Xylazine Hydrochloride

The mice brains were collected in a separate suite at the same time of the day during their active cycle following four different anesthesia and euthanasia methods: (1) Ketamine/Xylazine: mice were decapitated 45 min following an intraperitoneal injection of 100 mg/kg (#VINB-KET0-7021, Henry Schein Animal Health, Dublin, OH, USA) with 10 mg/kg xylazine hydrochloride supplement (#X1251-1G, Sigma-Aldrich, St. Louis, MO, USA). Serum ketamine levels are highest at 10–20 min post-injection (Ganguly et al., 2018 (link)); therefore to reduce the impact of ketamine on MAPK activity we chose the 45 min time-point, also because that is around the time a perfused brain would be collected. The anesthetic effect of a mixture of 100/10 mg/kg ketamine/xylazine is known to last up to 80 min with reflex suppressions and produces stable heart rates 40 min post-injection in mice (Erhardt et al., 1984 (link); Xu et al., 2007 (link)); thus the mouse is still sedated at our chosen time point; (2) Isoflurane: mice were placed in a plexiglass chamber with 5% isoflurane, USP (#NDC 13985-046-60, VetOne, Boise, ID, USA) for 5 min, and decapitated when fully sedated, as measured by a lack of active paw reflex; (3) Carbon Dioxide Asphyxiation: mice were placed in a new cage with corn cob bedding, and immediately euthanized by displacement of air with 100% carbon dioxide, within 5 min and decapitated for tissue collection; and (4) Decapitation: mice were gently restrained and decapitated in a new cage to minimize the exposure to blood from conspecific mice.

Two topical preparations (10 mg/g and 20 mg/g) were prepared containing the crude ethanolic extract from A. chica leaves mixed with Lanette® cream, as a water-soluble anionic vehicle.
Forty 4-to-6 week old male Swiss Webster mice from the animal facility of the Universidade Estadual do Maranhão (São Luís, Maranhão, Brazil) were used in this experiment. The animals were randomly divided into two groups (n = 20 each). The control group was treated with Lanette® cream and the experimental group was treated with Lanette® cream containing the crude ethanolic extract of A. chica.
For creation of a dorsal skin wound, the animals were anesthetized with an intraperitoneal injection of xylazine hydrochloride (10 to 15 mg/kg) and ketamine hydrochloride (100 to 150 mg/kg). Trichotomy of the dorsal region and demarcation of the skin with a punch was then carried out. Resection of a circular segment of approximately 6 mm diameter was performed, exposing the dorsal fascia. Hemostasis was carried out by digital compression with sterile gauze, with treatment initiated immediately afterwards.
The therapeutic protocol was performed twice a day throughout the experimental period. Transverse and longitudinal diameters of the lesions were measured daily, using a digital caliper, and monitored through photography. Skin wound areas were calculated using the formula A = π x R x r, in which A represents the area, π is equal to 3.14, R represents the largest diameter and r the smallest diameter of the wound.

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.

A total of six mini-pigs (Sus scrofa; Medi Kinetics Co. Ltd., Pyeongtaek, Korea) were used for the in vivo experimental study (Supplementary Figure 1). The mean age of the mini-pigs were 14 months and each subject weighed approximately 30 kg. All animals were quarantined and acclimated in a vivarium for one week before the experiments. They were kept in specific pathogen-free facilities with complete substrate feeding. All animals fasted overnight and were given only water for 24 hours before the endoscopic procedure. Pre-anesthesia sedation consisted of an intramuscular injection of tiletamine/zolazepam (5mg/kg), xylazine hydrochloride (2mg/kg), and atropine sulfate (0.04 mg/kg). The animals were subsequently intubated, and general anesthesia was achieved with 1.5% isoflurane (Foran^®; JW Pharmaceutical Corp., South Korea). During the procedure, heart rate, respiratory rate, and oxygen saturation were monitored continuously. The pigs resumed their usual diet (1000 kcal/day) on the day after the procedure.