The successful establishment and maintenance of pregnancy is achieved by well-coordinated interactions between the maternal uterus and the implanting conceptus. In pigs, the conceptus undergoes dramatic morphological and functional changes, and secretes various biological products such as estrogens and cytokines, interleukin-1beta (IL1B), interferon-gamma (IFNG), and IFN-delta (IFND) during the implantation period. The uterine endometrium in response to the conceptus-derived molecules and ovarian progesterone becomes receptive to the conceptus by changing cell adhesion molecule expression, epithelial cell depolarization and secretory activity. Conceptus-derived estrogen acts as the maternal pregnancy recognition signal which changes the direction of endometrial prostaglandin (PG) F2 secretion from the uterine vasculature into the uterine lumen. Estrogen also induces the expression of a variety of endometrial genes, including AKR1B1, FGF7, LPAR3, and SPP1. The function of cytokines, IL1B, IFNG, and IFND, in the endometrium is not fully understood, but some recent work shows that IL1B is involved in the synthesis and transport of endometrial PGs by regulating endometrial expression of PG-synthetic enzymes, PTGS1, PTGS2, and AKR1B1, and PG transporters, ABCC4 and SLCO2A1. Estrogen and IL1B also stimulate endometrial expression of IFN signaling molecules, suggesting that estrogen and IL1B act cooperatively on priming the endometrial function of conceptus IFNG and IFND. In turn, IFNG derived from the elongating conceptuses, induces many endometrial genes, including CXCL9, CXCL10, CXCL12, and SLA-DQ. The role of IFND at the maternal-conceptus interface is not well understood yet. Further analysis of the molecules derived from the endometrium and conceptus will provide insights into the cellular and molecular basis of maternal-conceptus interactions for the establishment of successful pregnancy in pigs.

Establishment of the Adherens junction (AJ) and Tight junction (TJ) are important steps in terms of morphological formation during preimplantation develoment. Particularly, TJ complex is crucial for cavitation in blastocyst. So far, many TJ protein/genes are revealed. However, the biological function and regulation of TJ were not elucidated during post implantation. We depleted several TJ and TJ associated genes using RNA interference, and examined preimplantation development with TJ. We tested functionality of paracellular sealing to determine integrity of TJ formation and examined TE differentiation indirectly using outgrowth assay in vitro. We observed defect of paracellular permeability in the TJ related genes knockdown(KD) blastocyst and abnormal outgrowth. Particularly, trophoblast cells were not stretched out in the KD groups. Finally, we did embryo transfer using the TJ genes KD and control blastocysts into surrogate mothers. We found lower of the implantation rates/ maintenance of pregnancy in the TJ KD groups (less than 40%) than in the controls (about 80%). In conclusion, TJ integrity is can be used as a selective marker for developmentally competent embryos and successful pregnancy.

The OPU technology has been largely used in order to enhance genetic improvement in domestic animals. This study demonstrated that OPU in Hanwoo can be used for the effective technique to improve the reproductive efficiency. This experiment showed the longest times of OPU ever carried out in Hanwoo. In this study four donors were selected from Hanwoo by using DNA extraction and SNP maker. Individual donors have genes which are CAPN1, CAST and FASN and that contain dominant position. CAPN1, CAST genes are often thought to be related with tenderness and FASN gene is thought to be associated with oleic acid which is identified a monounsaturated fatty acid found naturally in many plant and animal products. In experiment 1, OPU technique was used to evaluate the influence of the number of oocytes recovery rate per session. Totally 50 times OPU sessions were performed and oocytes recovery at every 10 times session was evaluated. In case of H4, the OPU session could be done around 30 times after her calving. Compared to the average number of oocytes recovery, H1 was more efficient than H3. Considering this results, the current study showed that animals have considerable individual variation in numbers of oocytes. In this study, the average recovery rate in Hanwoo is similar to the recovery rate of the Bos Taurus. Individual donors have no significant difference among group for recovery rate during 30 sessions. However, Thoese showed significant decrease in the number of oocytes recovery rate after 40 session of OPU treatment. Therefore we conclude that the Hanwoo donor is considered to be suitable for 30 times of OPU treatment. In experiment 2, OPU technique was used to evaluate the influence of the number of recovery rate monthly. The average number of oocytes recovery rate showed no significantly difference for five months. However, the number of oocytes recovery rate decreased significantly during three months after first five months experiments. In experiment 3, whether donors are parity or non-parity, the average number of oocytes recovery rate was checked. However, we could not find any significant result from this experiment. In experiment 4, the developmental rate of in vitro produced embryos from OPU was compared with that from slaughterhouse. From this, cleavage rate of oocytes from OPU is significantly less than that from slaughterhouse. In conclusion, this study included a thorough analysis of oocytes and embryo production by OPU from Hanwoo. OPU can be successfully performed under a continuous regime for 8 month in Hanwoo. The current study shows the clear proof that OPU could be adopted to produce oocytes and embryos of better quality as an advanced technique replacing the usage of MOET in Korea. Finally, elucidation the basis for numerous oocytes obtained from Hanwoo may contribute to a better understanding of reproductive physiology in cattle.

In vitro maturation (IVM) systems have become indispensable for the production of large numbers of competent oocytes in domestic species. The quality of in vitro matured oocyte is one of the important factors determining the success of assisted reproductive technologies (ARTs) including intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) in human and livestock. Incomplete cytoplasmic maturation of oocytes can lead not only to a failure of fertilization but also to a developmental arrest after ARTs. Thus, establishment of a stable IVM system to produce a large number of high quality oocytes, especially in domestic animals, is essential for improvement of ARTs efficiency by producing high quality embryos. The morphological characteristics are commonly used to predict the developmental potential of oocytes and embryos. Usually, normal oocytes shrink when exposed to a hypertonic medium, and recover their morphology when returned to an isotonic medium. During this process, oocytes show various morphologic changes, such as shrinkage in spherical (SSP) or irregular shapes (SIR). In the first study, we investigated whether the shrinkage pattern of oocytes that was observed after hyperosmotic treatment could be used as a morphologic characteristic to predict the quality of IVM oocytes in pigs. We found that SSP oocytes showed improved developmental competence after PA and SCNT. This improved embryonic development was most likely because of the more advanced nuclear and cytoplasmic maturation in SSP oocytes compared with SIR oocytes. Pig oocytes shows a wide variation in the size of perivitelline space (PVS) after IVM. Based on this finding, we examined in the second study whether or not there was any correlation between the PVS size of IVM oocytes and their developmental competence after PA and SCNT. Our results demonstrated that in vitro developmental competence to the blastocyst stage positively correlated with the size of the PVS of oocytes after IVM. In addition, we observed that mature oocytes with a larger PVS showed higher levels of intracellular GSH content and transcription factor expression. Furthermore, enlargement of the PVS by culturing in reduced NaCl medium improves the embryonic development after PA and SCNT. In the third study, we investigated the effects of a hypotonic medium with reduced NaCl (61.6 mM) compared with an isotonic medium (108.0 mM NaCl) on oocyte maturation and embryonic development after PA and SCNT. In addition, we attempted to optimize our IVM system using a hypotonic maturation medium by examining the effects of hypotonic medium during various stages of IVM on oocyte maturation and subsequent embryonic development. Our results demonstrated that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 hr of IVM increased the developmental competence of oocytes after PA and SCNT. These beneficial effects was also shown in a commercial medium (a minimum essential medium; aMEM) in which the NaCl concentration was reduced to 61.6 mM. In addition, IVM of oocytes in medium with reduced NaCl increases the proportion of SSP oocytes in pigs. In summary, our results demonstrate that IVM of pig oocytes in a hypotonic medium with low-NaCl is better able to support embryonic development after PA and SCNT, most likely by improving the cytoplasmic maturation via increased intraoocyte GSH content and widened PVS. Based on these results, the newly developed IVM system using a hypotonic medium with reduced NaCl can produce high quality oocytes and be considered a new strategy for improving ARTs efficiency in pigs.

Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

돼지 난자의 체외배양 과정에서 배양액의 삼투압의 차이 및 glycine과 alanine 단독 또는 병행 첨 가가 단위발생 및 체세포 핵이식 난자의 배 발육에 미치는 영향을 검토하였다. 난자 의 체외성숙 에는 10% 돼지 난포액, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin이 첨가된 TCM-199을 이용하였다. 체외성숙 42∼44시간 후 제1극체가 방출된 난자만을 선별하여 단위발생 및 체세포 핵이식 난자를 생산한 후 modified porcine zygote medium (mPZM)-3 배양액에서 7일간 배양하였다. 실험 설계에 따라 체외배양 7일 또는 체외배양 초기 48시간 동안 280 또는 320 mOsm의 삼투압에서 난자를 배양하였다. 실험1에서 체외배양 7일 동안 280 또는 320 mOsm의 배 양액 내 glycine 또는 alanine 첨가 효과를 검토한 결과 삼투압 차이에 따른 분할률 및 배반포 발달 률에는 차이를 보이지 않았으나 320 mOsm에서 배양된 난자 및 280 mOsm에 glycine을 첨가한 군 은 280 mOsm에서 배양된 난자에 비해 높은 배반포 세포수를 보였다 (36.5-38.0 vs. 31.1, P<0.05). 실험2에서는 체외배양 초기 48시간 동안 280 또는 320 mOsm의 삼투압에서 배양하였고, 그 후 280 mOsm 배양액으로 옮겨 5일간 추가로 배양하였다. 또한 체외배양액 내 4.1 mM glycine 첨가가 배 발육에 미치는 효과를 검토하였다. 단위발생 난자를 배양초기 48시간 동안 320 mOsm에서 glycine이 첨가된 배양액에서 배양한 군이 280 mOsm에서 배양한 군에 비해 유의적으로 높은 배반 포 발달율을 보였다(36.5-50.4% vs. 25.9-27.9%, P<0.05). 또한 배양 초기 48시간 동안 320 mOsm 배 양액에 glycine을 첨가하여 배양한 난자 (50.4%)가 다른 처리군의 난자(25.9-36.5%)에 비해 유의적 으로 높은 배반포 발달율을 보였다(P<0.05). 실험3에서는 실험2와 동일한 실험설계로 체세포 핵이 식으로 작성된 배반 포의 inner cell mass (ICM)와 trophectoderm (TE) 세포수를 조사하였다. 실험결과 초기 46시간 동안 glycine이 첨가된 320 mOsm 처리군에서 배양된 난자의 ICM 비율이(26.0%) 280 mOsm 삼투압에 glycine을 첨가한 군(17.8%)에 비해 유의적(P<0.05)으로 증가하였다. 본 연구결과 단위 생식 및 체세포 핵이식 난자의 체외배양에서 glycine의 첨가 및 체외 배양초기 48시간 동안 320 mOsm 삼투압 처리는 배 발육과 배반포 세포수를 증가시키는 것으로 확인되었다.

Monosodium glutamate (MSG)는 아미노산의 일종인 글루탐산에 나트륨이 결합된 것으로 구수한 맛, 감칠맛을 내는 인공조미료로 사용되고 있다. 이전 연구에 의하면 쥐에서MSG의 섭취가 난모세 포의 발달에 유해하다는 결과가 보고되었다. 본 연구에서는 돼지 난자의 체외성숙 및 체외 배양액 에 MSG 첨가가 난자의 성숙 및 배 발육에 미치는 영향을 검토하였다. 난자의 체외성숙배양액으 로는 10% 돼지 난포액이 첨가된 Medium-199 (positivecontrol) 또는 비필수아미노산이 제거된 Porcine zygote medium (PZM)-4를 기본배양액으로 하여 여기에 cysteine, pyruvate, epidermal growth factor, kanamycin, insulin 및 호르몬을 첨가하여 사용하였다. 실험설계에 따라 MSG를 각각 0, 0.1, 1, 10 mM 농도로 체외성숙 배양액에 첨가하였다. 체외성숙 난자는 전기자극을 통해 단위발생을 유도하였고 PZM-3배양액에서 7일간 체외 배양하였다. 체외배양에서의 MSG 효과를 알아보기 위하 여 돼지 난포액 첨가 Medium-199에서 체외성숙된 난자들 중 제1극체가 방출된 난자만을 선별하여 단위발생 유도 후 PZM-3 (positive control) 또는 비필수아미노산이 제거된 PZM-4에 0,0.1, 1, 10 mM의 MSG를 첨가하여 배 발육에 미치는 영향을 조사하였다. 체외성숙 단계에서 MSG 효과를 조 사한 결과 MSG 농도에 따른 체외성숙률(61.3-70.3%)에는 차이를 보이지 않았지만 10 mM의 MSG 처리군(83.5%)이 0.1 mM 처리군(93.7%)에 비해 유의적으로 낮은 분할률을 보였다. 또한 MSG 처리 군들이 (14.5-25.5%) 무처리군(30.0%)에 비해 낮은(P<0.05) 배반포 발달률을 보였다. 체외배양 단계 에서 MSG 효과를 조사한 결과 10mM MSG 처리군(90.8%)이 1 mM 처리군(96.5%)에 비해 유의적 으로 낮은 분할률을 보였으며, 10 mM 처리군(32.8%)이 0.1과 1 mM MSG 처리군에(55.1, 51.4%)에 비해 유의적으로(P<0.05) 낮은 배반포 발달률 보였으나 무처리 대조군(46.6%)과 유의적 차이는 없 었다. 이러한 결과를 보아 체외성숙 배양액 내 MSG 첨가는 난자의 핵 성숙률에는 영향을 주지 않지만 고농도의 MSG 처리는 단위발생 후 분할률을 억제하며 배반포 발육능을 억제하는 것으로 확인되었다.

돼지 난자의 체외성숙 배양액에서 다양한 농도의 육탄당 처리는 난자의 성숙률과 배 발 육능, 세포수 증가에 영향을 주는 것으로 보고되고 있다. 본 연구에서는 육탄당의 일종인 fructose가 돼 지 난자의 체외성숙에 미치는 영향을 조사하였다. 난자의 체외배양액으로는 cysteine, pyruvate, epidermal growth factor, kanamycin, insulin 및 호르몬이 첨가된 porcine zygote medium (PZM)-4 또는 10% 돼지 난포액이 첨가된 PZM-3을 이용하였고 실험설계에 따라 다양한 농도의 fructose 및 5.5 mM의 glucose를 첨가하여 44시간 동안 배 양함으로써 난자의 성숙을 유도하였다. 첫 번째 실험에 서는 1.5, 3.0 및 5.5 mM의 fructose 와 5.5 mM의 glucose가 첨가된 성숙배양액에서 배양된 난자의 성숙률 및 단위발생 후 배 발육능을 조사하였다. 실험 결과 1.5, 3.0 및 5.5 mM fructose를 첨가한 배양액에서 성숙된 난자는 5.5 mM glucose가 첨가된 배양액에서 성숙된 난자와 비교하였을 때 유 사한 핵 성숙 률(81.9-91.5% vs. 94.2%), 단위발생 후 분할률(89.4-92.4% vs. 90.1%), 배반포 발달률 (39.3-41.1% vs. 39.4%) 및 배반포 세포수(30.8-34.2% vs. 31.8%)를 보였다. 두 번째 실험에서, 10% 돼지 난포액이 첨가된 PZM-3 배양액에서 무처리 및 3.0 mM fructose, 5.5 mM glucose 및 3.0 mM fructose와 5.5 mM glucose 병행 첨가 성숙배양액에서 성숙된 난자의 성숙률 및 단위발생 후 배 발 육률을 조사하였다. 실험 결과 3.0 mM fructose (93.1%), 5.5 mM glucose(91.7%) 및 3.0 mM fructose 와 5.5 mM glucose 병행 처리군(93.5%)이 무처리군(74.4%)에 비해 유의적으로(P<0.05) 높은 핵 성 숙률을 보였다. 또한 5.5 mM glucose(90.1%) 및 병행 처리군(97.2%)은 대조군(67.6%)에 비해 유의 적으로(P<0.05) 높은 분할률을 보였다. 배반포 발달률은 3.0 mM fructose(52.6%) 및 병행 처리군 (58.8%) 에서 무처리군(44.4%) 및 5.5 mM glucose (51.0%)에 비해 유의적으로(P<0.05) 증가하였다. 본 실험 결과로 보아 체외성숙 합성배양액 내 fructose 첨가는 glucose와 유사한 수준의 배 발육률 을 보임으로써 glucose를 대체할 수 있는 에너지원으로 이용 가능함을 확인하였다.

Hyaluronan은 난포액, 나팔관과 자궁에 존재하는 물질로 돼지의 다정자 수정을 억제하고 체외배 양액에 첨가시 배 발육을 향상시키는 것으로 알려져 있다. Glucuronic acid와 N-acetyl-D-glucosamine (GlcNAc)은 이중결합하여 hyaluronan을 구성하는 물질이다. 본 연구에서는 체외성숙 배양액 내 Glucuronic acid 및 GlcNAc 첨가가 돼지 난자의 성숙 및 단위발생 난자의 배 발육에 미치는 영향 을 조사하였다. 난자의 체외성숙 배양액으로는 0.1% PVA (polyvinyl alcohol)가 첨가된 Medium-199 을 기본배양액으로 이용하였고, 여기에 cysteine, pyruvate, epidermal growth factor, kanamycin, insulin 및 호르몬을 추가하여 44시간 동안 난자를 배양하여 체외성숙을 유도하였다. 실험설계에 따라 glucuronic acid 및 GlcNAc를 각각 0, 0.005, 0.01, 0.05, 0.1 mM의 농도로 체외성숙 배양액에 첨가 하였다. 체외성숙된 난자는 전기자극을 통해 단위발생을 유도하였고 porcine zygote medium-3에서 7일간 체외 배양하였다. 실험 결과 난자의 체외성숙 배양액 내 glucuronic acid 첨가는 난자의 핵 성숙률(91.3-94.4%), 단위발생 후 분할률(85.5-93.6%) 및 배반포 발달율(42.0-51.0%)에 영향을 미치지 않았으나 배반포 세포수는 0.05 mM glucuronic acid 첨가 군에서 38.0개로 대조군의 31.5개에 비해 유의적으로(P<0.05) 증가하였다. 난자의 체외 성숙 배양액에 GlcNAc를 첨가하였을 때 난자의 핵 성숙률(94.3-97.2%) 및 단위발생 후 배반포 세포수(40.0-43.1)는 대조군 및 첨가 농도의 차이에 따라 유의적인 차이를 보이지 않았으나 분할률은 0.05 mM 처리군에서 91.8%로 대조군(85.0%) 및 0.005 mM 처리군(84.6%)에 비해 유의적(P<0.05)으로 증가하였다. 또한 0.05 mM 처리군의 배반포 발달 율은 59.6%로 대조군(46.3%), 0.005 mM 처리군(44.3%) 및 0.1 mM 처리군(45.2%)에 비해 유의적으 로 높았다. 이상 결과로 보아 체외성숙 배야액 내 0.05 mM glucuronic acid 및 GlcNAc 첨가는 돼 지 난자의 단위발생 후 배 발육을 증가시키는 것으로 사료된다.

Several species show low sensitivity to IVM, and the development of optimized medium possible oocyte quality and stable growth. Furthermore, adding additive to the medium can effectively reducing development cost and leads to easy handling of oocytes. Isoliquritigenin and formononetin are extracts found in licorice. Previous studies reported that isoliquritigenin and formononetin affected the activity of sperm, but the oocytes are unknown. This study adds isoliquritigenin or formononetin to αMEM to mature oocytes under simple IVC conditions. Recovered oocytes are cultured in αMEM, isoliquritigenincontaining medium and formononetin-containing medium. In study we proved that in addition to the medium, above the quality of oocytes cultured when specific additives were added, more stable growth is possible. collection and IVM of oocyte. SD rats at 6 to 8 wks of age are injected is intraperitoneal with 30 IU/mL of PMSG and 48 hrs later, HCG 50 IU/mL is intraperitoneal injected. Oocytes are collected ovary after 17 hrs. Collected oocytes are cultured for 16 hrs with 200 μL αMEM and 200 μL αMEM containing isoliquritigenin or formononetin at 0, 0.01, 0.02, 0.04, 0.1 mg/mL. Also, isoliquritigenin and formononetin were mixed with 200 μL αMEM at a ratio of 0.25: 0.75, 0.50: 0.50, and 0.75: 0.25 mg/mL respectively. Oocytes supplemented with isoliquritigenin and Formononetin had high quality than oocytes cultured with αMEM and showed an increase in the IVF fertility rate. Our experimental results indicate that using isoliquritigenin, formononetin when cell culture, rather than used only in medium, more effective oocyte quality and stable growth.

Superovulation is a technique to acquire rats to produce many eggs than normal rats. Superovulated eggs were used to make cloned animals through somatic cell nuclear transfer technology (SCNT). Healthy and valuable oocyte retrieval is essential for successful somatic cell nuclear transfer. Superovulation is also essential to maximize to the yield of IVF-derived rat eggs. Osmotic pumps (Alzet®) are miniature in order to provide research with a convenient, and reliable alternative to chronic injections. Acquiring superovulated oocytes through osmotic pump in minimizing irritation to the uterus and ovaries are competitive. We investigated the effects of pregnant mare serum gonadotropin (PMSG) and human Chorionic Gonadotropin (hCG) using osmotic pump in sDM rat. Adult female rats at 11 wks of age were used for superovulation. The response to PMSG and hCG were examined by osmotic pump of 150 IU/kg PMSG + 75 IU/kg hCG or 150 IU/kg PMSG + 150 IU/kg hCG or 300 IU/kg PMSG + 150 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. HCG was administered 48 hrs later after administration of PMSG. Oocytes were collected from the oviducts 16–18 hrs after hCG administration. Superovulation was significantly higher in rats administrated 150 IU/kg PMSG + 75 IU/kg hCG. This study demonstrated that healthy oocytes were produced in DM rat by PMSG and hCG that flowed through the osmotic pump ameliorate on uterus and ovaries.

Ganglioside GT1b, glycosphigolipids with three sialic acid, is known to play an important role in signal transduction such as epidermal growth factor receptor (EGFR). EGF is also known to induce resumption of meiosis and cumulus cells expansion during porcine oocyte maturation. Therefore, this study was conducted to evaluate the effects of ganglioside GT1b on resumption of meiosis and cumulus cells expansion in porcine oocyte maturation. First, porcine cumulus-oocyte complexes were cultured in NCSU-23 medium supplemented with GT1b (0, 1, 2 and 4 μM) at 44 h. We observed that the proportion of the metaphase II (M II) stage was significantly increased in the 2 μM GT1b (78.0 ± 2.3) treated group than in the other groups. Furthermore, expression of cumulus cells expansion factor genes (Has2, TNFAIP6, Ptx3) were significantly increased in the 2 μM GT1b treated group than in the other groups. Next, we investigated the meiotic maturation and the expressions of cumulus cells expansion factor genes after GT1b and/or EGF treatment. The proportion of the M II stage was significantly higher in the GT1b+EGF (90.1 ± 2.3) treated group than in the other groups. Moreover, expressions of cumulus cells expansion factor genes were significantly increased in the GT1b+EGF treated group than in the control group. After in vitro fertilization, fertilization rate, preimplantation development competence and quality of blastocyst were improved in oocytes derived from GT1b+EGF treated group. Taken together, these results suggest that exogenous ganglioside GT1b improving the developmental competence of porcine embryos via increase of resumption of meiosis and cumulus cells expansion during in vitro maturation of porcine oocytes.

한우의 암소 개량을 목적으로 다양한 번식우의 활용 기술이 개발되어 오고 있다. 특히 수정란 을 이용한 개량 연구 중 생체난포란 (ovum pick-up, OPU) 방법은 체내수정란 생산을 위한 과배 란처리 방법 보다 공란우의 활용성을 높이기 위해서 사용되고 있는 기법이다. 따라서 본 연구에 서는 한우의 생체에서 난포란의 채란의 효율성에 관한 연구를 수행하였다. 생체난포란 채란을 위한 한우 공란우는 한우연구소에서 사육중인 한우에서 실시하였다. 생체 내 난포란의 관찰은 MyLabTM30VETGOLD (Esaote, Genova, ITALY) 및 탐촉자 (EC123; Micro-Convex 9∼3 MHz, Esaote, Genova, ITALY)는 6.6 MHz convex scanner를 사용하였고, 난포란 채란에 사용된 주사침은 19G 주사침을 사용하였다. 초음파상에서 2mm 이상의 난포 수량을 확인하고 난포란을 흡인하였다. 흡입된 난포란은 2∼3회 washing으로 혈액 등의 이물질을 제거하여 실체현미경 하에서 회수하였 고, 난포란의 등급분류는 세포질 균일도와 난구세포의 부착 정도에 따라 평가기준을 1등급에서 3 등급까지 분류하였다. 체외발달을 유도하기 위해서 회수된 난포란의 체외성숙배양은 TCM 199 기 본배양액에 소 태아혈청 (Fetal Bovine Serum) 0.5%와 LH, FSH, FGF, EGF 첨가하여 22시간 동안 배양하였고, 체외수정은 IVF 100 (IFP, Japan) 배양액 50μl 미소적에 성숙 난포란 20개씩 넣어서 체 외수정을 실시하였다. 체외수정을 위해서 KPN 동결정액을 사용하였고 수정을 위한 정자의 최종 농도는 2x106/ml로 6시간 동안 체외수정을 유도하였다. 체외발달유도는 SOF 배양액으로 5% O2, 5% CO2, 그리고 90% N2 와 38.5°C 인큐베이터에서 8일째 까지 배양하였다. 4두의 공란우로부터 개체당 8session 을 실시하였다. 개체별 회수효율은 평균 6.6±1.30, 7.5±2.20, 6.13±1.13, 4.9±1.36개의 결과를 보였다. 평균 1등급 43개, 2등급 9.5개, 3등급4.8개로 나타났다. 회수된 난자의 체외발달율 에서 분할율은 68.8%, 배반포 발생율은 28.4%의 결과를 보였다. 따라서 본 연구 결과로 미루어 볼 때 사료급여 방법에 따른 연구도 병행하여 추진해야 할 것으로 사료된다.

Although assisted reproductive technology (ART) has been developed in many mammalian species including cows, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to preserve embryos at room temperature. The basic medium for embryo preservation consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum (FBS). To maintain survival and prevent damage during embryo storage, three candidate small molecules were selected—CHIR99021, Y-27632 and Thiazovivin—and their concentrations were optimized. Then, the embryos in the small molecule supplemented preservation medium were stored at room temperature. The viability and hatching rate of embryos stored at 10°C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after storage at 20°C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The mechanism by which small molecules enhance survival of embryos during storage was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

The present study was conducted to investigate the effect of different heights from liquid nitrogen (LN2) vapor on sperm motility and morphology after frozen-thawing. Two ejaculates were collected from 2 fertile Hanwoo bulls (A and B) by using artificial vagina at Hanwoo Research Institute. After collection, ejaculates were transferred to laboratory immediately and diluted with semen extender (Optixcell, France). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and cooled at 4°C for 4 h and loaded to 0.5 ml straws. The straws were divided into 2 groups. Straws were placed in 3 or 9 cm of LN2 vapor for 14 min and then plunged into LN2 tank and cryopreserved until evaluation. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain) after frozen-thawed. In bull A, 3cm group showed higher percentages of total motility, VSL with 25μm and VAP compared those with 9cm group (98.0 vs. 93.4%, 62.4 vs. 54.0% and 98.6 vs. 93.2%, 3 vs. 9 cm, irrespectively; p<0.001). In bull B, frozen-thawed sperm of 3cm group showed higher percentages of VSL with 25μm, VCL, VSL, VAP and BCF compared with those of 9cm group (43.5 vs. 26.0%, 123.8 vs. 111.6 μm, 62.9 vs. 57.3 μm and 81.5 vs. 72.5 μm; 3 vs. 9 cm, irrespectively; p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). In bull A, frozen-thawed sperm of 3 and 9cm groups showed no significant difference in LIA, LDA, DIA and DDA. In bull B, 3 cm group showed higher LIA and lower DIA compared with those of 9 cm group (73.2 vs. 23.7% and 23.7 vs. 32.2%, 3 vs. 9 cm, irrespectively; p<0.001). We suspected that 3 cm vapor on LN2 vapor might be affected positively spermatozoa viability and acrosomal integrity compared with 9 cm group. In conclusion, semen freezing procedure in the present study will improve sperm quality after frozen thawing.