• Korean Journal of Animal Reproduction
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• v.11 no.1
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• Animal Bioscience
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• v.34 no.4
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• Animal Bioscience
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• v.36 no.3
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• pp.385-403
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• 2023

Male fertility is affected by multiple endogenous stressors, including reactive oxygen species (ROS), which greatly deteriorate the fertility. However, physiological levels of ROS are required by sperm for the proper accomplishment of different cellular functions including proliferation, maturation, capacitation, acrosomal reaction, and fertilization. Excessive ROS production creates an imbalance between ROS production and neutralization resulting in oxidative stress (OS). OS causes male infertility by impairing sperm functions including reduced motility, deoxyribonucleic acid damage, morphological defects, and enhanced apoptosis. Several in-vivo and in-vitro studies have reported improvement in quality-related parameters of sperm following the use of different natural and synthetic antioxidants. In this review, we focus on the causes of OS, ROS production sources, mechanisms responsible for sperm damage, and the role of antioxidants in preserving sperm fertility.

• Development and Reproduction
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• v.3 no.1
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• pp.39-44
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• 1999

In the present study, we investigated the effect of maturity and motility of spermatozoa on the formation of pronuc-leus and subsequent developmental capacity of the human embryo in vitro. The fertilization was performed by means of intracytoplasmic sperm injection (ICSI) in HEPES-buffered m-TCM-199 medium. In the first part of the experiment, motile or im-motile human spermatozoa ejaculated were injected into cumulus-enclosed human oocytes matured in vivo. Significantly (p<0.002) higher proportion of oocytes that was injected with motile spermatozoa formed 2 pronuclei than the oocytes injected with immotile spermatozoa (79.8% vs 51.7%). In the second part of the experiment, cumulus-enclosed human oocytes matured in vivo were injected with motile or immotile spermatozoa collected from testes. There was no difference between motile and immotile spermatozoa. In the third part of the experiment, using modified Tyrode's medium containing 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin and 10% human follicular fluid, we found that the development of oocytes that formed 2 pronuclei were able to develop to 9-16 cells regardless of maturity and motility of spermatozoa.

• The Korean Journal of Zoology
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• v.15 no.1
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• pp.25-33
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• 1972

Two-Cell mouse embryos were incubated in the anterior chamber of the rat eye, which has been known as the best place among other animals' for the mouse ovum maturation, in order to observe the capability of their early development. Within 120 hours after incubation, 71.0% of two-cell embryos have developed to the blastocysts in the male rat eye, while only 38.5% in the eye of the same mouse as donated two-cell embryos. Thus, the rat eye chamber provides more favourable environment to the embryos than the mouse itself. The results are consistent with those of the previous studies comparing the maturation of the mouse follicular oocytes in the mouse and the rat eye chamber. Although the aqueous humor which is filled in the anterior chamber of the eye is characterized by its specific properties, being of higher osmolarity, higher concentrations of ascorbic acid, pyruvate and lactate, but lower of proteins and lower temperature than those in blood or lymph serum, The embryos are able to under-take their cleavage as normal as in vivo or in vitro. Concerning with a number of studies in vitro on the development of the mouse embryos which are requiring a very limited condition, the fact that they are able to manage their further development under very different enviroment from our knowledges would provide us a moment to understand their behavior during the early development. The difference of the proportion of the developed blastocysts between in the mouse eye chamber and in the rat can possibly be resulted from the species specific difference in the physicochemical properties between their eye chambers. This assumption is based upon the findings by many investigators who chmpared the nature of the eye chamber of various animals. As a consequence, the rat eye chamber might consist of better properties for the embryonal growth than the mouse eye chamber. The mouse embryos cleaved with a delayed period. In normal development they complete almost the cleavage within 94 hours after fertilization. However, in the present studies, 81.1% of two-cell embryos developed to the blastocysts and the morula in 120 hours in the eye chamber, assumed to be about 154 hours after fertilization. Such delay in development would be caused mainly by the low temperature of the eye chamber. At present we can make two assumptions to explain the capability of the emtryonal development in the eye chambers. One is that the embryos would possess an ability to adapt themselves to the environment which provides unfavourable conditions. The other is that the embryos might remain for a certain duration in the eye chamber, which is filled with a new body fluid produced immediately after the loss of the aqueous humor and the fluid of which becomes similar to blood serum in component. The first assumption is highly reliable since the embryonal cells are mostly at the undifferentiated state and so they probably engage a simple metabolism during their early period. The second assumption is induced by the fact that the rabbit eye chamber produces a plasmoid humor which has mostly similar components to blood serum after loss of aqueous humor through cornea by puncturing. However, the plasmoid humor is substituted by the initial aqueous humor in eight hours. Even though this finding, production of the new fluid, could be applied to the rat eye, it is hardly reliabel that the plasmoid humor remains for such a long period as 120 hours. Consequently, the development of the embryos is more likely due to their adaptability to the new environment during their early developmental stages.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.1-8
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• 2005

This experiment was carried out to investigate the effects of saccharide in the lactose-egg yolk(LEY) extender for freezing of boar semen on the viability, normal acrosome, fertilizable of in vitro or in vivo oocyte after thawed. Normal acrosome post-thawed spermatozoa was higher when increasing of glucose concentration in LEY extender with 3 or $4\%$ glycerol, but viability was not significant. Viability of the post-thawed spermatozoa was higher when fructose or fructose and glucose were added to LEY extender with $3\%$ glycerol than glucose and sucrose or fructose, glucose and sucrose(P<0.05). Rate of normal acrosome of post thawed spermatozoa was higher when both fructose and glucose$(81.4{\pm}2.3\%)$ were added to the LEY extender than saccharide not added$(41.6\pm0.6\%)$ to it(P<0.001). The percentage of fertilization, cleavage and development to blastocyst of oocytes fertilized with post-thawed spermatozoa from freezing by LEY extender were $70.8\~80.7\%$, $44.6\~45.7$ and $13.6\~16.0\%$, respectively. Conception rate by artificial insemination with frozen boa. semen was higher$(83.1{\pm}0.3\%)$ than commercial frozen semen from SGI company$(50.0{\pm}0.1\%,\;P<0.05)$, but litter size were no significant differences between frozen by LEY extender$(9.4{\pm}1.7\~10.4{\pm}0.7head/sow)$ and SGI semen$(8.0{\pm}1.1 head/sow)$.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.157-162
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• 2006

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs (cumulus oocytes complexes) in vitro. Attempts had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of FGF (fibroblast growth factor) on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in HPM 199 (Inst. of Functional peptide, Japan) containing 0.1, 1 and 10 ng/ml FGF for 24 hr, maturation rates to metaphase II ($70.0{\sim}75.0%$) were significantly higher (p<0.05) than that of control group (0 ng/ml FGF, 37.5%). When matured COCs with FGF were cultured in maturation medium after in vitro fertilization, developmental rates to blastocysts were 9.5, 0 and 2.9%, respectively, compared to 25.0% of the control group (p<0.05). When the matured COCs with FGF were cultured in HPM 199 (IFP971, Inst. of Functional peptide, Japan) containing 10% FBS, 0.8% BSA or 0.1% PVA (polyvinyl alcohol), the blastocyst formation rates were 12.4, 12.8 and 8.5%, respectively, while the rates of matured COCs with FGF and cultured with IVMD and IVD (Inst. of Functional peptide, Japan) without serum were 38.4% and 34.8%, respectively (p<0.05). These results suggested that FGF is available for in vitro maturation of bovine COCs and is not suitable for in vitro development, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.