• Journal of Embryo Transfer
• /
• v.20 no.1
• /
• pp.55-62
• /
• 2005

This study was undertaken to access the effect of collection methods on the collection efficiency, blastocyst rate and pregnancy rate after IVP embryo transfer. The ovaries of Hanwoo were obtained from an abattoir and kept on 25 to $28^{\circ}C$ and transported to laboratory within 4 hrs. The oocytes were collected by aspiration of follicles $(2\~6\;mm)$ with or without slicing of ovaries after aspiration. The oocytes were matured in vitro (IVM) for 20 to 24 hrs in TCM-199 supplemented with $10\%$ fetal bovine serum at $39^{\circ}C$ under $5\%\;CO_{2}$ in air. Following routine IVM/IVF procedure, the oocytes and presumed zygotes were cultured for three day in CRlaa medium with BSA. The cumulus cells at 2 to 8-cell stage of embryos removed then the embryos and were cultured in CRlaa medium containing $10\%$ fetal bovine serum in $5\%\;CO_{2}$ at $39^{\circ}C$. The fresh blastocysts cultured for 7 to 9 days were transferred into recipients. The numbers of oocytes recovered form two different methods, the aspiration and slicing after aspiration, were compared to know what. The number of oocytes per ovary was 8.2 and 6.5 in aspiration combining slicing, and aspiration groups, respectively (p<0.05). The cleavage rate in aspiration method are significantly (p<0.05) high than those in slicing post aspiration $(27.9\%)$, and aspiration $(25.5\%)$. The pregnancy .ate in aspiration method $(62.5\%)$ was high than that in slicing method after aspiration $(54.4\%)$. The pregnancy rates of aspiration method and slicing method after aspiration in nullipara $(58.1\%\;vs\;68.2\%)$ was high than that in pluripara $(49.5\%\;vs\;53.2\%)$. The results obtained that the increased number of oocytes per ovary in slicing method after aspiration could be better than that in aspiration method. Pregnancy rate in aspiration method was slightly higher in than that in slicing method after aspiration.

• Korean Journal of Animal Reproduction
• /
• v.21 no.2
• /
• pp.147-156
• /
• 1997

Ultrasound-guided follicular aspiration was performed in Holstein heifers once weekly with or without pretreatment of single or multiple decreasing doses using a total of 400 mg FSH. Oocytes were aspirated with a 6.5 MHz convex-array ultrasound trasducer designed for intravaginal use. All the visible follicles larger than 4 mm in diameter were punctured with a 17 gauge, 55 cm needle at each aspiration session and the follicular fluids containing oocytes were obtained by vacuum suction. The results obtained were as follows: As a preliminary experiment, the recovery rates of folicular oocytes by ultrasound-guided aspiration from the isolated ovaries of Korean native cows were compared between suction methods using manual syringe or vacuum pump. The recovery rate of oocytes using vacuum pump (80.7%) was significantly (P<0.05) higher than that using manual syringe (47.1%). The follicles were counted by their size in diameter with ultrasound image, and recovery rates and grades of follicular oocytes collected by ultrasound-guided aspiration were investigated in Holstein heifers pretreated with or without FSH. A group of heifiers were injected with multiple decreasing doses (twice a day for 3 days) of a total of 400 mg FSH. The other 2 groups were injected with a single dose of 400 mg FSH mixed with 25% PVP. Ultrasound observation of follicle population and/or ultrasound-guided transvaginal oocyte aspiration were performed 12 hrs following the last FSH injection in the multiple dose group, and 48 or 60 hrs after FSH injection in the single dose groups. Most of the visible follicles had small size of less than 3 mm in diameter in unstimulated heifers (71.0%), but medium size in all the heifers treated with FSH. (70.5 to 92.8%). The number of OPU follicles per session (4.6$\pm$1.9) were much less, compared to the vilsible follicle counts (9.7$\pm$2.2), in the nustimulated heifers due to the small dominant follicles. Among 4 goups of heifers the most visible as well as OPU follicles were observed in the heifers at 60 hrs following treatment of a single dose of 400 mg FSH (21.2$\pm$2.3 and 21.0$\pm$2.0), and the differences in both the follicle counts between the groups was found significant (P<0.05) The rates of oocyte recovery from the follicles by ultrasound-guilded aspiration were varied 46.3 to 75.0% in the heifers unstimulated and treated with a single dose of 400 mg FSH, but the group difference was not significant. The number of recovered oocytes per session a, pp.ared to be highest at aspiration at 60 hrs following single FSH (10.6$\pm$2.2) than at aspiration at 48 hrs after single FSH (7.8$\pm$2.7) or in the unstimulated heifers (3.4$\pm$3.0). The proportion of grade I and II oocytes to all oocytes collected was varied 31.8 to 64.0% between the groups. However, there was found no significant difference in both the number of oocytes recovered per session and the percentage and the percentage of grade I and II oocytes. From the above results it was concluded that the more oocytes of superior quality might be recovered economically by ultrasound-guided aspiration at 60 hrs following the pretreatment of a single dose of 400 mg FSH and by suction using a vacuum pump system of about negative pressure of 75 to 85 mmHg.

• Journal of Embryo Transfer
• /
• v.29 no.1
• /
• pp.35-41
• /
• 2014

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot ($20mg{\times}6/300IU$) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ${\pm}$ SEM) observed which developed to larger than 2 mm in diameter were $1.6{\pm}2.5$, $4.3{\pm}3.1$, $5.5{\pm}4.2$, $6.6{\pm}2.1$ and $8.8{\pm}7.8$, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + $10{\mu}g/ml$ FSH + $1{\mu}g/ml$ $17{\beta}$-estradiol for 27 h at $39^{\circ}C$ in 5% $CO_2$ in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6-DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.8
• /
• pp.1127-1133
• /
• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• The Korean Journal of Physiology and Pharmacology
• /
• v.15 no.1
• /
• pp.17-22
• /
• 2011

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-$HT_{3A}$ and ${\alpha}7$ nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal ${\alpha}3$ and ${\beta}4$ subunits. Treatment with acetylcholine elicited an inward peak current ($I_{ACh}$) in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited $I_{ACh}$ in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptors. The inhibition of $I_{ACh}$ by quercetin was reversible and concentration-dependent. The half-inhibitory concentration ($IC_{50}$) of quercetin was $14.9{\pm}0.8\;{\mu}M$ in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor. The inhibition of $I_{ACh}$ by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

• Journal of Embryo Transfer
• /
• v.10 no.2
• /
• pp.121-129
• /
• 1995

To examine the critical effect of oxygen concentration on embryonic development, in vitro fertilized embryos were cultured in media(TCM199 vs. SOF) supplemented sera(1O% FCS vs. 10% HS) with and without bovine oviduct epithelial cells under two gas atmosphere (5% $CO_2$ in air vs. 5% $CO_2$, 5% $O_2$, 90% $N_2$). Oocytes, obtained from abattoir ovaries, were matured in EGF containing TCM199 medium co-cultured with BOEC for 24 hours, followed by exposure to frozen-thawed, heparin4reated spermatozoa in TALP for 30 hours. And then early embryos(1~2 cell) were cultured in both TCM199 and SOF supplemented with 10% FCS or 10% RS under 5% $CO_2$ in air or 5% COi, 5% $O_2$, 90% $N_2$. Development to morulae and blastocysts was recorded on days 7, after the start of in vitro fertilization. The developmental rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC under 5% $CO_2$, 5% $O_2$, 90% $N_2$(24.4%) were significantly(p<0.05) higher than cultured in SOF with BOEC under 5% $CO_2$ in air(14.1%) at seven days after in vitro fertilization. When early bovine embryos were cultured in TCM 199 and SOF under two different gas atmosphere, there were no significant differences in the developmental rates to morulae and blastocysts between supplements of 10% FCS and 10% HS. The rates of development to morulae and blastocysts were significantly(p<0.01) higher in TCM 199 with BOEC(24.7%) than TCM199 without BOEC(10.9%) under 5% $CO_2$ in air, otherwise SOF without BOEC(36.4%) were significantly (p<0.05) higher than in SOF with BOEC (24.4%) under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In summary, these experiments have proved that the culture system in SOF supplemented 10% ES is effective on in vitro development of early bovine embryos under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In addition, it is effective to development of bovine embryos that TCM 199 should be co-cultured with BOEC and SOF should be cultured without somatic cells under two different gas atmosphere.

• Asian-Australasian Journal of Animal Sciences
• /
• v.9 no.5
• /
• pp.583-590
• /
• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.101-111
• /
• 2001

The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P ＜ 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

• Journal of Embryo Transfer
• /
• v.14 no.1
• /
• pp.31-37
• /
• 1999

The objective of this study was to optimize the selection of sperm sources, optimal culture systems and vitrification method depends on sperm sources. The oocytes were inseminated with either KPN 105, 114, 191, SNU 101, 102, 103 or epididymis and then embryos inseminated were cultured in oviductal cell co-culture or HECM-6 as defined me dium. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The results obtained were as follows: 1. The cleavage(86.2 or 84.7%) and development rates to blastocyst (30.6 or 32.0%) were not significantly different between oviductal cell co-culture or HECM-6 culture systems(P<0.05). 2. To determine the optimal sperm sources for using IVF in this system, cleavage rates in KPN 191 and SNU 101 (74.2, 55.8%) were significantly lower rather than those in KPN 105, 114, SNU 102, 103 or epididymis (86.7, 85.1, 89.8, 85.5 or 81.2%), but development rates to blastocyst in KPN 114, SNU 103 or epididymis sperm (30.0, 33.0 or 28.6%) were significantly higher rater than those in KPN 105, 191, SNU 101, 102(21.4, 15.4, 14.9 or 25.4%), respectively (P<0.05). 3. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The survival rates were not significantly different among sperm sources (89.6%: 43/48 ; 90.1%: 46/51 ; 83.3% : 20/24). These results obtained indicate that the defined medium, HECM-6, could be use to produce of IVP bovine embryos. Since the frozen semen must be required to maintain of unvariation data in IVP embryo production system, KPN 114 and SNU 103 produced in our laboratory were useful for this purpose. The blastocysts produced by different sperm sources as KPN, SNU or epididymis were vitrified by OPP vitrification method and survived very high rates. The OPP vitrification method could be susceptibility to use of IVP bovine blastocyst embryos.

• Korean Journal of Veterinary Research
• /
• v.37 no.4
• /
• pp.917-924
• /
• 1997

Estrus cycle of cow, season and follicldar stimulating treatment, having an effect on the number of follicle, are investigated for the oocyte recovery rate in ovum pick-up(OPU). The number of follicle aspirated and oocyte collected on the different days of estrus cycle(D 4~5, D 9~10 and D 14~15) were not significantly different among the groups. The higher number of viable oocytes were produced on Jan-May(79.0%) than Jun-Aug(33.3%) by OPU in cow. The number of follicle and aspirated oocyte in cows treated FSH or PMSG combined with bovine somatotropin(bST) were 1.2~1.5 times higher than in cows treated alone follicular stimulating hormone(FSH) or pregnant mare serum gonadotropin(PMSG). In conclusion, OPU can be repeatedly practiced 2 or 3 times in an estrus cycle. In addition, the high environmental temperature is not good for ovarian function of cow and the bST co-treatment with FSH or PMSG is increasing the number of aspiratable follicle.