• Asian-Australasian Journal of Animal Sciences
• /
• 제26권12호
• /
• pp.1680-1688
• /
• 2013

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly ($88.9{\rightarrow}114.4$). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

• Clinical and Experimental Reproductive Medicine
• /
• 제24권2호
• /
• pp.261-265
• /
• 1997

본 연구는 EGF가 체외수정 및 체외배양에 의해 생산된 생쥐배의 체내 발달에 미치는 영향을 조사하고자 실시하였다. 본 실험에 사용된 난자는 체외수정 후 얻어진 2-세포기배를 EGF 첨가유무에 따라 배양 (5-6 embryos/25${\mu}l$/drop)하여 얻어진 4일령의 배반포기배로서, 각 처리군의 배반포기배는 가임신 3일된 대리모의 자궁내에 이식되어졌다. 그 결과를 요약하면 다음과 같다. 1,2-세포기배를 EGF의 첨가유무에 따라 배양하여 배반포기배로의 발달율과 세포수를 조사하였던 바, 처리군간의 유의한 차이는 나타나지 않았다. 2. 하지만, 각 처리군에서 회수된 배반포기배의 체내 발달을 조사하였던 바, 총 수태율의 결과에 있어서는 대조군과 EGF 처리군 각각 64.4%와 69.8%로서 두 군간에 유의한 차이를 나타내지 않았지만, 정상태아 발생율에 있어서는 EGF 처리군 (51.2%)이 대조군 (31.1%)보다 매우 높게 나타냈다. 따라서, 비록 EGF 처리군이 대조군과 비교하여 볼 때 체외수정 및 체외배양에 의해 생산된 난자의 유의한 발달은 나타내지 않았지만 난자의 질적인 향상을 통해서 체내발달을 증진시킬 수 있을 것으로 사료된다.

• 한국수정란이식학회지
• /
• 제26권3호
• /
• pp.159-164
• /
• 2011

This study was performed in order to determine optimum flushing solution using the direct embryo collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3$^{rd}$ day administration of FSH, 25 mg $PGF_2{\alpha}$ was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1$^{st}$ insemination and embryos were recovered 8 days after the 1$^{st}$ insemination. Embryo collection from superovulated donors were performed to flushing by DEC and conventional method. As a results, the average number of recovered embryos were significantly higher as 19.1${\pm}$1.40 with DEC method than 12.0${\pm}$0.44 with conventional embryo collection method, respectively (p<0.05). Also, The average number of transferable embryos were significantly higher (p<0.05) as 15.8${\pm}$1.72 with DEC method than 6.9${\pm}$0.35 from conventional embryo recovery procedures. Meanwhile, number of recovered embryos and number of recovered transferable embryos following the number of flushing times until 6${dr}$ flushing were significantly higher as 8.6${\pm}$0.53 and 8.6${\pm}$0.53 from 2$^{nd}$ flushing time than other groups (p<0.05). No. of Ear. B stage embryos were significantly higher as 3.9${\pm}$0.90 and 3.9${\pm}$0.90 with 2$^{nd}$ flushing time in total collected embryos and transferable embryos (p<0.05). Com M stage embryos were significantly higher as 3.7${\pm}$1.00 in 2$^{nd}$ flushing time and as 2.2${\pm}$0.76 in 3$^{rd}$ flushing time for recovered embryos (p<0.05). In transferable embryos, Com. M stage embryos were significantly higher (p<0.05) as 3.7${\pm}$1.00 in 2$^{nd}$ flushing time and as 2.2${\pm}$0.76 in 34$^{dr}$ flushing time, also. No. of degradation embryos was significantly higher as 2.2${\pm}$0.72 in 5${rd}$ flushing time, On the other hand, degradation embryos was not observed in transferable embryos (p<0.05). In conclusion, these results suggest that DEC method should effective methods for production of in vivo embryos using less flushing solution following perform until 4$^{rd}$ flushing time than conventional embryo collecting method. Also, it might be effectively collection of transferable embryos following more less procedure times compared to conventional embryo recovery methods.

• 한국동물학회지
• /
• 제8권2호
• /
• pp.37-41
• /
• 1965

The frequency and pattern of sex chromatin in primary cultured cells of kidney cortex of cats and guinea-pigs, and muscle of chick embroys were examined and compared to those of in vivo condition, with special reference to the various cultured stages. 1. In cat, the frequencies of sex chromatin positive I of peripheral position were average 62.7% in female, and 15% in male, whereas those of non-peripherla position were 5.8% in female and 0.1% in male. The incident proportion between them showed a marked difference-approximately 10 times higher in female than male. These results failry indicated that a distinct nuclear dimorphism with regard to the sex chromatin positive I was established in cultured cells. The position of sex chormatin was usually peripheral location. The tendency of frequencies , with reference to the cultured stages, was low count in primary extracted and initial stage cells , but it showed a peaked frequency in 10-13 days after primary culture, and after that the frequencies were decreased gradually. Compared between I vitro and in vivo condition of the same tissues, the cells in vivo exhibited the sex chromatin in high frequency at the peak showed stage. 2. In guinea-pig , the frequencyies of peripheral positive I were 36.8% in female and 6.3% in male, while non-peripheral positions were 6.1% in female and 3.5% inmale. Its incident was a rate of nearly 4 times higher in female than male. The nuclear dimorphism was also established in cultrued cells of guinea-pig. The position and the incident frquency showed a similar pattern as in cat except the primary extracted cells. 3. In chick embryo, the frequencies of sex chromatin positive I of peripheral position were 38.2% in female, and 18.3% in male, non-peripheral position, however, was hardly to find. These results suggest that the definite sexual dimorphism was unable to find in chick embryo cultured cells. The position and the incident tendency were a similar pattern as in above mammals and the frequency was higher in vitro cells.

• Asian-Australasian Journal of Animal Sciences
• /
• 제16권7호
• /
• pp.1071-1086
• /
• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• 한국수정란이식학회지
• /
• 제26권3호
• /
• pp.153-158
• /
• 2011

This study was performed in order to simplify the operation and minimize stress of donor and be readily available in the field with low cost and high quality embryos using the Direct Embryo Collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3rd day administration of FSH, 25 mg $PGF_2{\alpha}$ was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1 st insemination and embryos were recovered 8 days after the 1st insemination. Embryo collection from superovulated donors was performed to flushing by non-surgical methods of 3-way, 2-way and DEC (l-way). The average number of recovered embryos were 11.25${\pm}$0.63, 12.5${\pm}$0.65 and 11.75${\pm}$0.48 from operations of 3-way, 2-way and DEC methods, respectively. There were no significant differences among the embryo collection methods. Also, The average number of transferable embryos were 6.25${\pm}$0.48, 7.25${\pm}$0.48 and 7.25${\pm}$0.63 from each embryo collection procedures. The number of transferable embryos was no differences among the 3-way, 2-way and DEC methods, respectively. Meanwhile, the ratio of transferable embryos for all recovered embryos from DEC methods was higher as 61.7 % than 55.6 %, 58 % from methods of 3-way, 2-way. And the flushing solution required for recovering embryos by DEC method was significantly lower as 0.28${\pm}$0.32 1 than 1.8${\pm}$0.12 1, 1.75${\pm}$0.10 1 from 3-way, 2-way methods (p<0.05). Also, the time required for recovering embryos by DEC methods was significantly lower as 27${\pm}$2 min than 51${\pm}$3, 45${\pm}$2 min, respectively (p<0.05). In conclusion, these results suggest that DEC method for embryo collection may be effectively used for production of in vivo embryos using less flushing solution and, it might be effectively available in the field compared to conventional embryo recovery methods using 3-way or 2-way balloon catheter.

• 한국수정란이식학회지
• /
• 제32권3호
• /
• pp.147-151
• /
• 2017

Commercial applications of OPU/IVP were to produce embryos and calves from high genetic cows. The aim of this present study was to compare the number of recovered oocytes and cultured In vitro produced embryos from Ovum Pick-up (OPU). OPU derived embryo production was carried out of oocytes by ultrasonographic guided follicular aspiration and then produced in vitro produced blastocysts by IVP culture system. In result, the rate of recovered oocytes was obtained 612 (57.2%) and 451(73.7) G1+G2 grade oocytes. No difference of recovered rate (51.1~62.1%) was seen in six donor. The rate of cleavage and blastocyst development were obtained 320 (70.9%) and 78 (24.4%) that was $3.3{\pm}0.4$ cleaved embryo and $0.9{\pm}0.2$ blastocysts per session. Cleavage rate of OPU oocytes in No. 6 donor was 90.6%, significantly (P<0.05) higher than that in the other donors, However, blastocysts was similar (25.8~30.0%). In conclusion, limited numbers of OPU oocytes had competent development when cultured in SOF culture medium.

• 한국수정란이식학회지
• /
• 제24권3호
• /
• pp.159-167
• /
• 2009

Ultrasonographic examination was performed to observe the ultrasonographic image of Korean native cows' normal uterus in condition of in vitro and in vivo. The experiment was done 28 slaughtered cows' uterus using immersed in water in vitro, and 41 healthy breeding cows taken rectal ultrasonography in vivo. Ultrasonographic examination of uterine was taken on the reference of cross section of intercornual ligaments' cranial. Each uterus on the experiments was compared by estrous cycle and ultrasonographic frequency. The uterine structure using ultrasonography was 5 layers of uterine horn in vivo as well as in vitro. Uterine horn was observed to be distinguished from inside to outside as endometrium to inner echogenic layer, circular muscle layer to slightly echogenic elliptical layer, stratum vasculare to central echogenic layer, longitudinal muscle layer to slightly echogenic arched layer, and perimetrium to outer echogenic layer, respectively. According to the observation of uterus related to estrous cycle and ultrasonographic examination, uterine endometrium in vitro was constantly founded irrespective of estrous cycle and ultrasonographic frequency. On the low frequency, endometrium and circular muscle layer in estrus were prone to distinguished than in diestrus. On the high frequency, endometrium and circular muscle layer were always distinguished regardless of estrous cycle. In vivo, uterine endometrium and circular muscle layer were observed regardless of estrus and ultrasonographic frequency. On the low frequency, stratum vasculare and longitudinal muscle layer were not likely to be distinguished in diestrus, but estrus. On the high frequency, stratum vasculare and longitudinal muscle layer were observed regardless of estrous cycle. Also, every uterine structure was easily distinguished on high frequency than low frequency owing to precision of distinction in layers. The difference of results followed by the experiments conditions between in vitro and in vivo was that uterine endometrium and circular muscle layer in diestrus in vitro were difficult to be distinguished and uterine lumen was observed during whole estrous cycle. In vivo, It was founded that the distinction of stratum vasculare and logitudinal muscle layer in diestrus was complicated and uterine lumen was observed during only estrus. In view of the result so far achieved, normal uterine structure divided in 5 layers on ultrasonography was accorded with microscopic organization, uterine structure was likely to be observed during estrus than diestrus, high frequency checkup than low frequency, and uterine endometrium, circular muscle, stratum vasculare was easily observed regardless of estrous cycle and ultrasonographic frequency.

• 한국수정란이식학회지
• /
• 제26권1호
• /
• pp.33-36
• /
• 2011

The objective of this work was to determine the effect of corpus luteum (CL) grade on pregnancy rate after embryo transfer in Korean cattle and we found that CL development was linked to pregnancy rate. The in vivo derived blastocyst-stage embryos were transferred to 15 recipients synchronized in the estrus cycles. Based on size and palpable characteristics, CLs were categorized into three grade. The grade three CL is not to be identified by rectal palpation. The pregnancy rates tended to increase with the increase in CL size of recipients. In grade one, two, and three, the pregnancy rates were 62.5%, 50.0%, and 0%, respectively. This result suggests that pregnancy rates after embryo transfer might be affected by the CL status of recipients.

• 대한의생명과학회지
• /
• 제14권1호
• /
• pp.47-53
• /
• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.