Male pig urethras share similar physical characteristics with those of adult human males, making them a potentially excellent model for experiments utilising whole tissue. Recently, pig urethras have been used to evaluate the mechanical properties of the urethra, particularly in simulating injuries from trauma or modelling the mechanics of catheterisation. 

However, no data currently exist in the literature regarding the structural and metabolic viability of this pig urethral tissue over time. This is particularly important, as any significant degradation of the tissue over time during an experiment could introduce an uncontrolled variable.

Moreover, with recent advances in our understanding of uropathogen penetration and intracellular biofilm formation using human urinary organoids, a more complete model is needed to further study the mechanisms behind embedded infections. 
This study therefore assesses both the structural integrity and metabolic activity of the male pig urethra over time.

Urethras from four- to six-month-old male pigs (average weight 60kg) were donated immediately after slaughter by a local abattoir. They were then placed at room temperature into a Cell Culture Media Mixture (CCMM) composed of 94% cell culture media (Dulbecco's Modified Eagle Medium), 5% Foetal Bovine Serum, and 1% Penicillin/Streptomycin (10,000 µg/mL) for two hours to sterilise the samples during transport to the laboratory. Structural and metabolic assessments were carried out at 0, 2, 4, 6, 8, and 24 hours.

Structural integrity was assessed by visual inspection under a high-powered light microscope. Tissues were prepared by fixing in formalin, embedding in paraffin wax, sectioning into 4µm slices using a microtome, deparaffinising, and rehydrating through a graded alcohol series before being stained with Haematoxylin and Eosin (H&E).

Metabolic activity was assessed using the PrestoBlue viability assay. At each time point, a section of urethra in CCMM was examined under a light microscope to exclude any concurrent bacterial infection. Then, 10% PrestoBlue was added to the media containing the urethra, as well as to a blank CCMM control, and placed into a BINDER incubator (37°C, 90% humidity, 5% CO2) for 60 minutes. 100µL supernatant was taken from each sample and placed into a plate-well for fluorescence measurement. Readings were taken using a microplate reader, with excitation set at 520nm and emission at 590nm.

The increase in fluorescence intensity is directly proportional to the number of metabolically active cells. Metabolic activity was calculated as: Metabolic activity = metA - metB (metA = sample fluorescence, metB = blank fluorescence).

The male porcine urethra demonstrated a gradual but diminishing loss of metabolic activity over 24 hours. 50% viability was reached at hour 7. However by 24 hours, 42% of metabolic activity remains. (Figure 1)

H&E histological analysis showed no loss of structural integrity at each time point except for the loss of the urothelial layer over time. (Figure 1)

The observed decline in metabolic activity (50% at 7 hours, 42% at 24 hours) is likely linked to the loss of the urothelium, as seen histologically. Despite this, the urethra's overall structure, including the crucial inner and outer muscularis layers, remained intact over the 24-hour period. 

This suggests that even with reduced metabolic function, the tissue remains viable ex vivo and suitable for longer experiments. This is particularly relevant for studies investigating the time course of uropathogen infection, which require bacterial growth and tissue penetration.

The male porcine urethra, with its preserved structure and metabolic activity, represents a valuable model for advancing research on the human male urethra.