• Korean Journal of Agricultural Science
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• v.18 no.2
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• pp.114-118
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• 1991

This study was conducted to find out the suitable in vitro media for fertilization and culture on the pig oocytes matured in vivo and in vitro. The results obtained were as follows; A comparison of fertilization media on the oocytes matured in vivo was made between M199 with 10% FCS versus TL Hepes with 1% BSA Fertilization rate was significantly higher in the TL Hepes medium. But polyspermic incidence did not favor in the TL Hepes medium. Embryos were cultured in TL Hepes wash and culture medium for 48h after insemination. A total of 53 embryos were cultured and 39(73.6%) cleaved. Of these 39 embryos, 31(79.5%) cleaved equally to the 2-8 cell stage. Immature oocytes cultured in Waymouth's maturation medium were much more able to induce sperm swelling than immature oocytes cultured in TL Hepes maturation medium, however, most ova were polyspermic and did not develope to the 4 cell stage during 48h culture in TL Hepes wash and culture medium.

• Journal of Veterinary Clinics
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• v.7 no.1
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• pp.419-427
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• 1990

Immature bovine oocytes were cultured to investigate whether the addition of FCS(10% or 20% ), CS (10%or 20% ) or BSA(5mg/ml) to culture medium with or without HEPES and co-culture with granulosa cells affect the frequency of in vitro maturation of extrafollicular bovine oocytes. After culture, the maturation rates were examined by the presence of 1st polar body and nuclear configuration. The maturation rate when FCS and CS as protein supplement were added to culture medium with or without HEPES was significantly higher than when BSA was added, and the maturation rate of extrafollicular bovine oocytes co-cultured with granulosa cells was higher than that cultured without granulosa cells, but there was no significant difference. FCS and CS were shown to be superior protein supplement when compared to BSA, and serum concentration, HEPES and co-culture with granulosa cells did not affect the in vitro-maturation of extrafollicular bovine oocytes.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.25-30
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• 1997

This study was done to find out the preservation possibility of liquid boar semen at variabel room temperature of 9 to 16$^{\circ}C$. The percentages of sperm motility and NAR acrosome were highest in B tschwiler extender compared to B tschwiler＋Hepes, Andro＋Hepes and Andro extenders. The extenders with Hepes buffer showed detrimental effect for preservation of liquid boar semen. The pH of ejaculated sperm-rich fraction was 7.5. The pH of B tschwiler＋Hepes, B tschwiler, Andro＋Hepes and Andro extenders was 6.9, 7.5, 7.1 and 8.1, respectively. The pH of liquid boar semen with B tschwiler＋Hepes, B tschwiler, Andro＋Hepes and Andro extenders was 6.6, 6.9, 6.7 and 6.9 at 1st day of storage, and 5.5, 5.7, 5.6 and 5.8 at 7th day of storage, respectively. Gilts and sows were inseminated twice with liquid boar semen stored at 9~16$^{\circ}C$ in B tschwiler extender for 3~4 days. Farrowing rate, litter size and average pig weight at birth between AI and natural service did not differ significantly in gilt and sow, respectively. However, sow showed higher farrowing rate and litter size compared to gilt both in AI and in natural service. As a result of this study, we found out that liquid boar semen can be stored for 5~7 days at uncontrolled room temperature of 9~16$^{\circ}C$ in B tschwiler extender.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.181-186
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• 2011

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.217-223
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula /blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, respectively. The above development rates to morula /blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TGM-HEPES without PEM(P<0.05). The in vitro development rates to the morula /blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without PEM were 0~1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.221-224
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• 1991

This study was undertaken to evaluate the effect of rabbit peritioneal fluid(rPF) on in vitro maturtion of porcine follicular oocytes. From does 20h after hCG injection, rPF was aspirated aseptically at laparatomy, and then centrifuged, filtrated, and preincubated immediately for 12h. Porcine follicular oocytes isolated from ovaries of slaughtered animals were incubated in TCM-HEPES＋10% FCS, TCM-HEPES＋rPF(v/v, 50/50), or rPE only and examined the nuclear maturation after aceto-orcein or hochest staining. After identifying the optimal incubation time, this experiment was repeated for 5 times. Under the TCM-HEPES containing hormones and serum codition, the time range of porcine follicular oocyte maturation was 38 to 44 hours and the optimal time of maturation of follicular oocyte in vitro was 42 hour cultivation, respectively. The maturatin rates(89.4% and 92.7%) of porcine follicular oocytes cultured in the media with 50% rPF or only rPF were signifciantly higher thanthat (84.6%) of oocytes cultured with TCM-HEPES, respectively. These results suggest that the unknown components(s) of rPF promoted in vitro maturation of porcine follicular oocytes.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.299-305
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula/blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine oviductal epithelial cell monolayers(POEC) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8∼16-cell and morula/blastocyst stage were 57.2, 48.2, 37.2 and 19.3% in Ham's F-10 with POEC, and 51.4, 41.2, 31.1, and 15.5% in TCM-HEPES with POEC, respectively. The above development rates to morula/blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TCM-HEPES with out POEC(P<0.05). The in vitro development rates to the morula/blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without POEC were 1.1∼1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with POEC in the two different media enhanced the development of fertilized eggs to morula/blastocyst stages in vitro. However, we didn't find out any difference for the in vitro development to morula/blastocyst stages between Ham's F-10 and TCM-HEPES media.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.289-297
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• 1996

This study was conducted to investigate the effects of maturaton medium and porcine follicular fluid on in vitro maturation of porcine follicular oocytes. The results obtained are as follows; 1. When the oocytes were cultured for 42 hours, the maturation rate was significantly (P<0.05) higher in TCM-HEPES(75.5%) than Ham's F-10(60.9%) and mKRB(60.7%) medium. The optimal medium for the maturation of porcine oocytes in vitro was the TCM-HEPES medium. 2. When the oocytes were cultured for 42 hours in TCM-HEPES medium with 10, 20 and 30% of porcine follicular fluid(pFF), the maturation rates of porcine oocytes were 69.1, 65.6 and 63.3%, respectively. The maturation rate(51.4%) of oocytes cultured without pFF was significantly(P<0.05) lower than that of oocytes cultured with pFF. 3. The maturation rates of porcine oocytes cultured for 42 hours in TCM-HEPEs medium with 3 different porcine follicular fluid treatments were 68.6%(centrifused), 72.3%(filtered) and 73.1%(heat treated), respectively. The maturation rate(49.4%) of control group without pFF treatment was significantly(P<0.05) lower than that of oocytes cultured with pFF treatment.

• Journal of Pharmaceutical Investigation
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• v.29 no.4
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• pp.337-341
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• 1999

We have studied the stability and transdennal flux of prostaglandin $E_1\;(PGE_1)$ from various donor solutions through hairless mouse skin. Stability in HEPES buffer or in propylene glycol (PG) solution where enhancer (oleic acid (OA), propylene glycol monolaurate (PGML), transcutol (TC), ethanol (EtOH))s dissolved was investigated. $$PGE_1 was not stable in HEPES buffer. The concentration of $$PGE_1 decreased continuously for 7 days, and the degradation rate constant was $0.0028\;h^{-1}$, assuming first order reaction. The effect of current or penetration enhancer on the degradation was minimal. Percutaneous transport from HEPES buffer by passive or iontophoretic delivery without enhancer was close to nil. When OA or PGML was used together with PG, both passive and iontophoretic flux increased. PGML showed better enhancing effect than OA. Flux by cathodal delivery was about 2 times larger than that by passive delivery. Flux by anodal delivery was lower than that by passive delivery. TC and EtOH also increased the transdermal flux, but the effect was not as good as that observed when OA or PGML was used. These stability and flux data provide important information on how to formulate the patch, which will be the next step of this work, and on the polarity of current to use during iontophoresis.

• Korean Journal of Agricultural Science
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• v.23 no.2
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• pp.206-211
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• 1996

This study was conducted to investigate the effects of medium and cumulus cell on in vitro fertilization of porcine follicular oocytes. The results obtained are as follows ; 1. The normal fertilization rates of in vitro matured follicular oocytes cultured in 00, mT ALP and TCM-HEPES medium were 14.0~24.3%, 30.8~32.7% and 21.4~23.9%, respectively. These data indicated that the optimal medium for fertilization of porcine oocytes in vitro was the mTALP medium 2. The normal fertilization rates of epididymal sperm were 24.3%(80), 30.8%(mTALP) and 23.9%(TCM-HEPES), and those of ejaculated sperm were 14.0%(B0), 32.7%(mTALP) and 21.4%(TCM-HEPES). 3. The sperm penetration rates of cumulus-enclosed and cumulus-free oocytes on in vitro fertilization were 54.0% and 72.0%. The normal fertilization rates of cumulus-enclosed and cumulus-free oocytes were 11.9% and 21.5%. The normal fertilization rate of cumulus-enclosed oocytes was significantly(P<0.05) higher than that of cumulus-free oocytes.