Breathe (Study 2 - in vitro)

Purpose

To quantify effects of Breathe Liquid on contraction of tracheal smooth muscle in vitro

Methods

Extraction of Breathe Liquid: 
Breathe Liquid was sterile filtered (0.2μm) and stored at room temperature until use. 

Tissue collection and tracheal strip preparation:
Ovine tracheal tissue was obtained from the University of Guelph’s meat lab and stored in potassium buffer saline until dissection. The tissue was then dissected and incubated in a 25 mL tissue bath containing Krebs-Heneseleit solution (KHS) and oxygenated with 95% O2 -5% CO2 at 37 °C. One section was hung in the vertical orientation (contraction shortens the length of the trachea) and the second section was hung in the horizontal orientation (contraction shortens the diameter of the trachea). The tissue was incubated in the tissue bath for 45-60 minutes with a preload of 4 g and isometric tensions were recorded by a force transducer connected to a computer data acquisition software. The preload of 4 g was adjusted every 15 minutes during the incubation period to determine a baseline force. After the stabilization period the tissue was contracted by increasing concentrations of acetylcholine (10-7 M to 10-4 M) in 10-minute intervals. The tissue baths were than washed out and replaced with fresh KHS.  Once the tissue stabilized, either KHS (control group) or Breathe Liquid was added to the tissue bath. The tissue was then allowed a 1-hour incubation period. Following the incubation period the tissue was contracted again with increasing acetylcholine concentrations. The wet weight and length of each tracheal smooth muscle extract was then taken and recorded. 

Results

At acetycholine concentration of 10-6 M, tracheal smooth muscle contraction was significantly lower in the Breathe baths compared with control. There was no significant increase in force in the Breathe group at this point, whereas control tissue produced a significant increase in contraction force. There was an increase in contraction force in the control tissue at a 10-fold lower dose of acetylcholine than the Breathe-treated tissue.

Fig 1: Contractile force of tracheal tissue exposed to increasing concentrations of acetylecholine (Ach). * denotes significant effect of Ach within a group. 

Conclusions:

Breathe Liquid reduced contractility of tracheal smooth muscle. This may contribute to the observed effect of the product in promoting healthy respiratory function in horses with heaves.