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

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).