난자의 성숙과정과 노화에 관한 이해는 인공수정과 체외수정 최적기를 판단하기 위하여 가장 중요한 연구내용으로 알려져 있다. 이러한 기작은 번식 호르몬들에 의하여 조절되는 것으로 알려져 있으나 난자 세포질 변화에 관한 내용은 잘 알려져 있지 않다. 본 연구에서는 산화질소물(nitric oxide, NO)이 난자 성숙과정에서 증가하는 것을 밝혔으며 난자의 미성숙단계(germinal vesicle stage, GV)와 난자핵막붕괴단계(germinal vesicle breakdown, GVBD) 및 성숙완료단계(metaphase II, MII)단계에서 생산되는 NO의 양을 비교하였다. 또한, 난자를 체외에서 배양할 때, MII단계로 성숙되지 않는 성장 단계의 난자에서는 NO의 증가 현상을 관찰할 수 없었고, 세포질이 불균일한 노화된 난자에서는 NO가 증가된 상태로 유지되는 특성이 있음을 밝혔다. 이러한 결과는 NO의 작용이 난자의 성숙과정과 난자 노화과정에서 중요한 기능을 담당하고 있음을 보여주고 있다.

본 연구는 파킨슨 질환(Parkinson’s disease) 마우스 모델을 대상으로 지구성 운동과 MitoQ 섭취가 뇌의 흑질의 미토콘드리아 기능에 미치는 영향을 확인하는데 목적이 있다. 파킨슨 질환을 유도하기 위해 C57BL/6 수컷 마우스를 대상으로 복강 내 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) 25mg/kg과 흡수를 돕기 위한 probenecid 250mg/kg을 이용하여 주 2회 5주간 총 10회 투여하였다. 실험 집단은 생리식염수를 투여하는 집단(Normal Conrol (NC), n=10), MPTP 투여집단(MPTP Control (MC), n=10), MPTP 투여 + MitoQ 투여집단(MPTP + MitoQ (MQ), n=10), MPTP 투여 + 운동집단 (MPTP + Exercise (ME), n=10), MPTP 투여 + MitoQ 투여 + 운동집단(MPTP + MitoQ + Exercise (MQE), n=10) 총 5 집단으로 구성하였으며, 운동집단은 지구성 운동을 실시하였고 MitoQ집단은 점진적으로 250μmol로 늘리면서 5주간 섭취하였다. 연구결과 Rotarod-test에서 MC 집단에 비해 처치 집단은 운동 기능 저하의 개선을 보였다. 또한 MC 집단에 비해 처치 집단은 tyrosine hydroxylase의 수준의 증가와 알파시누클린(α-synuclein) 단백질 축적을 감소시켰다. 그리고 미토콘드리아 생합성에 주요조절 인자인 PGC-1α와 항산화 효소인 Catalase 발현이 MC 집단에 비해 처치 집단에서 증가해 미토콘드리아 기능을 개선했으며, 세포사멸 조절인자인 Bcl-2의 증가와 Bax의 감소를 통해 세포사멸을 완화했다. 따라서 5주 간의 지구성 운동과 MitoQ 섭취는 파킨슨 질환에서 나타나는 병리학적 특징을 완화하고 운동기능을 향상 시키는데 효과적인 것으로 나타났다.

본 연구는 알츠하이머(Alzheimer’s disease: AD) 형질전환 생쥐를 대상으로 저항성 운동 (resistance exercise: RE)이 해마의 베타 아밀로이드(β-amyloid: Aβ) 단백질 대사, 신경세포사멸 및 인지기능에 미치는 영향을 확인하는데 목적이 있다. AD 비 형질전환 생쥐(non-transgenic: non-tg, n=14) 와 형질전환 생쥐(transgenic: Tg, n=14)를 무선 배정하여 비 형질전환 생쥐 대조군 (non-tg-control: NTC, n=7), 비 형질전환 생쥐 저항성 운동군(non-tg-RE: NTRE, n=7), 형질전환 대조군(tg-control: TC, n=7) 및 형질전환 저항성 운동군(tg-RE: TRE, n=7)으로 구분하였다. RE는 특수 제작한 사다리 저항성 운동 기구를 사용하여 점진적으로 set 수를 증가시켜 총 8주간 실시하였다. 운동 후 인지기능 능력을 평 가하기 위한 수중미로검사와 Aβ 단백질 대사, 신경세포사멸 지표 및 SIRT1/PGC-1α 단백질 발현 수준 을 확인하였다. 수중미로검사 결과 거리와 시간 모두 TC 집단에서 유의하게 증가 되었지만 RE를 실시한 TRE 집단에서 거리와 시간이 감소 되어 인지능력이 개선된 것으로 확인되었다. 또한, TC 집단에서 증가 된 Aβ 단백질 발현은 RE를 통해 감소하는 것으로 나타났다. 신경세포사멸 관련 단백질인 Bcl-2/Bax ratio는 TC 집단에서 유의하게 감소되어 신경세포사멸이 증가 된 것으로 나타났지만 RE는 Bcl-2/Bax ratio을 증가시켜 신경세포사멸을 감소시킨 것으로 확인되었다. TC 집단에서 증가된 BACE1 및 ROCK1 과 감소된 ADAM10과 RARβ 단백질 발현은 RE를 통해 감소되거나 증가 된 것으로 나타났고, SIRT1/ PGC-1α 단백질 발현은 TC 집단에서 감소 되었지만 RE를 통해 증가 된 것으로 나타났다. 따라서 8주간 의 RE는 AD의 병리학적 특징인 Aβ 단백질 발현을 감소시키고 관련 생성 기전들을 조절하여 (SIRT1/PGC-1α 기전 활성, 아밀로이드 생성기전 억제, 비-아밀로이드 생성기전 활성) 신경세포사멸 억제시키고 결과적으로 인지기능을 개선 시킬 수 있는 효과적인 운동 방법이라고 생각된다.

Atopic dermatitis (AD) usually develops in patients with an individual or family history of allergic diseases, and is characterized by chronic relapsing inflammation seen specially in childhood, association with IgE hyperproduction and precipitation by environmental factors. and wished to examine closely effect that Polygonum multiflorum isolated PM-E and PM-70M orally adminstration used to atopy dermatitis disease patient get in atopy eruption control experimentally. Atopic dermatitis is a chronically relapsing inflammatory skin disease. Animal models induced by relevant allergens play a very important role in the elucidation of the disease. This study was investigated the anti-allergic effect of PM-E and PM-70M on BMAC induced atopic dermatitis in NC/Nga mice. We summerized as the follow. PM-E and PM-70M significantly reduced the skin number of total cell number, CD4+ and CD11b+/Gr-1 cell compared with positive control and decreased the invasion of CD4+ cell in dorsal skin tissue compared with positive control group by using immunohistochemical staining and chemokine such as eotaxin and CCR3 compared with positive control group. PM-E and PM-70M markedly suppressed invasion and edema of leukocytes and mast cell in dorsal skin. Taken together, these findings suggested that PM-E and PM-70M has an anti-allergic activity and this might be useful for the clinical application to treat allergic diseases such as atopic dermatitis.

Lipopolysaccharide (LPS)는 염증유발 cytokine 분비를 자극하고 염증을 유발하는 그람음성균의 내독소이다. 본 연구에서는 LPS가 신경아교세포 활성과 해마에 있는 nuclear factor kappa B (NF-κB) 매개 염증유발 요소를 조절하는지를 조사하였다. 성체 수컷 쥐를 대조군과 LPS를 투여한 실험군으로 무작위로 나누어 vehicle 또는 LPS (250 μg/kg)를 5일 동안 복강투여하고 무게를 측정했다. 해마의 활성산소와 과산화지방질 수준을 분석하고, 형태학적 연구를 위해 Hematoxylin and eosin 염색을 시행하였다. 또한, 해마에서 ionized calcium-binding adapter molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), NF-κB, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α)의 발현을 확인하기 위해 Western blot 분석과 immunofluorescence 염색을 시행하였다. 그 결과, LPS를 투여한 쥐들의 체중이 감소하였다. LPS 투여는 활성산소와 과산화지방질 수준의 증가를 유발하였고 LPS를 투여한 쥐의 해마에서 심각한 조직병리학적 변화를 확인했다. 또한 LPS 투여는 신경교세포와 별아교세포의 표시물인 Iba-1과 GFAP의 발현을 증가시켰고, NF-κB의 발현과 IL-1β와 TNF-α와 같은 염증성인자의 발현을 증가시켰다. 이러한 결과들을 통해 LPS 투여는 해마손상과 염증반응을 유도한다는 것을 알 수 있고 LPS 투여가 해마조직에서 신경아교세포와 NF-κB에 매개된 염증인자들을 활성화시킨다는 것을 확인하였 다. 따라서, 본 연구는 LPS 투여는 해마조직에서 산화적 스트레스 증가와 염증인자 활성을 증가시켜 신경손상을 유도함을 보여준다.

Previous studies have suggested that rice bran oil (RBO), an edible oil from the byproducts of rice milling, has antiinflammatory effects in inflammation inducing macrophages, known as M1 subsets. Yet the effects of RBO on the counterpart M2 subsets, the “healing” macrophages, were poorly investigated to date. In this regard, recent studies on the molecular/cellular anti-inflammatory mechanisms of dietary components have demonstrated that mitochondrial respiration contributes to macrophage functioning. Therefore, the current study examined whether RBO regulates cytokine secretion by modulating mitochondrial metabolism in wound healing M2 subsets. Palm oil (PO), enriched with medium-chain fatty acids, served as a positive control. C57BL/6 mice were fed a diet containing either corn oil (CO), PO or RBO for 4 weeks, followed by purification of bone marrow-derived macrophages (BMDM) from their tibias and femurs. Cells were further polarized to M2-BMDM, and the expression of M2 marker (CD206) on cellular surfaces were not affected by dietary intervention. In addition, the secretion of anti-inflammatory cytokine (IL-10) in the culture supernatant was not affected by dietary lipids. Oxygen consumption rate, the indicator of mitochondrial respiration in M2-BMDM was not regulated by RBO intervention and PO treatment. Taken together, this study imply that RBO did not intervene both the regulation of inflammatory responses and mitochondrial respiration in M2 macrophages.

This study was performed to investigate the body fat-lowering effect of garlic powder in peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α)-luciferase transgenic mice (TG). In this study, we generated transgenic mice with a PGC-1α promoter (—970/+412 bp) containing luciferase as a reporter gene. Mice were fed a 45% high-fat diet for 8 weeks to induce obesity. Subsequently, mice were maintained on either a high-fat control diet (CON), or high-fat diets supplemented with 2% (GP2) or 5% (GP5) garlic powder for an additional 8 weeks. Dietary garlic powder reduced the body weight in the GP2 and GP5 groups, compared to the CON group. Furthermore, garlic supplementation significantly decreased the plasma levels of triglycerides, total cholesterol, and leptin in the GP5 group, compared to the CON group. Specifically, luciferase activity in liver, white adipose tissue (WAT), and brown adipose tissue (BAT) was increased by garlic supplementation in a dose-dependent manner. These results suggest that the body fat-lowering effect of garlic powder might be related to PGC-1α by the increase in luciferase activity in liver, WAT, and BAT. Furthermore, transgenic mice might be useful for evaluating the body fat-lowering effect of various health functional foods.

The objective of this study was to investigate the effects of a lettuce (Lactuca sativa L.) extract on the inflammation of human umbilical vein endothelial cell (HUVEC) and blood lipid improvement in hypercholesterolemic mice fed a high cholesterol diet. The lettuce extract (100% ethanol extract) inhibited the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in HUVEC treated with tumor necrosis factor-α (TNF-α). The lettuce extract suppressed the adhesion of THP-1 to TNF-α-treated HUVEC. The lettuce extract decreased the TNF-α-stimulated production of proinflammatory cytokine interleukin-6, interleukin-8 and chemokine monocyte chemotactic protein 1. In hypercholesterolemic mice, the lettuce extract reduced serum total cholesterol, triglyceride, and low-density lipoprotein-cholesterol level, while the lettuce extract elevated high-density lipoprotein-cholesterol level, resulting in the decrease of atherogenic index and cardiac risk factor level. These results suggested that lettuce extract can be an useful resource to show an anti-inflammatory effect and improve lipid metabolism

Bitter melon (Momordica charantia) is used in traditional herbal medicine in many Asian countries for the treatment of several diseases such as diabetes, eczema, night blindness, psoriasis, and rheumatism. Especially, most reports concerning the biological activities of bitter melon have focused on its effects on diabetes and hyperglycemia. Also, bitter melon is regarded as a longevity food, suggesting that it has several beneficial effects on anti-aging and the maintenance of a healthy state. Thus, we investigated whether bitter melon could increase the capacity of exercise in this study. Interestingly, bitter melon fruit extract activated AMP-activated protein kinase (AMPK), which is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. In addition, bitter melon extract increased the expression of enzymes involved in fatty acid oxidation such as mitochondrial uncoupling protein 3 (UCP3), carnitine palmitoyl transferase 1b (CPT1b), and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4). Moreover, exercise tolerance was much more enhanced in bitter melon treated animals compared to the non-treated control group. These results suggest that bitter melon is a promising candidate for the development of functional foods beneficial for physical strength and the enhancement of exercise capacity.

Makgeolli is a health beneficial food for diabetes compared to other alcoholic beverages. We examined the effect of Makgeolli on blood glucose level and survival rate in a streptozotocin (STZ)-induced diabetic mouse model. We force fed 30 male STZ-induced diabetic ICR mice Makgeolli consisting of 6% alcohol (DM-MAK), 6% ethanol (DM-EtOH), or distilled water (DM-DW) for 4 weeks. In the DM-MAK group, food intake and water intake were higher than those of other groups after 4 weeks. Body weight, however, was not different among the experimental groups. We also found no significant difference in blood glucose level among the experimental groups. In normal ICR mice fed Makgeolli for 1 week, the area of the blood glucose curve was higher than those of other groups fed 6% ethanol, 2% glucose, or distilled water. Survival rates of STZ-induced diabetic mice fed Makgeolli, 6% ethanol, or DW for 4 weeks were 100%, 25%, and 62.5%, respectively. In conclusion, Makgeolli had no beneficial effect on blood glucose in a STZ-induced diabetic mouse model, although their survival rate was high. These results show that Makgeolli has an effect on type 1 diabetes through other mechanisms than blood glucose control.

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

Programmed cell death or apoptosis is associated with changes in K+ concentration in many cell types. Recent studies have demonstrated that two-pore domain K+ (K2P) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of K2P channels, were found to be critical for cell death. This study was performed to identify the role of K+ channels in the H2O2-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to H2O2 for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited H2O2-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (β-mercaptoethanol) with 25 mM KCl significantly decreased the rate of H2O2-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of K+ channel efflux for a short-time reduces H2O2- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of K+ channels which are highly expressed in a given cell type.

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhi-bition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

HX106N은 용안육, 맥문동, 단삼 및 천마 등의 4가지 식물로 구성된 추출물로서, 선행 연구에서 amyloid β peptide에 의한 생쥐의 기억력 저하 및 산화 손상을 억제하는 것으로 밝혀졌다. 이 연구에서는 HX106N이 비선택적 무스카린 수용체 길항제로 잘 알려진 스코폴라민(scopolamine)으로 유도한 콜린성 건망증(cholinergic amnesia)에 어떤 영향을 미치는지를 평가하였다. ICR 생쥐에게 스코폴라민(1 mg/kg body weight, i.p.)을 주입하기 1시간 전에 HX106N(100 mg/kg body weight, p.o.)을 투여하였다. 30분 후 수행한 Y-미로 시험(Y-maze test) 및 수동 회피 시험(passive avoidance test)에서 HX106N는 스코폴라민에 의해 감소되는 자발적 변경 행동(spontaneous alternation) 및 지체시간(step-through latency)을 유의미하게 억제하여 건망증을 개선시키는 것으로 나타났다. 또한 HX106N을 투약 1시간 후 생쥐의 해마와 대뇌피질 부위의 아세틸콜린에스테라제(acetylcholinesterase; AChE)의 활성을 측정한 결과 통계적으로 유의미한 정도의 활성 감소가 관찰되었다. 이러한 결과들을 종합할 때 HX106N은 AD에서 관찰되는 콜린성신경전달 장애로 인한 기억력 저하 억제에 사용될 수 있는 가능성을 가진 것으로 판단된다.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

본 연구는 아토피 피부염을 갖는 질환동물에서 어성초를 비롯한 다양한 식물추출물을 이용하여 제조한 크림의 항아토피 피부염증에 대한 효능을 평가하였다. BALB/c 마우스 를 이용하여 아토피 피부염 유발 대조군 및 크림 처리군으로 완전 임의 배치하였다. 혈액 IgE와 히스타민 농도는 크림 처리군이 DNCB 유도 아토피 대조군에 비해서 유의하게 감소하였으며, 아토피 피부염이 심각한 상태의 마우스에서 정상적인 피부상태로 회복되었음을 관찰하였다. 본 결과는 아토피질환 모델 BALB/c 마우스에서 어성초 혼합 식물추출물의 투여가 혈액 IgE를 낮춤과 동시에 비만세포에서 생성되는 히스타민의 배출량을 억제함으로써 항아토피 효능을 갖는다는 점을 시사 해준다.

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ( <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ( <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl2 in Ca-free CZB medium in the presence of 5 μg/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6～7 week-old mice(53.3%) than in oocytes from 8～9(80.9%) and 10～11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium (51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6～7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.