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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240017	1/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Follicular Fluid Sample origin Growing follicle Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 173.78 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.93E+09 EM EM-type Transmission-EM Image type Close-up

	EV240017	2/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Follicular Fluid Sample origin Dominant follicle Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 225.76 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.17E+09 EM EM-type Transmission-EM Image type Close-up

	EV240017	3/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Follicular Fluid Sample origin Pre-ovulatory follicle Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 237.41 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.05E+09 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV240017
species	Ceratotherium simum simum
sample type	Follicular Fluid
condition	Growing follicle	Dominant follicle	Pre-ovulatory follicle
separation protocol	dUC/ Filtration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Size-exclusion chromatography (non-commercial)
Exp. nr.	1	2	3
EV-METRIC %	56	56	56