129 Embryo Production from Fully Grown and Growing Stage Oocytes in Japanese Black Calves

2018 ◽  
Vol 30 (1) ◽  
pp. 204
Author(s):  
S. Matoba ◽  
K. Takeda ◽  
Y. Ohkubo ◽  
M. Hirako ◽  
Y. Hirao

Before fattening of Japanese Black female calves, the ovaries are sometimes removed and discarded. Production of embryos from the oocytes residing in such ovaries is beneficial for the rescue of genetic resources. The aim of this study was to establish an embryo production system using oocytes collected from the ovaries of calves just before fattening and to investigate the correlation between the developmental competence of oocytes and the onset of puberty. Ovaries were collected from Japanese Black calves (9.5 ± 0.1 months old, n = 30, 3 replicates) in a fattening farm and separated according to the presence or absence of corpus luteum as the indicator of puberty (CL+ and CL– groups, respectively). Immature fully grown oocytes (IM oocytes), ~120 μm in diameter, were aspirated from follicles of 2 to 6 mm in diameter (CL+; n = 132, CL–; n = 41) and cultured for 22 to 23 h for maturation (IVM). After in vitro fertilization (IVF) for 6 h (designated Day 0), the oocytes were cultured for 9 days (in vitro culture, IVC) (Matoba et al. 2014 J. Dairy Sci. 97, 743-753). Growing oocytes, ~100 μm in diameter, were also collected by dissecting the follicles smaller than 1 mm in diameter. The growing oocytes were cultured for 14 days on membrane inserts for in vitro growth (IVG) (Hirao et al. 2013 Biol. Reprod. 89, 1-11). Then, IVG oocytes (CL+; n = 29, CL–; n = 32) were subjected to IVM, IVF, and IVC. Presumptive zygotes were cultured individually in microwells in culture dishes. Mitochondrial DNA (mtDNA) copy numbers (COX1 gene) of oocytes were examined (Takeda et al. 2010 Mitochondrion 10, 137-142). A comparison was made between the oocytes derived from calf ovaries and those of oocytes collected from cow ovaries by transvaginal ovum pick-up or by aspiration of the ovaries obtained at a local slaughterhouse. In IM oocytes, the rate of embryos developed to the blastocyst stage on Day 7 to 9 was higher in the CL+ group than in the CL– group (38.9 ± 2.6 v. 10.0 ± 7.1%, respectively; P < 0.05, t-test). However, IVG oocytes were compared, there was no significant difference in the blastocyst formation rate between the CL+ or CL– groups (34.7 ± 5.2 v. 19.8 ± 10.1%, respectively). The mtDNA copy numbers of matured oocytes were similar between IM and IVG oocytes irrespective of the maturity of the donor animals. In conclusion, we demonstrated the possibility of embryo production by IVM/IVF/IVC using fully grown and growing oocytes that are present in the ovaries of calves before fattening. Puberty positively affected the developmental competence of IM oocytes but the effect was not significant in IVG oocytes. Utilisation of both fully grown oocytes and growing oocytes may double the chance of rescuing genetic resources of high-breeding-value calves. This study was partly supported by grants from the Ito Foundation. We thank staff at Mie-Katoubokujou for allowing access to calves’ ovaries.

2014 ◽  
Vol 26 (1) ◽  
pp. 191
Author(s):  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
E. Kim ◽  
...  

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.


2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.


2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.


2012 ◽  
Vol 24 (1) ◽  
pp. 191
Author(s):  
K. Imai ◽  
S. Sugimura ◽  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  

More than 300 000 embryos have been transferred all over the world (Stroud 2010 IETS Newsl. 27(4), 11–21). We have reported that embryos that showed the abnormal cleavage pattern at the first cell division can develop to the blastocyst stage (Somfai et al. 2010 J. Reprod. Dev. 56, 200–207). However, we have limited knowledge about the consequences of the pattern of first embryonic cleavage on their post-transfer developmental competence. The present study was conducted to determine the developmental competence of bovine blastocysts showing different cleavage patterns at their first cell division. Cumulus–oocyte complexes were collected by ovum pickup from Japanese Black cows and were subjected to in vitro maturation and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl). Inseminated oocytes were cultured in CR1aa medium supplemented with 5% calf serum covered by mineral oil at 38.5°C in 5% CO2 in air with micro-droplets or 5% CO2, 5% O2 and 90% N2. The kinetics of embryo development were analysed by time-lapse cinematography for 168 h after IVF by using a Cultured Cell Monitoring System (CCM-M1.4ZS, Astec, Fukuoka, Japan). A total of 673 photographs of each embryo were taken (1 photograph in every 15 min) during in vitro culture. Image stacks were analysed by the CCM-M1.4 software. Embryos were classified in 5 groups according to the pattern of first cleavage as normal cleavage (NC), direct cleavage from 1 cell to 3 to 4 blastomeres (3–4BL), unequal blastomeres (UB), multiple fragments (MF) and protrusion formation (PT). Blastocysts developing from each group were transferred into the ipsilateral uterine horn of each synchronized recipient on Day 7 or 8 after oestrus. Data on conception at Day 60, abortion and delivery were then recorded. Data were analysed by chi-square test and Student's t-test. In total, 43 embryos were transferred, 17 conceptions (39.5%) were established and 16 recipients (94.1%) were delivered. Only 1 abortion was detected at Day 223 in the NC group. The highest conception rate was observed in the NC group (55%, n = 20) and the 3–4BL (n = 12), UB (n = 6) and PT (n = 3) groups showed similar conception rates of 33.3% (1 implanted embryo belonged to 2 classes in UB and PT) and none of the embryos derived from the MF group (n = 3) could cause conception. There was a significant difference (P < 0.05) in conception rates between the NC group and totals of each of the other cleavage groups. No significant difference was found in gestation lengths and birth weights between the NC group (282.2 ± 4.4 days, 30.6 ± 3.8 kg, respectively) and totals of each of the other cleavage groups (282.8 ± 5.3 days, 30.3 ± 1.9 kg, respectively). These results indicate that embryos showing abnormal cleavage patterns at first cell division can develop to normal calves with normal gestation lengths and birth weights; however, their post-transfer viability is lower than for NC embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.


2014 ◽  
Vol 26 (1) ◽  
pp. 158
Author(s):  
J.-H. Shang ◽  
H.-Y. Zheng ◽  
C.-Y. Yang ◽  
F.-X. Huang ◽  
B.-Z. Yang ◽  
...  

The efficiency of oocyte maturation and embryo production in vitro in buffalo is relatively poor when compared with that in cattle. The percentage of oocytes selected by pb1 (the 1st polar body) presence for somatic cell nuclear transfer (SCNT) ranged from 50 to 70% in our laboratory, which meant that 30 to 50% oocytes have been abandoned. The present study was designed to identify the effect of cumulus cells removal and pb1 presence or absence before the IVF of matured buffalo oocytes on cleavage rate and subsequent embryo development and to try to reuse those oocytes without pb1 for embryo in vitro production. In vitro-matured oocytes enclosed with cumulus cells were randomly selected and denuded mechanically, then the denuded oocytes (DO) were divided into 3 groups by non-selection (pb1 ± ), selection of pb1 presence (pb1+) and absence (pb1–). Intact cumulus–oocyte complexes (COC, control) and pb1 ± , pb1+, and pb1– DO (treatments) were inseminated with motile buffalo sperm in Tyrode's medium for 24 h. The presumed zygotes were washed 3 times and transferred into 50-μL droplets of IVC medium (TCM 199 + 10% fetal bovine serum) and co-cultured with buffalo cumulus cells monolayer for more than 10 days to evaluate the developmental ability of embryos. Cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 h and 240 h after fertilization (0 h). The results indicated that CR and BR for COC (61.69 ± 9.22% and 34.07 ± 7.61%) and pb1+ (66.59 ± 15.50% and 35.96 ± 10.87%) were significantly higher (P < 0.01) than those for pb1 ±  (49.11 ± 6.83% and 21.88 ± 8.17%) and pb1– (35.09 ± 9.17% and 13.16 ± 5.38%). In addition, there was a significant difference (P < 0.05) in the CR and BR between pb1 ±  and pb1– but no difference (P > 0.05) between COCs and pb1+ DO. These data show that removal of cumulus cells before IVF significantly reduces the overall developmental competence to cleavage and blastocyst stage and this negative effects mainly caused by the immature oocytes (indicated by the absence of pb1), but there was no effect on mature oocytes (presence of pb1). However, the oocytes without pb1 can still be used for in vitro embryo production even with lower efficiency when compared with intact COC. This research was supported by grants from the National Natural Science Foundation of China (31160456), the Natural Science Foundation of Guangxi, China (2011GXSFB018045, 2013GXNSFAA019075).


2018 ◽  
Vol 30 (1) ◽  
pp. 200 ◽  
Author(s):  
A. Lucas-Hahn ◽  
B. Petersen ◽  
M. Nowak-Imialek ◽  
U. Baulain ◽  
R. Becker ◽  
...  

Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10 IU, pregnant mare serum gonadotropin (PMSG) 10 IU mL−1, fibroblast growth factor (FGF) 40 ng mL−1, leukemia inhibitory factor (LIF) 2000 U mL−1, IGF-1 20 ng mL−1, epidermal growth factor (EGF) 10 ng mL−1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10 IU mL−1 PMSG and hCG, 50 ng mL−1 EGF, 100 ng mL−1 IGF1, and 5 ng mL−1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44 h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000 mL−1) of a multi-transgenic boar (GGTA1-KO/hCD46/hCD55/hCD59/hHO-1/hA20) by co-incubation with the matured oocytes in PGMTac4 medium for 4 h. Zygotes were washed twice and then cultured for 6 days in PZM3 medium. Development to the blastocyst stage was recorded at Day 6 of culture. Blastocysts were fixed and Hoechst33342 stained for counting the nuclei. Each of the experiments was repeated 3 times. In a second step, Day 5 blastocysts derived from the FLI medium were transferred to synchronized pubertal gilts to test the in vivo developmental competence of the IVF embryos. Maturation of oocytes in FLI medium resulted in a significantly higher blastocyst rate (49.3 vs. 13.5; P ≤ 0.001, Chi-squared test) and nuclei number (41.3 ± 12.2 vs. 35.3 ± 10.8; P ≤ 0.001, one-way ANOVA) compared with the standard medium, whereas the cleavage rate was not affected. Transfer of Day 5 blastocysts (average 35 embryos/recipient) derived from the FLI system using 8 recipients resulted in 7 pregnancies (87.5%) as determined by ultrasound scanning on Day 25 of gestation. At the time of writing, one recipient had delivered 5 healthy piglets after a gestation length of 114 days. Results indicate that the FLI medium significantly improves blastocyst rates and the cell number of the resulting blastocysts (Table 1) and yields pig IVF embryos with a high developmental capacity in vivo. By producing high-quality porcine embryos, this FLI-based IVF system provides an efficient method to modify the porcine genome by cytoplasmic microinjection of CRISPR/Cas molecules into IVF-derived zygotes. Table 1.Results of maturation of oocytes in FLI medium compared with DMEM


2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Joanna Romanek ◽  
Joanna Jurkiewicz ◽  
Agnieszka Wierzchoś-Hilczer ◽  
Jolanta Opiela

AbstractThe aim of this study was to examine the effect of thymosin beta 4 (Tβ4) during in vitro maturation (IVM) of oocytes and subsequent embryonic development after in vitro fertilization as well as to assess the quality of obtained blastocysts. COCs were matured in vitro in 4 different media: 1. control medium; 2. control media supplemented with 50 ng/mL Tβ4; 3. control media supplemented with 0.5 mg/mL Tβ4; and 4. control media supplemented with 1 mg/mL Tβ4. The quality of the developed blastocysts was analysed by the TUNEL method. The number of cleaved eggs was significantly higher (P < 0.05) when gametes were matured in the presence of 50 ng/mL Tβ4 than it was using the other types of media. Additionally, the largest number of blastocysts was observed when 0.5 mg Tβ4 was added to the medium (P < 0.05). No significant difference was noted in the mean number of apoptotic nuclei per blastocyst or in the mean number of nuclei per blastocyst in any of the analysed groups. In conclusion, Tβ4 supplementation (50 ng/mL) in maturation medium increased the number of cleaved oocytes, and the number of blastocysts obtained increased when 0.5 mg/mL Tβ4 was used. This positive effect was not observed when higher a higher concentration of Tβ4 (1 mg/mL) was used.


Author(s):  
Fatemeh Montazeri ◽  
Ali Mohammad Foroughmand ◽  
Seyed Mehdi Kalantar ◽  
Abbas Aflatoonian ◽  
Ali Mohammad Khalili

Background and Aims: One of the major subjects for improving in vitro fertilization (IVF) outcome is the quantity and quality of retrieved oocytes. In vitro maturation (IVM) provides an opportunity for using immature oocytes routinely discarded in clinics. This study aimed at evaluating the quality of embryos derived from in vivo and rescue in vitro matured oocytes. Materials and Methods: Totally, 462 immature oocytes as cases and 466 mature (MII) oocytes as controls were included for study of their developmental competence. Oocytes underwent intracytoplasmic sperm injection insemination and then denuded oocytes were microscopically assessed regarding cytoplasmic and nuclear maturity and quality. Results: The morphological assessments showed fertilization rate of 60.9 and 61.4%, the embryo formation rate of 86.7% and 90.9% and arresting rate of 27.3% and 25.6% for the case and control oocytes, respectively. Evaluating embryo quality in the cleavage stage indicated that 63% of the embryos in the case group and 68% of the embryos in the control group were of good quality. There was no significant difference between fertility rate and arresting rate of oocytes matured in both groups, although the embryo formation rate and the quality of embryos differed significantly. Conclusions: Our findings suggest that IVM is a valuable and practical option for patients who had to cancel IVF treatment cycles because of severe responses or resistance to routine hormonal therapies or those with low functional ovarian reserve.


Reproduction ◽  
2003 ◽  
pp. 629-637 ◽  
Author(s):  
M Tamassia ◽  
Y Heyman ◽  
Y Lavergne ◽  
C Richard ◽  
V Gelin ◽  
...  

There have been few studies on a possible maternal influence on in vitro embryo production in cows. The objective of this study was to evaluate the maternal influence on oocyte production and in vitro blastocyst formation rate using repeated ovum pick-up and in vitro fertilization. Six contemporary cows raised on the same farm and with varied genetic origins were submitted to 42 weeks of ovum pick-up organized into four series. Collected oocytes were fertilized in vitro with spermatozoa from a different bull for each series. In total, 1933 oocytes were recovered from 3936 follicles with a recovery rate of 57.2% and a mean oocyte collection of 4.6+/-0.2 (mean+/-SEM) per animal per session. Animals were ranked according to their oocyte production. The best oocyte donor was the same female in all four series. No relationship was identified between oocyte production and blastocyst production rate (r=-0.08). The mean blastocyst rate was 28.8% with significant variation among animals. The best and the worst blastocyst producers were always the same animals independent of the semen used. The results of the present study support the hypothesis that in cattle, the oocyte donor influences the production of blastocysts. Furthermore, they demonstrate that oocyte and embryo production are independent factors. Further studies are necessary to identify the maternal or oocyte factors responsible for such differences.


MedPharmRes ◽  
2018 ◽  
Vol 2 (2) ◽  
pp. 5-20
Author(s):  
Vu Ho ◽  
Toan Pham ◽  
Tuong Ho ◽  
Lan Vuong

IVF carries a considerable physical, emotional and financial burden. Therefore, it would be useful to be able to predict the likelihood of success for each couple. The aim of this retrospective cohort study was to develop a prediction model to estimate the probability of a live birth at 12 months after one completed IVF cycle (all fresh and frozen embryo transfers from the same oocyte retrieval). We analyzed data collected from 2600 women undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) at a single center in Vietnam between April 2014 and December 2015. All patients received gonadotropin-releasing hormone (GnRH) antagonist stimulation, followed by fresh and/or frozen embryo transfer (FET) on Day 3. Using Cox regression analysis, five predictive factors were identified: female age, total dose of recombinant follicle stimulating hormone used, type of trigger, fresh or FET during the first transfer, and number of subsequent FET after the first transfer. The area under the receiver operating characteristics curve for the final model was 0.63 (95% confidence interval [CI] 0.60‒0.65) and 0.60 (95% CI 0.57‒0.63) for the validation cohort. There was no significant difference between the predicted and observed probabilities of live birth (Hosmer-Lemeshow test, p > 0.05). The model developed had similar discrimination to existing models and could be implemented in clinical practice.


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