• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.882-897
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• 1998

This experiment was conducted to predict the effects of motional characteristics on the fertility of Korean native cattle(KNC) by using CASA technology and in vitro fertilization system. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system(SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber(Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The oocytes collection, in vitro maturation, IVF, in vitro culture and determination of the cleavage rate were performed by the technique, as described by Hwang et al (1997). Statistical analysis was done by linear regression with use of the Sigma plot program on a IBM personal computer. The cleavage rate in vitro fertilized oocyte was significantly correlated(P<0.05) with MOT, VCL, VSL, VAP, ALH, BCF and MAD, but not CON, LIN, STR, WOB, DNM, DNC and HYP in regressional analysis. The results show that some kinematic characteristics of frozen-thawed semen by CASA can be predict the fertility in in vitro model system.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.61-65
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• 2002

This study was carried out to study the viability of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified at 10, 14 and 20 hrs after the onset of maturation, respectively). The oocytes remained vitrified for 24 hrs, and then were thawed in 3$0^{\circ}C$. Survival and cleavage rates were investigated by results of in vitro culture and aceto-orcein staining or FDA test. No difference in the incidence of diploid oocytes was observed among the control, non-vitrified group(3.6%) and oocytes vitrified at 14 hrs(6.7%) or 20 hrs(1.7%). However, more diploid oocytes were detected after vitrification at 0 hr.(26.7%) and 10 hrs(21.7%) post maturation. The survival rate of all vitrified immature oocytes(12.0~38.0%) was low, 48.0% of unvitrified oocytes and oocytes vitrified before maturation or 0~ 10 hrs after the onset of maturation were higher than that of other groups. The overall fertilization and cleavage rates of vitrified immature oocytes (32.3 ~ 64.6% and 4.6 ~ 32.3%) were low, and 55.0% of unvitrified oocytes and the rate of immature oocytes were very higher than that of mature oocytes.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.27-36
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• 1997

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.119-126
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• 2003

Objective: This study was carried out to compare the clinical outcomes of intracytoplasmic sperm injection (ICSI) in patients with obstructive azoospermia according to sperm retrieval site and technique; microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular sperm extraction by open biopsy (TESE). Methods: The outcomes of ICSI and IVF-ET were evaluated and compared among 3 groups. Seventy three men suffering from infertility due to obstructive azoospermia had 107 ICSI cycles using MESA (21 cycles in 15 patients), PESA (26 cycles in 17 patients) and TESE (60 cycles in 41 patients). Results: In the clinical outcomes in patients undergoing ICSI with epididymal or testicular sperm, there were no significant differences in fertilization rate (66.1% vs. 60.5%), cleavage rate (94.9% vs. 97.6%), cumulative embryo score (CES) (51.3 vs. 58.8), implantation rate (7.9% vs. 6.1), and clinical pregnancy rate per ET (30.4% (14/46) vs. 25.4% (15/59)) between both groups. Also, in the clinical outcomes in ICSI patients using MESA, PESA, TESE, there were no significant differences in fertilization rate (61.8%, 69.4%, 60.5%), cleavage rate (92.1%, 97.3%, 97.6%), CES (38.1, 52.0, 58.8), implantation rate (9.5%, 6.6%, 6.1%), and clinical pregnancy rate per ET (35% (7/20), 26.9% (7/26), 25.4% (15/59)) among 3 groups. Conclusion: When compared with MESA or TESE, PESA, the clinical outcomes were similar in ICSI patients with obstructive azoospermia whatever the origin or the technique of sperm retrieval. However, we considered PESA is more time-saving and cost effective for ICSI in patients with obstructive azoospermia.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.3
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• pp.175-184
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• 2022

Objective: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. Methods: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. Results: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. Conclusion: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.311-318
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• 1997

Bypassing acrosome reaction and fusion process in intracytoplasmic sperm injection(ICSI), most of injected spermatozoa still contain intact acrosome contents and plasma membrane. It Is not known yet what acrosome contents and plasma membrane of spermatozoa have effect on the development of embryo. For further understanding of fertilization process after ICSI, we studied the time of pronucleus formation, disappearance and first cleavage in human zygote, and pregnancy rate in relation to acrosome reaction rate of spermatozoa after ICSI. Seventy cycles undergoing ICSI program were randomly selected. Sperm suspension from 38 cycles were treated 50% human follicular fluid(hFF) for 3 hours in order to induce acrosome reaction, others were not treated as control. Acrosome reaction in hFF treated and non-treated group was assessed by fluorescein isothiocyanate(FITC)-conjugated Arachis hypogea(PNA) and Pisum sativum agglutinin(PSA). Oocytes were classified into 'good' and 'poor' according to their morphology. After ICSI, fertilization of oocytes were assessed by detection of two pronuclei at 16 hours. The pronuclei disappearance and first cleavage of zygotes were observed at 24 hours, and then embryos were transferred to uterus after culture for 72 hours. The rate of acrosome reaction of spermatozoa in hFF treated group was significantly higher than that in control(p<0.01). Fertilization rates of good oocytes were not different both control and hFF treated group(81.3%(174/206) vs. 72.1%(102/130)). But, in poor oocytes, the fertilization rates in hFF treated group(72.1%(149/183)) were increased compared than those of control group (63.6%(98/140), p<0.01). In either good or poor oocytes, the rates of pronuclei disappearance in hFF treated-spermatozoa injected oocytes were higher than control (59.1%(103/174), 56.4%(84/149) vs. 32.4%(33/102), 37.8%(37/98), p<0.01). Also, the rates of thirst cleavage were increased in hFF treated group (31%(54/174), 24.1%(36/149)) compared than those of control group (10.8%(11/102), 13.2%(13/98), p<0.01). The pregnancy rates of hFF treated group (42.1%(16/38)) were slightly higher than control group (28.1%(9/32), p>0.05). But, the pregnancy rate of group which possessed more than one cleavaged zygote at 24 hours was higher than group which did not (45.2%(19/42) vs. 21.4%(6/28), p<0.05). From these results, the development of zygotes were faster in higher acrosome reacted sperm group than lower acrosome reacted sperm group after ICSI. Our results may be explained that acrosomal membrane and plasma membrane are easily detached from spermatozoa in acrosome reacted spermatozoa compared with acrosome intact sperm in the cytoplasm of oocyte during pronuclear formation. We conclude that the injection of acrosome reacted spermatozoa will increase the pregnancy rate as they can induce fast embryonic development in ICSI.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.