• Fisheries and Aquatic Sciences
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• v.19 no.1
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• pp.5.1-5.7
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• 2016

The study was conducted to know the cyclic changes in gonadal maturation and to investigate the developmental stages of oocytes and testicular germ cells of an air-breathing fish, Channa striata. Fish were sampled monthly from lentic and lotic environments of three geographical locations of Bangladesh from December to November and the histological analysis of their gonad was done to evaluate the objectives. The highest mean GSI was $5.95{\pm}0.20$ for female in July and $0.14{\pm}0.01$ for male also in July showing that the gonadal development reached its peak during this month. The highest mean oocyte diameter was $1257.50{\pm}24.17{\mu}m$ observed in July implying that the oocyte reached maturity in this month. Histological study of ovary revealed the evidence of early yolk granule stage and late yolk granule stage from April to July. In case of male four stages of spermatogenesis were distinguished and spermatozoa were highly abundant in June and July. So the monthly pooled values of GSI and the analysis of gonadal histology indicated that the peak breeding season of C. striata occurred in July in the lentic and lotic environments. Samples collected from lentic and lotic habitats are suggestive of no difference in the development of the gonad. The results of the present study will be useful for selective breeding programme, conservation and sustainable fishery management of C. striata in its natural habitat.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.4
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• pp.148-154
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• 2013

Objective: To compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested. Methods: Mature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed. Results: The modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts. Conclusion: Closed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

• Molecules and Cells
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• v.40 no.8
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• pp.558-566
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• 2017

Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

• Development and Reproduction
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• v.2 no.2
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• pp.213-221
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• 1998

Intracytoplasmic sperm injection (ICSI) has been widely used to treat couples with infertility due to severely impaired sperm charateristics and for whom conventional in-vitro fertilization (IVF) had failed. The extent to which the morphology of the oocyte at the light microscopy level is related to the results of ICSI vis controversial. In this study, oocytes from 44 patients were reviewed. The ICSI procedure was recorded through CCD camera. The oocytes were divided into five groups according to the presence of cytoplasmic inclusions, the width of perivitelline space (PVS), the presence of cell debris in PVS, the status of first polar body and the flexibility of oolemma. The results showed that the fertilization rate and embryonic development were not associated with the morphological criteria of oocyte. The degeneraton rate of oocytes after ICSI was significantly higher (P<0.001) in the oocytes whose membranes were broken at the moment of insertion (17.7%) than the oocytes whose membranes were broken by aspiration of cytoplasm (1.6%). More oocytes with cytoplasmic inclusions (48.4% vs. 25.1%, p<0.001), wide PVS (35.2% vs. 19.0%, p<0.001), or cell debris in PVS (53.3% vs. 38.4%, p<0.05) were observed in patients with female factor infertility compared to patients with male factor infertility. These results .suggest that the fertilization rate and embryonic development after ICSI are not correlated with oocyte morphology based on the presence of cytoplasmic inclusions, size of PVS, the presence of cell debris in PVS and the status of polar body. And the degeneration rate of oocytes after ICSI was associated with the flexibility of oolemma.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

• Clinical and Experimental Reproductive Medicine
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• v.15 no.1
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• pp.17-24
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• 1988

Follicular fluid(FF) prostaglandin $E_2$(PG$E_2$) and $PGF_{2{\alpha}}$ levels were compared in 3 groups of spontaneously ovulatory women undergoing ovulation induction with clomiphene citrate (CC) alone or with human menopausal gonadotropin(hMG) (14 patients), hMG(9 patients), or pure FSH/hMG(11 patients) for the purpose of in vitro fertilization. FF volume aspirated did not differ significantly according to the maturity of the oocyte. According to hyperstimulation regimens, the volume of FF from which preovulatory occytes were obtained was significantly less in the hMG-treated group than in the other groups. In follicles of preovulatory oocytes, FF PG$E_2$ values were significantly lower in the FSH treated group than in the Cc.treated or hMG-treated group, and FF $PGF_{2{\alpha}}$ values were significantly higher in the hMG-treated group than in the CC-treated or FSH-treated group. In follicles of immature or atretic oocytes, there was no significant difference in FF PG$E_2$ and PG$F_{2{\alpha}}$ concentrations of the similar morphology of the oocyte according to hyperstimulation regimens. In all cycles, FF PG$E_2$ and PG$F_{2{\alpha}}$ values of preovulatory oocytes were not significantly different from those of immature oocytes, but those of atretic oocytes were relatively lower than those of fertilizable oocytes and it was statistically signifincant in PG$E_2$ values of CC-treated group. In all treatment groups, FF PG$E_2$ and PG$F_{2{\alpha}}$ levels did not show and close relationship with the success of fertilization in vitro and of pregnancy after embryo transfer. Above results suggested that FF PG$E_2$ and PG$F_{2{\alpha}}$ be involved in oocyte maturation and ovulation, but their relationship with the success of in vitro fertilization was not found.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.195-201
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• 2002

This study was to assess the developmental capacity of oocytes matured in vitro after 20, 15, 10, 5 and 0 days of organ culture when ovaries were isolated from juvenile mice at 0-, 5-, 10-,15- and 20-day old, respectively, and to develop in vitro culture system that observed a view to morphology of ovaries and nucleus maturation of oocytes. The size of ovaries decreased 35.9%, 8.7%, 1.2% and 14.4% after 20, 15, 10, 5 days of organ culture when the ovaries were isolated from 0-, 5-, 10 and 15-day old mice, respectively. After organ culture, the recovery rates, diameters of oocytes and the number of oocytes progressed from GV to MII were increased as increasing age of mice.