proceeds with C-O bond cleavage $(k_{obs}=3{\times}10^{-7}s^{-1}\;at\;150^{\circ}C)$. The rate constant for P-O bond cleavage of dimethylphosphate is estimated at $1{\times}10^{-11}s^{-1}\;at\;150^{\circ}C,\;({\Delta}H{\neq}=36\;kcal,\;{\Delta}S{\neq}=25.5\;eu)$. A phosphodiesterase catalyzed hydrolysis of dimethylphosphate is $10^{17}$ times faster than the simple water rate. The observed rate of hydrolysis of hydroxyethylmethylphosphate is comparable to that of dimethylphosphate indicating C-O bond cleavage $(k_{obs}=6{\times}10^{-7}s^{-1}\;at\;150^{\circ}C)$.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.142-148
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• 2020

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode's albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.