• Journal of Embryo Transfer
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• v.26 no.2
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• pp.123-128
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• 2011

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.2
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• pp.111-117
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• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.295-302
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• 2000

The objective of this study was to compare different superovulation treatments using PMSG or PG600$^{ }$ and to determine the optimal time of oocyte recovery after hCG administration. A total of 90 prepubertal Yorkshire x Landrace gilts crossed with Duroc, 6~7 months old and 100~120 kg of body weight, were used. PMSG (1,500 IU/head) or 5~7.5 ml of PG600$^{ }$(400 IU of PMSG and 200 IU of hCG) were administrated subcutaneously, and then 1,000 IU of hCG were administered intramuscularly at 72 hours after PMSG or PG600$^{ }$ injection. At carious time of 44, 46, 48 and 50 hours after hCG injection, superovulated gilts were slaughtered in a local abattoir. Ovaries together with oviducts were excised from the body immediately after slaughtered and transported to laboratory in 39$^{\circ}C$ saline. Ovaries were examined fur the number of corpus hemorrhagicum and unovulated follicles present in the surface of ovary. The unovulated follicles were categorized into small (1~3 mm in diameter) and large (4~8 mm) groups according to their diameter. Oocytes were recovered by flushing both oviducts with micropipette tip (1~100 $\mu$l) attached to a 10-ml disposable syringe. The number of CH on ovary and recovered oocytes at 46, 48 and 50 hr after hCG injection in PG600$^{ }$ treated groups were significantly higher than the other group. Group of phCG 50 hr among PMSG treated groups had a greater number of CH and recovered oocytes(P<0.05). The number of CH on ovary and recovered oocytes at 50 hr after hCG injection in 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated groups was significantly higher than 1 vial(5 ml) of PG600$^{ }$ treated group(P<0.05). In conclusions, considering a number of corpus hemorrhagicum and recovered oocytes after superovulation in gilts, effective time of oocyte recovery by treatment with PMSG and hCG was post-hCG 50 hr and with PG600$^{ }$ plus hCG was post-hCG 46, 48 and 50 hr. Also, admini-stration of 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated group had a great number of CH and recovered oocytes.covered oocytes.

• 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

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.99-105
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• 2006

The growing oocytes become progressively capable of resuming meiosis, and full meiotic competence appear when they are about 80% of the size of fully grown oocytes. As hormonal influences vary at different stages of reproductive cycle, the size of oocytes may vary according to the reproductive stages. The present study was designed to compare the diameter between the ovulated and freshly collected immature canine oocytes. The ovulated oocytes were collected 72 hr after ovulation by oviductal tube flushing by laparotomy under general anesthesia. Immature oocytes were collected by ovarian slicing method. Diameter of all oocytes was measured directly using epiflurescence microscope with a calibrated micro-eyepiece micrometer at ${\times}200$ magnification. The thickness of zona pellucida and diameter of cytoplasm were measured separately and recorded. A total of 2209 zona intact oocytes were collected, among them 628 from anestrus, 675 from follicular, 838 from luteal and 68 by fallopian tubes flushing methods. The average number of oocytes was 104.7, 168.8, 119.7 and 11.3 for anestrus, follicular, luteal and fallopian tubes flushing methods, respectively. The average diameters of the ooplasm and oocyte were significantly varied in different reproductive stages as well as with ovulated oocytes (P<0.05). The average diameter of ooplasm and oocyte was 115.6 and 127.7, 143.0 and 162.0, 134.6 and 150.6, 159.6 and 185.6 for anestrus, follicular, luteal and ovulated oocytes, respectively. Highest number of oocytes with larger diameter could be collected from the follicular and luteal stages. In conclusion, the follicular and luteal ovaries are the best sources of oocytes for canine IVM.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.488-500
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• 2013

The objective of this study was to compare the effectiveness of the protocols for superstimulation of follicular growth in Thai native heifers. Heifers (n = 20) were randomly divided into four groups of five heifers/group. Heifers were given a single dose by i.m. administration of 100 mg Follicle Stimulating Hormone dissolved in polyvinylpyrrolidone (FSHp) at 24 h. Ovum pick up (OPU) occurred at 72 h ($F_{24}O_{72}$ protocol; Group 1) or 96 h ($F_{24}O_{96}$ protocol; Group 2), and at 36 h and OPU at 72 h ($F_{36}O_{72}$ protocol; Group 3) or 96 h ($F_{36}O_{96}$ protocol; Group 4) after follicular ablation. The dynamics of ovarian follicular growth were monitored by twice-daily ultrasonographic examinations. Blood sample collections were performed every 12 h after initiation of treatment for assessment of FSH, E2 and P4 profiles. All heifers were subjected to eight repeated sequential sessions of OPU. The follicular deviation commenced $24{\pm}5.32$ h after follicular ablation in all groups. The circulatory FSH surged quickly from 24 to 36 h (>0.8 ng/ml) after follicular ablation and circulatory estrogen levels steadily increased from 36 h until OPU in all groups. At the end of the OPU sessions, the mean number of aspirated follicles/heifer/session in $F_{36}O_{72}$ protocol (Group 3) and $F_{36}O_{96}$ protocol (Group 4) were higher than in the two other groups (p<0.05). The number of cumulus-oocyte complexes (COCs), cleaved and day 8 blastocysts rates in the $F_{36}O_{72}$ protocol (Group 3) were higher than in the other groups (p<0.05). It can be concluded that a single dose i.m. administration of 100 mg FSHp at 36 h and OPU at 72 h after follicular ablation ($F_{36}O_{72}$ protocol; Group 3) was the most effective protocol for superstimulation of follicular growth for repeated OPU and subsequent in vitro embryo production in Thai native heifers.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.39-45
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• 2009

This study was carried out to evaluate the nuclear, cytoplasmic maturation and developmental potential of bovine oocytes selected by brilliant cresyl blue (BCB) as indirect measurement of oocytes growth phase. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm follicles from slaughterhouse Hanwoo ovaries. The COCs were divided into stained cytoplasm to blue (BCB+) and unstained (BCB-) according to their ooplasm BCB coloration stained by $26{\mu}m$ of BCB after 90 min. Selected COCs were cultured in a TCM 199 for 18 to 26 h. Nuclear maturation and total cell number was evaluated after in vitro maturation (IVM) or in vitro culture (IVC) using $10{\mu}g/ml$ Hoechst 33342, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay before (0 h) and after (24 h) IVM. The oocyte diameters were not differed significantly between BCB+ ($157.4{\pm}5.8{\mu}m$) and BCB+ ($149.0{\pm}31.0{\mu}m$) groups (p>0.05). However, the proportion of metaphase II oocytes in BCB+ group was significantly higher than BCB- group after IVM (p<0.05). GSH content of BCB+ group oocytes was significantly higher than that of BCB- group just after collection ($7.3{\pm}0.6$ vs. $4.8{\pm}0.6\;pmol/oocyte$, p<0.05), but not varied after IVM($13.1{\pm}0.9$ and $12.6{\pm}2.5\;pmol/oocytes$ for BCB+ and BCB- respectively; p>0.05). The proportion of blastocyst formation and total cell number in BCB+ group (23.5% and $105.5{\pm}28.6$) was significantly higher than that in BCB- (9.8% and $72.4{\pm}26.1$; p<0.05). The results indicate that BCB+ group oocytes may provide a cellular and functional basis for the greater developmental competence in Korean Native Cow (KNC) oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.173-184
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• 1990

At infertility clinic, department of Obstetrics and Gynegology, Kyung Hee Medical Hospital, 80 patients who underwent IVF-ET from January to July, 1989 were evaluated for the prediction of endogenous LH Surge and its effects on outcome of controlled ovarian hyperstimulation (COH) were compared among LH Surge group without hCG given (N=18), with hCG given (N=5), and no-LH Surge group with hCG given (N=57). LH Surge were occurred in 23(28.7%) out of 80 patients studied. Serum E2 levels on Day-1, Day 0, Day+1, were no significant different among three groups. When basal serum LH/FSH ratio is above 1.0, the possibility of endogenous LH Surge is much higher (56.3% in LH Surge group without hCG given). Serum P4 levels on Day 0 were significantly increased in LH Surge group without hCG given. Cycles which serum P4 level is higher than l.0ng/ml were 70.6% of LH Surge group without hCG given. But there was no significant interrelationship between endogenous LH Surge and serum P4 rising rate as an efficient predictor of the occurrence of endogenous LH Surge in COH for IVF. There was no significant differences in number of follicles, follicular size on Day-1, Day 0, Day+1, and number of oocyte collected per cycle. The oocyte fertilization rate of No-LH surge group with hCG given was significantly higher than LH Surge group without hCG given. There was no significant difference in oocyte cleavage rate among three groups.

• Applied Microscopy
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• v.20 no.2
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• pp.117-126
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• 1990

Structural changes of the vitelline envelopes during oogenesis of a polychaete, Nectoneanthes oxypoda, were examined with a scanning electron micrscope. Oocytes grow in the same coelomic fluid to the final stage, but the surface appears to change in the structure during oogenesis. Projections, which were identified to be microvilli, change in shape, number and size. Short microvilli, which cover the surface of oocyte of $33{\mu}m$ diameter densely, grow in length, reaching a maximum at the stage of $73{\mu}m$. The number of microvilli increases with the stages of oogenesis, reaching a plateau at the stage of $82{\mu}m$. The observations suggest that control of material transport including yolk precursor proteins may be correlated with the structural changes in the microvilli.