• 농업과학연구
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• 제18권2호
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• pp.114-118
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• 1991

본(本) 연구(硏究)는 생체(生體) 및 실험실(實驗室)에서 성숙(成熟)된 돼지 난모세포(卵母細胞)의 체외수정(體外受精) 및 배양(培養)에 적합(適合)한 배지(培地)를 찾기 위하여 실시(實施)한 바, 그 얻어진 결과(結果)는 다음과 같다. 생체내(生體內)에서 성숙(成熟)된 난모세포(卵母細胞)에 대하여 10% FCS을 함유(含有)한 M199수정배지(受精培地)와 1% BSA를 함유(含有)한 TL Hepes 수정배지(受精培地)를 비교(比較)한 결과(結果), 수정율(受精率)은 TL Hepes 수정배지(受精培地)가 M199 수정배지(受精培地)보다 우수(優秀)하였으나, 다정자침입율(多精子侵入率)이 다소 좋지 않은 편이었다. 체외수정후(體外受精後) TL Hepes 세척(洗滌) 및 배양배지(培養培地)에서 48시간(時間) 배양(培養)한 결과(結果), 배양(培養)된 난자(卵子) 53개중(個中) 39개(個) (73.6%)가 분할(分割)되었으며, 분할(分割)된 수정란(受精卵) 39개중(個中) 31개(個) (79.5%)가 2~8세포기(細胞期)까지 균등(均等)하게 분할(分割)되었다. 미성숙난포란(未成熟卵胞卵)을 Waymouth 성숙배지(成熟培地)에서 배양(培養)했을 때가 TL Hepes성숙배지(成熟培地)에서 배양(培養)했을 때 보다 수정후(受精後) 더 많은 확장(擴張)된 정자두부(精子頭部)를 유도(誘導)할 수 있었으나, 대부분 다정자침입(多精子侵入)이 되었다. 48시간(時間) TL Hepes 세척(洗滌) 및 배양배지(培養培地)에서 배양(培養)한 결과(結果) 4세포기(細胞期)까지 발육(發育)을 시킬 수가 없었다.

• 한국임상수의학회지
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• 제7권1호
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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.

• 한국가축번식학회지
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• 제21권1호
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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.

• 한국수정란이식학회지
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• 제26권3호
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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.

• 한국수정란이식학회지
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• 제11권3호
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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.

• 한국가축번식학회지
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• 제15권3호
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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.

• 한국가축번식학회지
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• 제20권3호
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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.

• 한국가축번식학회지
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• 제20권3호
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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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• 제29권4호
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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.

• 농업과학연구
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• 제23권2호
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• pp.206-211
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• 1996

본 실험은 수정배양액 및 난구세포가 돼지난포란의 체외수정에 미치는 영향을 구명하고자 실시하였는 바, 그 결과는 다음과 같다. 1. 체외수정배양액에 따른 수정률은 BO, mTALP 및 TCM-HEPES 배양액에서 각각 14.0~24.3%, 30.8~32.7% 및 21.4~23.9%의 정상수정률을 나타내서 mTALP 배양액이 가장 적합한 수정배지였다. 2. 수정에 이용된 두 종류 정액간의 정상수정률은 정소상체미부정액이 BO, mTALP 및 TCM-HEPES 배양액에서 각각 24.3%, 30.8% 및 23.9%이었고, 사출정액이 14.0%, 32.7% 및 21.4%로 나타나서 mTALP 배양액을 수정배지로 하여 사출정액을 사용한 경우가 가장 높은 정상수정률(32.7%)을 나타냈다 3. 난자-난구세포복합체와 난구세포를 제거한 나화난자의 정자침투률은 각각 54.0% 및 72.0%로 나화난자에서 높게 나타났다 그러나, 정상수정률은 난자-난구세포복합체 및 나화난자에서 각각 11.9% 및 21.5%로 난구세포를 제거하지 않은 난자-난구세포복합체에서 유의적(P<0.05)으로 높은 성적을 나타냈다.