137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Bos Indicus ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Quality ◽  
Phenazine Ethosulfate

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P > 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P < 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P < 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P > 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

scholarly journals 160 IN VIVO-CULTURE OF BOVINE EMBRYOS: TRANSFER OF SEMEN PRE-INCUBATED OOCYTES, ZYGOTES AND 4 TO 8 CELL STAGE EMBRYOS INTO THE BOVINE OVIDUCT

2005 ◽  
Vol 17 (2) ◽  
pp. 231
Author(s):  
V. Havlicek ◽  
F. Wetscher ◽  
T. Huber ◽  
M. Gilles ◽  
D. Tesfaye ◽  
...  
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Group Iii ◽  
Group I ◽  
In Vivo Culture ◽  
Group Ii

Oviduct as well as oocyte and embryo development are subject to developmental changes which have crucial effects on the application of in vivo culture. The present study aimed at optimizing in vivo culture of IVP bovine embryos at different developmental stages in the bovine oviduct. Cumulus oocyte complexes (COC) were collected from slaughterhouse ovaries, matured in vitro for 22 h and assigned to four groups. In groups I and II, oocytes were pre-incubated for 3 to 4 h with 5 × 106 sperm/mL, and then immediately transferred to recipients, which had just completed ovulation (group I), or kept in vitro for a further 12 to 18 h and transferred to Day 1 synchronized recipients (group II). In groups III and IV, COC were subjected to standard IVF/IVC; then embryos were either transferred at the 4- to 8-cell stage on Day 3 into the oviducts of Day 3-synchronized recipients (group III) or kept in vitro for a further 4 to 5 days (group IV). Thirty-four 18- to 30-month-old temporary recipients were synchronized using a standard Ovsynch protocol. COC and embryos were transferred and re-collected by transvaginal endoscopy. COC or embryos were loaded into a 180° curved glass capillary, which was inserted via the infundibulum 5 to 8 cm deep into the ampulla ipsilateral to the CL. On recipient Day 7, a 90° curved metal canula served for tubal flushing prior to conventional uterine embryo flushing. Sixty mL of PBS containing 1% fetal calf serum were rinsed through the oviduct into the uterus and a further 400 mL of medium were finally used for flushing of the uterine horn and collected via an embryo filter. Embryo development was evaluated directly after flushing (Day 7) and on Day 8. For statistical analysis (ANOVA), the blastocyst rates (Days 7 and 8) in group III were related to COC corrected by the collection rate. In group I, 575 COC were transferred to 11 recipients and 420 (73%) were re-collected as oocytes or embryos. The blastocyst yields on Day 7 and Day 8 were 23% (97) and 25% (104), respectively. In group II, the transfer of 489 presumptive zygotes into 13 heifers resulted in only 175 re-collected (36%), of which 15% developed into blastocysts (Day 7: 26; Day 8: 27). Ten heifers (group III) served for in vivo culture of 643 embryos at the 4- to 8-cell stage. On Day 7, 568 (88%) embryos were flushed and 171 (30%) reached the blastocyst stage. A further 24 h culture in vitro finally resulted in 244 (42%) blastocysts. The complete in vitro production system delivered 13% (63/477) blastocysts on Day 7 and 34% (161/477) blastocysts on Day 8. The collection rates (P < 0.001) and the blastocyst rates on Day 7 (P < 0.05) and Day 8 (P < 0.001) differed significantly in all groups. The present data demonstrate that the developmental stage of transferred complexes has an influence on embryo recovery as well as an embryo development. This work was supported by Austrian BMBWK and BMLFUW (#1227).

89 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECT IN THE ULTRASTRUCTURE AND DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 157 ◽  
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. F. Crocomo ◽  
M. D. Guastali ◽  
...  
Keyword(s):  
Lipid Content ◽  
Lipid Droplets ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Culture Methods ◽  
Mitochondrial Number ◽  
Phenazine Ethosulfate

Over the past decades, there have been great advances in in vitro production (IVP) systems, with improved culture methods and new knowledge regarding embryo physiology, ultrastructure and morphology. Currently, the major obstacle associated with the extensive use of this technology is the great sensitivity of IVP embryos to cryopreservation. According to the literature, the reduced cryotolerance of IVP embryos is frequently associated with their high lipid content. Although is not clear until now how the lipid accumulation occurs, it may be influenced by the use of undefined culture media, supplemented with fetal calf serum (FCS); or as a result of embryo energy metabolism abnormalities that affect mitochondrial function, leading to the decrease in both the embryo quality and survival after cryopreservation. In this context, phenazine ethosulfate (PES), a reducer of NADPH electrons, which favours pentose–phosphate pathways and also inhibits the fatty acids synthesis, has been used to increase IVP embryo cryotolerance (Sudano et al. 2011 Theriogenology 75, 1211–1220). The aim of the present study was to evaluate the phenazine ethosulfate and FCS effect in the ultrastructure of IVP bovine embryos. A 2 × 2 factorial experiment design was used to test 2 FCS concentrations (0 or 10%) and the addition of PES (without or with PES) in the culture media. Slaughterhouse ovaries were used to obtain oocytes which were matured and fertilized in vitro (Day 0). Presumptive zygotes (n = 1440) were divided in 4 culture media: SOFaa without FCS; SOFaa without FCS + 0.3 μM PES (started on Day 4); SOFaa + 10% FCS; SOFaa + 10% FCS + 0.3 μM PES (started on Day 4). Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2 and 90% N2). Transmission electron microscopy (TEM) was performed on Day-7 blastocysts from each group (n = 5) through standard protocol. For the statistical analysis, the arcsine transformation was applied to blastocyst percentage data and submitted to the ANOVA, followed by Tukeys' test through PROC GLM (SAS Institute Inc., Cary, NC, USA). In the absence of significant interactions, only main effect means are presented. The blastocyst production was not affected (P = 0.47) by the use of PES (42.7 ± 3.2 vs 39.3 ± 3.2, respectively for control and PES Day 4). The addition of 10% of FCS increased (P < 0.0001) the percentage of blastocysts (48.9 ± 3.2 vs 33.0 ± 3.2, respectively, for 10% and 0% of FCS). The ultrastructure analysis showed similar features in embryos from all studied groups. However, embryos cultured in the absence of FCS presented fewer and smaller lipid droplets. Moreover, embryos cultured without FCS presented more cellular debris in the perivitelinic space and in the blastocoele, indicating loss of blastomeres. The use of PES was able to reduce lipid droplets and increase the mitochondrial number in serum-produced embryos. Therefore, the PES decreased lipid content and increased mitochondrial number without affecting the development and ultrastructure of IVP bovine embryos. FAPESP 09/54513-3, 10/09922-0.

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

1998 ◽  
Vol 49 (6) ◽  
pp. 1239-1249 ◽  
Author(s):  
J.G Thompson ◽  
N.W Allen ◽  
L.T McGowan ◽  
A.C.S Bell ◽  
M.G Lambert ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Bovine Embryo ◽  
Development In Vitro

140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
I. P. Emanuelli ◽  
E. Razza ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Calf Serum ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Advanced Stages

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O2 and 5% CO2). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Fetal Calf Serum ◽  
Formation Rate ◽  
Calf Serum ◽  
Basic Medium ◽  
Intracellular Camp ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
High Concentration

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.

92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes

2019 ◽  
Vol 31 (1) ◽  
pp. 172
Author(s):  
T. Yamanouchi ◽  
H. Matsuda ◽  
K. Ogata ◽  
Y. Hashiyada
Keyword(s):  
Culture Medium ◽  
Liquid Paraffin ◽  
Calf Serum ◽  
Petri Dish ◽  
Culture Method ◽  
Optimal Number ◽  
Blastocyst Development ◽  
Long Distance

In vitro-produced (IVP) embryos are more easily damaged by cryopreservation than in vivo-derived embryos. Therefore, transportation of fresh IVP embryos in a manner that can maintain viability is necessary. This study was conducted to determine the preferable culture conditions for transport of embryos at 5 days post-insemination (dpi) in 1.5-mL microtubes. Cumulus-oocyte complexes derived from an abattoir were matured and then inseminated with frozen-thawed semen. Presumptive zygotes were cultured in mCR1aa (CR1)+5% calf serum (CS) until use. In Exp. 1, embryos with 5 blastomeres at 5 dpi were randomly assigned to 1 of 3 groups: 25mM Hepes-CR1aa (H-CR1)+5% CS or 25mM Hepes-M199 (H-M199)+5% CS in air, or CR1 in 5% CO2. Embryos were cultured in microdrops overlaid with liquid paraffin in a petri dish for 48h at 38.5°C. In Exp. 2, the optimal number of embryos to culture per microtube was assessed. Presumptive zygotes were cultured in groups of 20, 40, or 80 in 1mL of CR1 covered with liquid paraffin in microtubes in an incubator at 38.5°C in 5% CO2 until 7 dpi. For Exp. 3, culture of embryos in microtubes in a portable incubator was tested. At 5 dpi, 5-cell embryos (n=17 to 36 per microtube) were statically cultured in 1mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48h. The CR1 was pre-equilibrated in an incubator in 5% CO2 for 24h before use. Embryos were harvested from microtubes after 48h and were then cultured in microdrops of CR1 overlaid with liquid paraffin in a petri dish in an incubator at 38.5°C in 5% CO2 until 8 dpi. In Exp. 4, embryos (n=29 to 39 five-cell embryos per microtube) were transported in a portable incubator by land for 1000km over a period of 44h using the same conditions as in Exp. 3. Control embryos were statically cultured in microdrops of CR1 in an incubator in 5% CO2. Statistical analyses were carried out by ANOVA (Exp. 1 and 2), t-test (Exp. 3), or Fisher’s exact test (Exp. 4). In Exp. 1, there was no effect (P&gt;0.05) of culture medium on blastocyst development at 7 dpi (27.6±2.3, 25.7±7.2, and 17.3±2.9% for CR1, H-CR1, and H-M199, respectively). In Exp. 2, blastocyst development at 7 dpi was not affected (P&gt;0.05) by the number of presumptive zygotes cultured per microtube (43.6±8.3, 42.4±4.0, and 39.9±2.9% for 20, 40, and 80 presumptive zygotes, respectively). In Exp. 3, blastocyst development at 8 dpi was not affected (P&gt;0.05) by culture medium (60.7±7.4 and 53.1±4.4% for CR1 and H-CR1, respectively); however, the pH of CR1 changed from 7.5 to 8.1 at 48h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P&gt;0.05) by transport (57.1, 64.4, and 75.5% for CR1, H-CR1, and control, respectively). These results indicate that IVP embryos harvested at 5 dpi can be transported by portable incubator with no effect on embryo development to the blastocyst stage. This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and the development for next-generation technology).

Sign in / Sign up

Export Citation Format

Share Document