106 THE WARMING PROCEDURE: A FIRST STEP FOR IMPROVING THE NONSURGICAL DEEP INTRAUTERINE TRANSFER OF SOPS-VITRIFIED PORCINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 165
Author(s):  
J. Gomis ◽  
C. Cuello ◽  
J. Sanchez-Osorio ◽  
M. A. Gil ◽  
I. Parrilla ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Petri Dish ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Viability ◽  
Step Method ◽  
One Step ◽  
The One

We previously reported successful nonsurgical deep intrauterine embryo transfer (ET) of fresh in vivo–derived porcine embryos. However, several trials from our laboratory demonstrated that when this procedure was used in combination with vitrified/warmed (VW) embryos, its efficiency was very low. Recently, we have shown that the one-step warming method in syringe, which was used in the earlier trials, compromises the in vitro embryo viability. The aim of this study was to confirm the negative effect of the direct warming in syringe and to evaluate the effect of alternative warming procedures on the in vivo development of VW embryos after nonsurgical ET. In Experiment 1, morulae and early blastocysts were collected on Days 5 to 6 (Day 0: onset of oestrus) and assigned to one of the following groups: 1) syringe group: vitrified embryos (n = 88) were warmed by the one-step method directly in a 1-mL syringe containing 300 μL of warming medium, which was connected to the ET catheter and then transferred to recipients (n = 6); 2) dish group: vitrified embryos (n = 194) were warmed with one-step warming method in a Petri dish containing 1 mL of warming medium, loaded into a Tom Cat catheter and transferred to recipients (n = 13); and 3) control group: fresh embryos (n = 129) were loaded in a 1-mL syringe in 100 μL of transfer medium and transferred to 9 recipients. An average of 15 embryos were transferred to each recipient on Day 4 or 5. Embryos were surgically recovered 24 h after ET. Data were analysed by ANOVA. The embryo recovery rate was similar among groups (range: 70.7 ± 4.8% to 77.2 ± 6.5%). The embryo survival (ES) and the hatching rate (HR) from the control group (94.0 ± 2.1% and 33.4 ± 7.6%, respectively) were higher (P ≤ 0.05) than those from the dish group (80.4 ± 4.6% and 14.5 ± 4.1%, respectively). All embryos from the syringe group were degenerated. Some viable recovered embryos (n = 135) were cultured for 48 h to evaluate their subsequent in vitro development. No differences were observed in ES between the control and the dish group (100.0 ± 0.0% vs 98.9 ± 1.0%). The HR in the control group (71.5 ± 2.1%) was higher (P ≤ 0.01) than that of the dish group (42.7 ± 6.1%). In experiment 2 we evaluated the reproductive performance of naturally cycling recipients after nonsurgical ET of vitrified embryos warmed with the one-step method in a Petri dish. An average of 35 VW morulae and blastocysts were transferred to each recipient (n = 10) on Days 4 to 6. Four recipients returned to oestrus at Days 21 to 22. The remaining 6 recipients were diagnosed pregnant by ultrasonography on Day 26. At Days 50 to 55, 5 recipients remained pregnant. In conclusion, the one-step in syringe warming method for vitrified porcine embryos had a completely adverse effect on the vivo viability, whereas nonsurgical ET of embryos warmed in a Petri dish allowed us to obtain promising reproductive performance. Supported by MICINN (AGL2009-12091) and SENECA (GERM 04543/07).

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

Fluorescent carbon dots from antineoplastic drug etoposide for bioimaging in vitro and in vivo

2017 ◽  
Vol 5 (38) ◽  
pp. 7796-7800 ◽  
Author(s):  
Bin Wu ◽  
Rongrong Zhu ◽  
Mei Wang ◽  
Peng Liang ◽  
Yechang Qian ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Antineoplastic Drug ◽  
Step Method ◽  
One Step ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Carbon dots for bioimaging in vitro and in vivo were synthesized from the antineoplastic drug etoposide by a one-step method.

G-CSF Activated Granulocytes Inhibit Experimental Graft-versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4989-4989
Author(s):  
Zilton F.M. Vasconcelos ◽  
Julia Farache ◽  
Bruna M. Santos Grad ◽  
Tereza S. Palmeira Grad ◽  
Luis Fernando Bouzas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Activity ◽  
Control Group ◽  
Low Density ◽  
Graft Versus Host ◽  
The One

Abstract Acute Graft versus host diseas (aGVHD) is a major complication of stem cell transplantation. The disease is mediated by T cells and a higher incidence/severity would be expected when higher numbers of T cells are inoculated. However, the incidence of aGVHD in PBST, which carries about 10 times more T cells then BMT, is not higher than the one found in later. This finding indicates a modulatory role for G-CSF over T cell activity. We had previously shown that T cells from G-CSF treated PBSC donors do not produce g-IFN nor IL-4 and that this inhibition was mediated by low density, G-CSF activated, granulocytes. In order to test if in fact G-CSF activated granulocytes could inhibit disease, we first checked if G-CSF could generate low density granulocytes, in vivo and in vitro. Indeed, either in vivo(21mg /day - 5 days) or in vitro (150 ng -12hs) with G-CSF generates low density granulocytes which co-purify with the mononuclear cells in the ficoll® gradient. Moreover, as we had shown in humans, these low density cells, inhibit the production of g-IFN by anti-CD3 activated T cells on flow cytometry studies (17%-T cells alone versus 3% T cells with granulocytes 1:1). Radiation quimaeras were set with (B6 X BALB/c)F1 as hosts reconstituted with T cell depleted C57Bl6 bone marrow, in the presence or absence of nylon wool selected spleen cells (NWSC), as T cell source, from normal or G-CSF treated mice. As previously shown by others, NWSC from G-CSF treated mice diminishes the incidence of acute disease on day 20 post-transplant, from 75 to 25%. In order to investigate if this inhibition was dependent on the activated granulocytes present in the NWSC from G-CSF treated mice, granulocytes were depleted with anti-GR1 and complement. In this case, the incidence of disease is the same or even higher (75% experiment#1 and 100% in experiment #2) than the one observed on the control group (NWSC from control mice). These results strongly suggest that activated granulocytes could indeed inhibit aGVHD. We then generated activated granulocytes in vitro, by treating spleen derived high density granulocytes with 150ng of G-CSF for 12 hs. After the incubation period, a new ficoll® gradient was performed and the low density cells were obtained. T cell contamination on the second gradient was eliminated by anti-CD4 and CD8 complement lysis. These activated granulocytes were inoculated together with NWSC from control mice in the radiation quimaeras at a 1:1 ratio. In this case 100% disease inhibition was observed when compared to the positive control group, where 75% of the animals got sick. Our data indicate that activated granulocytes are the major mediators of the G-CSF immunossupressive effects and that these cells can be used as a novel immune modulator in clinical transplantation to prevent acute GVHD.

scholarly journals Brief Vibrotactile Stimulation Does Not Increase Cortical Oxygen Consumption When Measured by Single Inhalation of Positron Emitting Oxygen

1999 ◽  
Vol 19 (3) ◽  
pp. 260-265 ◽  
Author(s):  
Shinsuke Ohta ◽  
David C. Reutens ◽  
Albert Gjedde
Keyword(s):  
Blood Flow ◽  
Oxygen Consumption ◽  
Sensory Cortex ◽  
Vibrotactile Stimulation ◽  
Step Method ◽  
Brain Oxygen ◽  
One Step ◽  
The One ◽  
Stimulation Of

Vibrotactile stimulation of the hand elicits no increase in oxygen consumption commensurate with the increase in blood flow measured in human sensory cortex. To test the hypothesis that previous failures to detect a proportionate increase in oxygen consumption could be an artefact of the sequential bolus, or three-step, method used to measure this parameter in the human brain in vivo, the authors compared the measurements with the results of a novel single bolus, or one-step, method of measuring oxygen consumption. The time of completion of the three-step method was 40 to 50 minutes, whereas the one-step method lasted only 3 minutes. The baseline whole-brain oxygen consumption averaged 185 ± 32 μmol hg−1 min−1 by the three-step method and 153 ± 15 μmol hg−1 min−1 by the one-step method. Vibrotactile stimulation did not elicit a significant increase in oxygen consumption measured by either method. This finding rejects the hypothesis that failure to detect an increase of oxygen consumption could be an artefact caused by limitations of the method used previously. Conversely, it also rejects the hypothesis that observations of an increase of oxygen consumption by the new method are artefacts caused by limitations of the one-step method.

Astatine-211 labelled a small molecule peptide: specific cell killing in vitro and targeted therapy in a nude-mouse model

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Weihao Liu ◽  
Yu Tang ◽  
Huan Ma ◽  
Feize Li ◽  
Yingjiang Hu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Cell Killing ◽  
The Body ◽  
Control Group ◽  
Specific Cell ◽  
Step Method ◽  
Nude Mouse Model ◽  
Tissue Samples

AbstractExtensive interest in the development of α-emitting radionuclides astatine-211 (211At) stems from the potential superiority for the treatment of smaller tumors, disseminated disease, and metastatic disease. VP2, a small molecule fusion peptide, can specifically bind to the VPAC1 receptor which is over-expressed in malignant epithelial tumors. In our recent study, we performed the preparation of 211At labelled VP2 through a one-step method. In this work, we explored the targeted radionuclide therapy with [211At]At-SPC-VP2 in vitro and in vivo. The cytotoxicity and specific cell killing of [211At]At-SPC-VP2 were evaluated using the CCK-8 assay. Compared with the [211At]NaAt, the VPAC1-targeted radionuclide compound [211At]At-SPC-VP2 showed more effective cytotoxicity in vitro. Targeted radioactive therapy trial was carried out in non-small-cell lung cancer (NSCLC) xenograft mice. For the therapy experiment, 4 groups of mice were injected via the tail vein with 370 kBq, 550 kBq, 740 kBq, 3 × ∼246 kBq of [211At]At-SPC-VP2, of which the second and third injections were given 4 and 8 days after the first injection, respectively. As controls, animals were treated with saline or 550 kBq [211At]NaAt. The body weight and tumor size of mice were monitored before the administration and every 2 days thereafter. Cytotoxic radiation of partial tissue samples such as kidneys, liver and stomach of mice were assessed by immunohistochemical examination. The tumor growth was inhibited and significantly improved survival was achieved in mice treated with [211At]At-SPC-VP2, two-fold prolongation of survival compared with the control group, which received normal saline or 550 kBq [211At]NaAt. No renal or hepatic toxicity was observed in the mice receiving [211At]At-SPC-VP2, but gastric pathological sections showed 211At uptake in stomach resulting in later toxicity, highlighting the importance of further enhancing the stability of labelled compounds.

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application

1991 ◽  
Vol 37 (7) ◽  
pp. 1235-1244 ◽  
Author(s):  
Gabriëlle E Ponjee ◽  
Huib L Vader ◽  
Net J de Wild ◽  
Ger W T Janssen ◽  
Fedde van der Graaf
Keyword(s):  
Partial Thromboplastin Time ◽  
Clinical Laboratory ◽  
Intrinsic Factor ◽  
Activated Partial Thromboplastin Time ◽  
Factor Vii ◽  
Factor Deficiency ◽  
One Step ◽  
The One

Abstract We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.

scholarly journals Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

2015 ◽  
Vol 27 (1) ◽  
pp. 114
Author(s):  
J. N. Caamaño ◽  
E. Gómez ◽  
B. Trigal ◽  
M. Muñoz ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Survival Rates ◽  
Field Conditions ◽  
Control Group ◽  
Embryo Survival ◽  
Grant Support ◽  
One Step ◽  
The One ◽  
New System

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

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