Effects of 2 preparation methods and long-term storage on structural integrity and bacterial loads of equine amnion

Objective: To determine the influence of tissue preparation and long-term storage methods on structural integrity and risk of bacterial contamination of equine amnion. Study design: Prospective experimental investigation. Sample population: Amniotic membranes from 8 healthy mares (n = 440 tested samples). Methods: Samples for baseline bacteriology and histology were taken after removal of debris. The remaining tissue was divided and processed with 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. Processed amnion samples were assigned to 1 of 9 combinations of storage media (saline, chlorhexidine, acetic acid) and temperature (4 °C, −20 °C, −80 °C). Samples were submitted for quantitative bacteriology and histopathology at 1 week, 4 weeks, and 3, 6, 9, and 12 months. Results: Baseline bacterial levels ranged from <200 to > 150 000 colony-forming units (cfu)/mL. None of the potentially pathogenic bacteria in baseline samples were subsequently cultured throughout the study. Nonpathogenic bacteria (median 20 cfu/mL), most commonly Bacillus sp, were cultured sporadically across storage conditions. Tissue architecture was minimally affected histologically by processing protocol, storage temperature, or storage duration. Conclusion: The 2 processing protocols tested here resulted in minimal bacterial contamination or loss of structural integrity of equine amnion stored for up to 12 months at 4 °C, −20 °C, or −80 °C. Clinical significance: Amnion collected during the foaling season may be stored for up to 12 months without significant bacterial contamination or structural alterations.