#METABOLOMICS WORKBENCH laura_lorenzorebenaque_20221205_010452 DATATRACK_ID:3626 STUDY_ID:ST002388 ANALYSIS_ID:AN003892 PROJECT_ID:PR001535 VERSION 1 CREATED_ON December 5, 2022, 1:09 am #PROJECT PR:PROJECT_TITLE Metabolomics in Small-spotted catshark reproduction PR:PROJECT_SUMMARY This study aimed to elucidate the influence of the environment (wild vs. PR:PROJECT_SUMMARY aquarium) on the metabolic signatures in the seminal plasma and peripheral blood PR:PROJECT_SUMMARY plasma in small-spotted catshark (wild-captured vs. aquarium-housed). PR:INSTITUTE Universidad San Pablo-CEU, CEU Universities PR:LAST_NAME Lorenzo Rebenaque PR:FIRST_NAME Laura PR:ADDRESS Ed. Seminario. S/n, Moncada, Valencia, 46113, Spain PR:EMAIL laura.lorenzorebenaque@uchceu.es PR:PHONE 615056012 #STUDY ST:STUDY_TITLE Metabolomics in small-spotted catshark reproduction- Blood plasma No-polar ST:STUDY_SUMMARY this study aimed to elucidate the influence of the environment (wild vs. ST:STUDY_SUMMARY aquarium) on the metabolic signatures in the blood plasma (wild-captured vs. ST:STUDY_SUMMARY aquarium-housed). ST:INSTITUTE Universidad San Pablo-CEU, CEU Universities ST:LAST_NAME Lorenzo Rebenaque ST:FIRST_NAME Laura ST:ADDRESS Ed. Seminario. S/n, Moncada, Valencia, 46113, Spain ST:EMAIL laura.lorenzorebenaque@uchceu.es ST:PHONE 615056012 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Scyliorhinus canicula #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - SO1 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO1.raw SUBJECT_SAMPLE_FACTORS - SO2 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO2.raw SUBJECT_SAMPLE_FACTORS - SO3 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO3.raw SUBJECT_SAMPLE_FACTORS - SO4 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO4.raw SUBJECT_SAMPLE_FACTORS - SO5 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO5.raw SUBJECT_SAMPLE_FACTORS - SO6 Group:aquarium-housed | Sample:blood plasma RAW_FILE_NAME=SO6.raw SUBJECT_SAMPLE_FACTORS - SS1 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS1.raw SUBJECT_SAMPLE_FACTORS - SS2 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS2.raw SUBJECT_SAMPLE_FACTORS - SS3 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS3.raw SUBJECT_SAMPLE_FACTORS - SS4 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS4.raw SUBJECT_SAMPLE_FACTORS - SS5 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS5.raw SUBJECT_SAMPLE_FACTORS - SS6 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS6.raw SUBJECT_SAMPLE_FACTORS - SS7 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS7.raw SUBJECT_SAMPLE_FACTORS - SS8 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS8.raw SUBJECT_SAMPLE_FACTORS - SS9 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS9.raw SUBJECT_SAMPLE_FACTORS - SS10 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS10.raw SUBJECT_SAMPLE_FACTORS - SS11 Group:wild-captured | Sample:blood plasma RAW_FILE_NAME=SS11.raw #COLLECTION CO:COLLECTION_SUMMARY Blood plasma samples were collected and immediately snap frozen for metabolomics CO:COLLECTION_SUMMARY analysis at the end of experiment CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Samples were collected from a total of 11 wild small-spotted catsharks from the TR:TREATMENT_SUMMARY Mediterranean Sea and 6 aquarium-housed small-spotted catsharks in collaboration TR:TREATMENT_SUMMARY with Oceanogràfic of Valencia. Location (wild vs. aquarium). Wild individuals TR:TREATMENT_SUMMARY were donated from local fisheries at the Valencian Community and were part of TR:TREATMENT_SUMMARY accidental captures intended to commercial fisheries ports at Valencia TR:TREATMENT_SUMMARY (39°26′45″N 0°19′12″O), Jávea (38°47′21″N 0°09′47″E) and TR:TREATMENT_SUMMARY Cullera port (39°09′58″N 0°15′10″O). Mediterranean Sea water TR:TREATMENT_SUMMARY parameters, measured by Valencia buoy (39º52′N0º20′E) were 14.6-19 °C TR:TREATMENT_SUMMARY temperature and 34-37 g/l salinity TR:TREATMENT_SUMMARY (http://www.puertos.es/es-es/oceanografia/Paginas/portus.aspx). Small-spotted TR:TREATMENT_SUMMARY catsharks under managed care were housed at Oceanogràfic of Valencia, Spain, TR:TREATMENT_SUMMARY in closed and recirculation system under controlled conditions, monitored water TR:TREATMENT_SUMMARY quality (17–21°C, 5.1 mg/l oxygen, 36 g/l salinity and 7.6–8.2 pH), fixed TR:TREATMENT_SUMMARY photoperiod (12:12 h) and disinfection using UV light and ozone. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Blood plasma were subjected to a comparative metabolomics analysis. For SP:SAMPLEPREP_SUMMARY aquarium-housed individuals’ 7 samples of both types (Seminal plasma and SP:SAMPLEPREP_SUMMARY peripheral blood plasma), were analysed to study the semi-polar and non-polar SP:SAMPLEPREP_SUMMARY fractions. For wild-captured individuals’ 12 samples of both types (Seminal SP:SAMPLEPREP_SUMMARY plasma and peripheral blood plasma), were analysed to study the semi-polar and SP:SAMPLEPREP_SUMMARY non-polar fractions. For semi-polar analysis, metabolites were extracted from SP:SAMPLEPREP_SUMMARY 100 μL of each Seminal plasma or peripheral blood plasma samples following a SP:SAMPLEPREP_SUMMARY published method with a little modification (Y. Yu et al., 2021). Briefly, SP:SAMPLEPREP_SUMMARY samples were dissolved in 200 μL of cold aqueous methanol (75 %), and 200 μL SP:SAMPLEPREP_SUMMARY of acetronitrile (75 %), spiked with 10 μg/ml formononetin as internal SP:SAMPLEPREP_SUMMARY standard. Then, the mixture was centrifugated at 20,000 xg for 15 min at 4 °C. SP:SAMPLEPREP_SUMMARY The supernate (200 μL) was gained, and dried under low-temperature vacuum SP:SAMPLEPREP_SUMMARY (Thermo Scientific, USA). The samples were redissolved resuspended with 100 μL SP:SAMPLEPREP_SUMMARY of methanol (10 %) and transferred to HPLC tubes and an aliquot of 3 μL was SP:SAMPLEPREP_SUMMARY injected for the analysis. For no-polar analysis, metabolites were extracted SP:SAMPLEPREP_SUMMARY from 50 μL of each Seminal plasma or peripheral blood plasma samples following SP:SAMPLEPREP_SUMMARY a published method with a little modification (Y. Yu et al., 2021). Briefly, SP:SAMPLEPREP_SUMMARY samples were dissolved in 300 μL of cold aqueous methanol (100 %), spiked with SP:SAMPLEPREP_SUMMARY 50 μg/ml alpha-tocopherol acetate as internal standard. Then, the mixture was SP:SAMPLEPREP_SUMMARY swirled for 120 seconds, and 900 μL MTBE and 250 μL ultrapure water were SP:SAMPLEPREP_SUMMARY added. After vortexed for 15 mins, the mixture was placed 30 min at 4 ℃. Then, SP:SAMPLEPREP_SUMMARY the supernatants (900 μL) were gained, and dried under low-temperature vacuum SP:SAMPLEPREP_SUMMARY (Thermo Scientific, USA). The samples were redissolved resuspended with 600 μL SP:SAMPLEPREP_SUMMARY of acetonitrile–isopropanol mixture and transferred to HPLC tubes and an SP:SAMPLEPREP_SUMMARY aliquot of 3 μL was injected for the analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Phenomenex Luna (100x2.1, 2.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE APCI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For the untargeted metabolomic analysis, Compound Discoverer software MS:MS_COMMENTS (Thermofisher Scientific) was used to identify the peaks, peak filtration, and MS:MS_COMMENTS peak alignment, and performed chromatogram alignment, peak picking, and public MS:MS_COMMENTS database (e.g., ChemSpider, KEGG, Metabolika) querying based on accurate masses MS:MS_COMMENTS (m/z). MS:MS_RESULTS_FILE ST002388_AN003892_Results.txt UNITS:m/z ratio Has m/z:Yes Has RT:Yes RT units:Minutes #END