Skip to main content
Dryad logo

Oxygen measurements in closed system respirometry using blubber explants from suckling and fasting grey seal pups

Citation

Robinson, Kelly; Bennett, Kimberley; Hall, Ailsa (2021), Oxygen measurements in closed system respirometry using blubber explants from suckling and fasting grey seal pups, Dryad, Dataset, https://doi.org/10.5061/dryad.wh70rxwnz

Abstract

Understanding physiological responses of wildlife and domesticated animals to environmental challenges can be difficult or impossible to investigate at the whole animal level. Tissue culture approaches open up experimental possibilities, but are difficult to undertake in remote environments where facilities to assess tissue viability and minimise or monitor microbial contamination may not be available. Here we used planar optodes in closed system respirometry to measure oxygen use by blubber explants from grey seal (Halichoerus grypus) pups, obtained in a remote field environment in 2017 and processed in a field laboratory with minimal equipment. These data show oxygen measurements at timed intervals and demonstrate that the tissues were respiring aerobically for the 24 h measurement period, compared to empty control vials. We also measured glucose, lactate and glycerol levels in culture media after 24 hours to allow correlation of glucose uptake, lactate accumulation and lipolytic rate, which are also useful markers of metabolic activity, with oxygen use data. Nutritional state of the pups (either suckling or fasting) and tissue depth (closer to muscle (inner) or closer to skin (outer)) were recorded to allow effects of tissue characteristics on metabolic parameters to be examined. We were also able to use the oxygen use profiles in explant-containing and control vials to identify those contaminated by microbes well ahead of any visible colour change in the media stocks. The use of planar optodes for oxygen measurement thus allows simultaneous physiological measurements and contamination monitoring to be undertaken in a minimally equipped field laboratory. Robinson et al. (2021) published in Methods in Ecology and Evolution provides the details of tissue collection and tissue culture. These data will facilitate comparison of metabolic activity of adipose tissue within and between species and assessment of efficacy of monitoring microbial contamination in tissue culture experiments, particularly in field laboratories where other methods may not be logistically possible.

Methods

This file contains data from 19 grey seal pups (Halichoerus grypus) captured on the Isle of May, Firth of Forth, Scotland (56°11′N, 2°33′W) during the breeding season (Oct-Dec) of 2017. Individuals were sampled for blubber tissue, which was used to generate blubber explants of both inner (close to animal's core) and outer (close to the skin) tissue that were monitored for a 24 hour period for oxygen consumption in sealed vials. Oxygen sensitive planar optodes (OSPO) were used to measure oxygen use (see usage notes for more details). The culture media was collected after the 24 hour period ended for analysis of glucose, glycerol and lactate. In some vials (n=49 of 103) an experimental exposure of PCBs were added (see usage notes for details) however this treatment had no impact on blubber explants, with no difference in metabolite or oxygen use data from controls. Some animals were sampled during the suckling period (n = 9) and some were sampled their natural postweaning fast (n = 13). Of these, 3 individuals were sampled during both nutritional states. Information on capture, handling and samples taken for this dataset are given in the publication Robinson, K. J., Armstrong, H. A., Moss, S. E. W, Oller, L., Hall, A.J., & Bennett, K. A. (2021). Signs of life: Oxygen sensors confirm viability, measure oxygen consumption and provide rapid, effective contamination monitoring for field-based tissue culture. Methods in Ecology and Evolution (Robinson et al. 2021).

Data were recorded in Microsoft Excel and was imported into R via R studio to allow data exploration and analysis. Oxygen sensors were supplied calibrated by the company that manufactures them, and do not need to be re-calibrated unless they exceed 10,000 reads, which did not occur in this study. Calibration to standardise oxygen reading for each optode at 35 degrees C is described in Robinson et al. 2021. Glucose, lactate and glycerol assays are performed in duplicate on all samples and are detailed in Robinson, K. J., Hall, A., Debier, C., Eppe, G., Thome, J-P., & Bennett, K. A. (2018). Persistent organic pollutant burden, experimental POP exposure and tissue properties affect metabolic profiles of blubber from grey seal pups. Environmental Science and Technology, 52(22), 13523-13534. https://doi.org/10.1021/acs.est.8b04240 (Robinson et al. 2018).

Usage Notes

Each row contains all the data for one vial containing 1.5ml of cell culture media and an oxygen spot sensor that measures oxygen concentrations in the media across a 24 hour period. Some vials contain 100mg explants of adipose tissue from the seal pups sampled (n=70) and some vials contain no tissue to act as controls for this validation study (n=33).

Variable List:

Pup_ID: one of 2 letters followed by 4 digits. Unique identifier for each pup in the study based on PHATS_letter for each year and the year in which it was born. This can be cross referenced with File_1_ile_1_Bennett_Grey_Seal_Pups_2015_to_2017_POPS_mass_TH
PHATS_letter: one or two letters. Pups are assigned a letter alphabetically within each year in the order in which they enter the study, which helps with keeping track of the sampling during the field season.
Vial_ID: Text string containing the pup ID plus an X where the animal was fasted and a unique number for the individual vial or C_ followed by a number for the controls that did not contain explants
Empty_filled: Single word, 'filled' indicates vials that contain explants, 'empty' indicates vials that do not contain explants.
Nutritional_state: Single word, 'feed' indicates vials contain explants from animals that were suckling at the time of sampling, 'fast' indicates vials contain explants from animals that had weaned and were in a fasting state at the time of sampling
Tissue_type: Single word, 'inner' indicates vials contain explants consisting of tissue from the inner portion of the biopsy sample which contains cells close to the body core, 'outer' indicates vials contain explants consisting of tissue from the outer portion of the biopsy sample which contains cells close to the skin
Microbe_contamination: Single word, 'clean' indicates vials that were not suspected to have been contaminated while in the experiment, 'infected' indicates vials that are suspected to have been contaminated while in the experiment
Treatment: Single word, 'control' indicates vials containing cell culture medium with no pollutants added, 'PCB' indicates vials containing cell culture media that has been spiked with 150 ng/mL POP mix (PCB Aroclor standard mix 1 (Sigma-Aldrich), see Robinson, K. J., Hall, A. J., Debier, C., Eppe, G., Thomé, J. P., & Bennett, K. A. (2018) Persistent organic pollutant burden, experimental POP exposure, and tissue properties affect metabolic profiles of blubber from gray seal pups Environmental science & technology 52, no. 22 (2018): 13523-13534 (Robinson et al. 2018))
GLU: number to 6 dp. Glucose removal rate in vials in µmol per 100 mg tissue per hour. Internal quality control measurements lay within < +/− 15%. Intra and inter assay variability for sample analysis was <15% and <10% respectively. For assay details and QC see Robinson et al. 2018 and Bennett, K.A., Robinson, K.J., Armstrong, H.C., Moss, S.E., Scholl, G., Tranganida, A., Eppe, G., Thomé, J.P., Debier, C. and Hall, A.J., (2021). Predicting consequences of POP-induced disruption of blubber glucose uptake, mass gain rate and thyroid hormone levels for weaning mass in grey seal pups. Environment international, 152, p.106506 (Bennett et al. 2021).
LAC: number to 6 dp. Lactate accumulation rate in vials in µmol per 100 mg tissue per hour. Internal quality control measurements lay within < +/− 15%. Intra and inter assay variability for sample analysis was <15% and <10% respectively. See Robinson et al. 2018 and Bennett et al. 2021 for assay details and QC
GLY: number to 6 dp. Glycerol accumulation rate in vials in µmol per 100 mg tissue per hour. Internal quality control measurements lay within < +/− 15%. Intra and inter assay variability for sample analysis was <15% and <10% respectively. See Robinson et al. 2018 and Bennett et al. 2021 for assay details and QC
O2_0Hr: number to 6 dp. Dissolved oxygen concentration in mg/L in the cell culture media in the vials measured by oxygen spot sensor. This column contains pre-explant addition readings, which were all taken at 17 hours after vial creation and 10 hours before explant addition. See Robinson et al. 2021 for further methods details.
O2_1Hr: number to 6 dp. Dissolved oxygen concentration in mg/L in the cell culture media in the vials measured by oxygen spot sensor one hour after explants had been added to begin an experiment. See Robinson et al. 2021 for further methods details.
O2_5Hr: number to 6 dp. Dissolved oxygen concentration in mg/L in the cell culture media in the vials measured by oxygen spot sensor five hours after explants had been added to begin an experiment. See Robinson et al. 2021 for further methods details.
O2_9Hr: number to 6 dp. Dissolved oxygen concentration in mg/L in the cell culture media in the vials measured by oxygen spot sensor nine hours after explants had been added to begin an experiment. See Robinson et al. 2021 for further methods details.
O2_24Hr: number to 6 dp. Dissolved oxygen concentration in mg/L in the cell culture media in the vials measured by oxygen spot sensor twenty-four hours after explants had been added to begin an experiment, and just prior to the experiment ending and all cells and culture media being collected for analysis. See Robinson et al. 2021 for further methods details.
Sensor_ID: Text string containing S and a unique number for the individual sensor ID in the vial used to obtain oxygen concentration. Sensors are oxygen sensitive planar optodes (OSPO), type SP-PSt3-NAU made by Presens (Precision Sensing GmbH). See Robinson et al. 2021 for further methods details.

Missing data codes:
null = not collected for that vial. Only present for glucose, glycerol and lactate values for control vials containing no explants, or vials that were suspected to be contaminated.
NA = Not applicable for that vial type.

Funding

Natural Environment Research Council, Award: SMRU 1001

Natural Environment Research Council, Award: NE/M013723/1

Natural Environment Research Council, Award: NE/M01357X/1