This experiment was carried out to study the determination of survival of vitrified and thawed mammal follicular oocytes by FDA-test. Oocytes were divided into 3 groups according to attachment of cumulus cell. Group A oocytes were tightly surrounded by cumulus cell, group B oocytes were partially surrounded by cumulus cell, and group C oocytes were poorly surrounded by cumulus cell. Vitrification solution developed by our previous study (Kim et al, 1992) which consisted of permeable agent (20 % glycerol + 10 % ethylene glycol) and nonpermeable agent (30 % Ficoll + 10 % sucrose). Oocytes (7~10) loaded into 0.25 ml straw after 10 min equilibration were plunged into liquid nitrogen (- 196) directly. The FDA-score of vitrified and thawed group A oocytes was higher in rat (4.2) than in rabbit (3.9), cow (3.8), mouse (3.4) and porcine (2.4), however that of cumulus cell was higher in rabbit (4.7) than in rat (4.1), cow (2.9), porcine (2.6) and mouse (1.4). The FDA-score of vitrified and thawed group B oocytes were 3.1 (cow), 2.9 (rabbit), 2.9 (mouse), 2.6 (rat) and 2.5 (porcine), respectively. However that of cumulus cell was higher in rabbit (3.7) than in porcine (2.6), rat (2.3), cow (1.7) and mouse (0.3). The FDA-score of vitrified and thawed group C oocytes was higher in mouse (4.1) than in cow (2.9), rabbit (2.6), rat (1.3) and porcine (1.1). As shown in the above results, The survival rates of oocytes were higher in group A than in group B and C except in mouse and cow. These results suggest that the survival of cumulus cell as well as follicular oocytes can be reliably judged by their fluorescence with FDA-test.

Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

One of the problems associated with in vitro culture of primordial gern cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of the porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, \ulcorner2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p<0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layer, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of \ulcorner2-macroglobulin and antioxidatns can increase the number of PGCs in vitro by suppressing apoptosis.

콩포장에 사용하는 제초제가 근류균의 생존률에 미치는 영향을 구명하기 위하여 제초제 Alachlor, Linuron, Simazine 및 Paraquat를 농도별로 YEMA 배지에 혼합하여 근류균을 접종, 배양하여 생존률을 조사하였다. 1. 콩근류균은 Alachlor와 Linuron의 사용권장 농도인 400ppm 처리에서도 각각 27.4％와 57.8％의 생존률을 보여 유의한 근류균의 감소를 보였다. 2. Simazine은 공시제포제중에서 가장 영향이 적었으며 Paraquat는 200ppm에서도 현저한 생존균수의 감소를 보였다. 3. Alachlor에 대한 근류균의 감수성은 균주에 따라 차이가 있었으며 I-122 균주가 가장 낮은 감수성을, I-145가 가장 높은 감수성을 보였고 중도 저항성인 K-5 균주는 사용권장농도에서는 높은 저항성을 보였으나 농도가 증가함에 따라 급격히 감소하였다. 4. 콩품종의 근류형성력은 균주, 품종에 따라 차이가 있었으며 Alachlor에 저항성인 균주 I-122, K-5 가 높은 근류형성력을 보여 근류형성력은 환경적응력과 밀접한 관계가 있는 것으로 보였다.