Among fatty acid families, the polyunsaturated fatty acids were demonstrated to be mediators in various reproductive processes as precursor of steroid hormone (via cholesterol) and prostaglandins (via arachidonic acid), and in the last decade, major research was focused on the effects of omega-6 and especially omega-3 fatty acid. Eicosapentaenoic acid, the longest members of omega-3 fatty acid family, can be produced by a series of desaturation and elongation reactions from shorter member such as α-Linolenic acid. However, very few studies have provided detailed descriptions of Eicosapentaenoic acid effects and mechanisms of action in mammalian oocytes. The purpose of this study was to evaluate the effect of Eicosapentaenoic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of Eicosapentaenoic acid was added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, nuclear maturation rate, blastocysts quality, and levels of prostaglandin E2, 17β-estradiol, progesterone in the spent medium. High doses (100 mM) of Eicosapentaenoic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 mM Eicosapentaenoic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of E2/P4 also significantly increased compared with control group (p < 0.05). However, Supplementation of 100 mM Eicosapentaenoic acid showed high apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol/progesterone also significantly decreased compared with control group (p < 0.05). Our results indicated that supplementation with appropriate levels of Eicosapentaenoic acid beneficially affects the change of hormone synthesis for controlling oocyte maturation, leading to improved embryo quality. However, high doses of Eicosapentaenoic acid treatment results in detrimental effects.

In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade 1⁺⁺, 1⁺, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded 1⁺⁺, 1⁺, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield 1⁺⁺, 1⁺ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield 1⁺⁺, 1⁺, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield 1⁺⁺ and 1⁺ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield 1⁺⁺and 1⁺ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, 1+ and 1++, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield 1⁺⁺, 1⁺ , 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin K1(vit K1) 첨가 농도, 시기 및 시간의 효과를 검토하였다. 1.0 μM, 3.0 μM 및 6.0 μM vit K1을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 14.5 ± 4.3, 0.0 및 0.0%로써 대조군의 35.5 ± 3.2%에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 31.8 ± 2.6%로써 시험군의 22.2 ± 2.9, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, 1.0 μM 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 26.5 ± 2.9% 및 47.2 ± 2.8%로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). 3.0 μM 농도에서는 대조군의 배발달율이 36.4 ± 3.1%로 가장 높았으나, 생존율은 3.0시간 첨가군이 41.7 ± 3.2%로 대조군에 비하여 유의하게 높았다(p<0.05). 6.0 μM 농도에서도 배발달율은 대조군(32.0 ± 2.8%), 생존율은 0.5시간 첨가군(42.9 ± 1.8%)이 가장 높았다. 각각의 vit K1 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, 1.0 μM 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, 3.0 μM 3.0시간 및 6.0 μM 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율(59.5 ± 4.1% 및 50.0 ± 3.6%)과 생존율(72.7 ± 5.4% 및 79.2 ± 4.0%)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit K1 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군(1.0 μM 6시간 배양 2일째, 3.0 μM 3.0시간 배양 4일째 및 6.0 μM 0.5시간 배양 6일째)이 53.4 ± 5.8, 49.4 ± 3.8 및 51.5 ± 4.5개로써 대조군의 40.2 ± 2.3개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 3.2 ± 0.9∼3.7 ± 2.1개로써 대조군의 4.2 ± 1.2개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, 6.0 μM 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit K1의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 (14.7±3.0 vs 30.3±4.8%, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

The objective of this study was to examine the effect of thymidine treatment during in vitro maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group (p<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group (p<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group (p<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups (p<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG (33.1±9.6 vs 21.7± 8.3%). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP- treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The m-RNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

Embryonic compaction is essential for normal preimplantation development in mammals. The present study was to investigate the effects of compaction patterns on developmental competence of pig embryos. The proportion of blastocyst formation derived from compacted morula was higher than those of compacting and pre-compacting morula (P<0.01). Nuclei numbers of inner cell mass (ICM), trophectoderm (TE), and total of blastocysts derived from compacted group were also superior to those of compacting and pre-compacting groups (P<0.05). Then, compaction patterns, developmental ability and structural integrity were compared between mono- and poly-spermic embryos. The rate of compacted morula in mono-spermic embryos was higher than that of poly-spermic embryos (P<0.05). Especially, the rate of blastocyst formation derived from compacted embryos in mono-spermic embryo group was higher than that of poly-spermic embryo group (P<0.05), although no difference was detected between the two groups in the structural integrity. Finally, we confirmed that beta-catenin was differentially expressed according to compaction patterns in morula and blastocyst stage embryos. In conclusion, our results suggest that the compaction patterns during preimplantation development play a direct role in developmetal competence and quality of pig embryos.

This study was performed to elucidate the effects of addition of and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell () and blastocyst stages () with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0mg/ml) than in the group of 1.0mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell () and blastocyst stages () with different concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0mg/ml(15.3%) than in the group of 0.5mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was (77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage () and blastocyst stages () with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage (), blastocyst () and hatching stages () were not different. The development rate to the 2 cell stage (), blastocyst () and hatching stages () at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production.

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were and respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at was significantly lower than that at 0 hr (<), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

본 연구에서는 수정란 이식을 위해 선발된 대리모에 이식 전 백신의 투여 시기가 임신과 유산에 미치는 효과와, 임신한 대리모의 분만 예정일 전에 백신의 투여가 송아지의 질병과 생존에 미치는 효과를 검토하였다. 실험 1에서는 한우 체외 수정란의 이식에 선발된 대리모의 백신 투여 시기가 임신과 유산에 미치는 영향을 검토하였다. 이식 전 주째 백신 투여시 임신율이 31.5%로서 대조군 및 주 투여군의 42.4 및 45.9%에 비하여 유의하게 낮았다(p<0.05

본 연구는 수정란이식의 현장 적용 측면을 중심으로, 한우 체외 수정란의 발생 단계, 일자 및 등급이 젖소 대리모에 이식 후 임신과 유산, 분만과 관련된 임신기간, 생시체중, 난산 및 쌍자율을 조사하였다. 또한 단자 및 쌍자 분만에 따른 임신기간, 생시체중, 성비 및 폐사율을 분석하였다. 발생 단계에 따른 임신율은 배반포 단계(64.4%), 유산율은 부화 배반포(HB) 단계(21.4%)가 유의하게 높았다(p<0.05). 발생 일령에 따른 임신율은 7일령

본 연구는 돼지 체외 수정란의 생산에 있어서 체외성숙 시간이 핵성숙, 다정자 침입율 및 배 발생과 배반포의 부화율 배반포의 부화에 미치는 효과를 검토하였다. 돼지 난포란의 핵성숙율이 체외성숙 36, 38, 40, 42 및 44 시간째에 각각 68.0, 78.0, 79.5, 73.8 및 81.8%로서 각 군간에 유사한 경향이었으며, 체외 수정 후 다정자 침입율도 각각 48.7, 36.0, 44.4, 38.9 및 31.8%로서 차이가 없었다. 체외 성숙 시

본 연구는 고품질의 돼지 체외 수정란을 생산하기 위하여 체외 성숙 배지에 첨가하는 polyvinylpyrrolidone(PVP)의 분자량, 첨가 농도 및 시간(실험 1)과 체외 성숙 수정 배양 단계에서 PVP의 첨가(실험 2)가 배 발생과 세포수에 미치는 효과를 검토하였다. 돼지 미성숙 난자의 체외 성숙은 NCSU 23 용액, 체외 수정은 mTBM 용액, 체외 배양은 PZM 3 용액을 이용하였다. 체외 성숙용 배지에서 PVP의 분자량, 농도 및 첨가 시

본 연구는 체외성숙 배지에 첨가하는 PVP의 농도, EGF, cysteine 및 PVP의 단독 또는 혼합첨가가 한우 체외수정란의 체외발생에 미치는 영향을 검토하였다. 체외성숙 배지에 PVP의 첨가농도(0.1~3.0%)에 따른 분할율은 차이가 없었으나, 배반포 발달율은 0.5% PVP 첨가군이 가장 높았다(P<0.05). PVP, EGF 및 cysteine의 단독 및 혼합 첨가에 따른 분할율은 cysteine 단독첨가군이 높았으나(P<0.05), 배반포 발달율은 차이가 없었다. Inner cell mass 수는 대조군과 cysteine 첨가군이 PVP 첨가군에 비하여 유의하게 높았고(P<0.05), 총 세포수도 cysteine 첨가군에서 가장 높았다. 수정란이식 결과는 대조군, EGF, cysteine 및 EGF+cysteine 군의 임신율은 46.1~63.6%로서 비슷하였으나, PVP 첨가군은 10%로서 다른 군에 비하여 유의하게 낮았다(P<0.05). 본 연구 결과는 체외성숙 배지에 PVP의 첨가로 배 발생은 가능하지만, 세포의 품질에는 악영향을 미치는 것을 보여준다.

본 연구는 체외수정란에서 유래한 한우 송아지의 생존에 미치는 각종 요인들에 대하여 분석하여 체외수정란을 이용한 수정란 이식과 송아지의 효율성을 향상시키고자 실시하였다. 분만된 송아지의 기형율은 전 시험군에서 비슷한 경향이었다. 질병 발생율은 임신 기간, 분만 유도, 분만처치 방법과 형태 및 난산 처리 방법에 따른 차이가 없었다. 그러나 경산우가 미경산우, 봄과 겨울(20.4 및 )이 여름과 가을(4.3 및 ), 단태가 쌍태 및 정상 분만이 난산에 비하여

본 연구에서는 한우 체외수정란의 이식에 있어서 수정란 측 요인들이 수란우의 임신과 유산에 미치는 영향을 검토하였다. 이식에 제공하는 배반포의 수량에 따른 임신율은 1개, 2개 및 3개 이식 군에서 각각 32, 44 및 였고, 유산율은 로서 2개 이식군의 임신율과 유산율이 가장 높았으나 유의차는 인정되지 않았다. 배반포 등급에 따른 임신율은 , 유산율은 로서 유사한 경향이었다. 배반포의 발생 단계에 따른 임신율은 로서 비슷하였으나, 유산율은 HB군이 LB