Pentobarbital Sodium

At the end of the experimental period, rats were anesthetized with pentobarbital sodium, and blood was immediately collected through cardiac puncture. Rats were euthanized, and the soleus muscles were excised, weighed, and used for ex vivo collection of muscle EVs (right) or dissected, weighed, frozen in liquid nitrogen, and stored at − 80 °C for later biochemical analyses (left). For immunohistochemistry (IHC) analyses, the soleus muscles were covered in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen-cooled isopentane, and stored at − 80 °C. The soleus muscles were used in these experiments because in rats, the soleus, which is almost exclusively type I, is especially susceptible to hindlimb suspension-induced muscle atrophy [25 (link), 26 (link)]. Furthermore, the smaller size of the soleus limits issues of oxygen diffusion during ex vivo muscle assessments [27 (link)]. The gastrocnemius muscles were used for single-cell RNA sequencing (scRNA-seq) as described in [28 (link)]. The serum was isolated by allowing blood to clot at room temperature for 30 min before centrifugation for 10 min at 2000g at 4 °C. The serum supernatant was collected and stored at − 80 °C until analysis. The number of animals used in each experiment is listed in the figure legends.

At the conclusion of the tick observations on day 8 post-attachment, fresh fecal samples were collected from each test deer pen. Additionally, internal tissues were collected from each deer in each treatment group. The deer were first sedated by injection of 1–2 mg/kg xylazine hydrochloride (100 mg/ml) into the large muscle bellies of the rump/rear limbs. While sedated, deer were euthanized by intravenous injection, administered via the jugular vein, of 86 mg/kg Euthasol (pentobarbital sodium, 390 mg/ml), resulting in pentobarbital sodium overdose. Death was confirmed by a combination of the following: (i) lack of heartbeat based on auscultation with a stethoscope; (ii) lack of respiration based on visual inspection of the thorax; (iii) lack of corneal reflex; and (iv) lack of response to firm toe pinch. All euthanasia was performed by the attending veterinarian exclusively.
Various tissues were collected from euthanized deer. The objective was to collect tissues similar to what would be collected by hunters when field dressing a killed deer. Thus, we focused on specific meat cuts, meat by-products and fatty tissues. Approximately 50 g of each tissue was surgically removed using disposable scalpels. Scalpels and surgical gloves were replaced between each individual tissue collection to minimize the risk of contamination. Each tissue was transferred to an individual biological specimen bag (Keefitt®), which was immediately stored at − 20 °C until analysis. In addition to collecting tissues from 16 deer in the treatment group, we collected tissues from two deer in the control group to establish a baseline and for analytical method development.
Tissues, plasma and feces were delivered to CSU for method development and analyses, and analyzed for the presence of fipronil and fipronil metabolites using validated methods of liquid chromatography/mass spectrometry (LC/MS). A list of tissue classifications, the maximum residue limits (MRL) listed by the US Environmental Protection Agency (EPA) for fipronil in cattle and the explicit tissue identifications are presented in Additional file 6: Table S2.
Critical study dates for each test deer (acclimation, exposure, post-attachment, capsule checks, tissue collection) are presented in Additional file 7: Table S3.