Processing and treatment of corncob bedding affects cage-change frequency for C57BL/6 mice.

The rodent microenvironment is a primary consideration for animal welfare. The Guide for the Care and Use of Laboratory Animals (The Guide) is the primary reference used for mice in AAALAC-accredited facilities and for those used in studies funded by the Public Health Service; this reference states that the cage environment should be dictated by performance standards, including temperature and humidity and concentration of gases and particulate matter.14 These parameters can alter metabolic and physiologic processes and disease susceptibility.14 Housing density, cage-change frequency, bedding material, and the use of ventilated caging contribute to the microenvironment.37,39 In addition to maximizing space and personnel resources, ventilated racks provide frequent HEPA-filtered air changes, remove excess humidity and waste gases from cages, and reduce disease transmission.8,37 Conversely, as cage density increases and cage-change frequency decreases, waste gases accumulate.39 The Guide states that rodents should be housed with bedding because it allows foraging, burrowing, digging, and nest building and absorbs urine and feces.14 Moreover, the type of bedding selected can influence animal wellbeing and experimental results.11,15,16,33 Several bedding materials are available for rodents, including corncob, wood chips, paper products, and grass fiber pellets. Bedding is evaluated for absorbency, biodegradability, toxicity, dust, palatability, comfort, cost, availability, damage to cage washers, and effect on research.43,50 Untreated softwood bedding can affect rodent metabolism,48,49 and the aromatic hydrocarbons of cedar shavings can induce hepatic microsomal enzymes and cytotoxicity.9,27 Aspen bedding is associated with sneezing and respiratory pathology in rats.7 In a study comparing aspen shavings, virgin pulp, recycled paper, corncob, reclaimed wood pulp, virgin cellulose, pine shavings, and hardwood chips, ammonia was detectable last in corncob.31 Rats have been shown to prefer paper bedding to corncob; however, corncob has been used to minimize dampness and ammonia concentrations.34 Mice prefer material suitable for nest building such as cloth, cotton, or paper.3,17,29,45 Although corncob bedding is not a sterile material,25,26,50 it is a common bedding choice due to its absorbency,26 biodegradability,26 and ability to control ammonia levels.43 In this study, corncob bedding was used as the control for comparison with a new processed corncob bedding material and because it is the most common bedding material used in our facilities. The Guide also states that bedding should be changed frequently enough to keep animals clean and dry and to maintain acceptable concentrations of pollutants, including ammonia.14 The maximal acceptable concentration of ammonia in rodent housing has not been determined definitively; however, the 8-h exposure limit for humans (25 ppm)1 has been used as a guideline.37,39 Although subjective, human perception of cage cleanliness is still a factor in determining the need to change cages despite the lack of correlation between cage appearance and mouse health.39 Intracage ammonia concentration is influenced by temperature, humidity, ventilation, urine, and bacteria. Temperature and humidity are controlled at the room level. Humidity is influenced by bedding material, with highly absorbent materials decreasing the intracage humidity levels. Corncob bedding has been shown to be significantly more absorbent than other products.5 Likewise, in ventilated rodent cages, increased air flow within the cage decreased both humidity and ammonia concentrations.19 Intracage ammonia is produced by the combination of mouse urine and urease-producing bacteria, sources of which may include mouse feces and bedding material. Urine provides the urea substrate for ammonia production. Urease from bacteria catalyzes the conversion of urea to ammonia by cleaving urea to form 2 molecules of ammonia. The initial bacterial content in bedding can be controlled by husbandry practices.37 Autoclaving is a common method of minimizing the bacterial content of rodent bedding. The combination of steam at high pressure sterilizes the bedding material, killing bacteria, viruses, and fungi. Disadvantages of autoclaving bedding include personnel time, autoclave energy consumption and maintenance costs, and potential loss of absorptive capacity of the bedding material. A novel, proprietary processed corncob bedding product (PCC; Green Pet Products, Conrad, IA) has been shown to contain fewer than 10 CFU per gram of Enterobacteriaceae, yeasts, molds, mesophilic spore, and thermophilic spores in the absence of autoclaving.13,24-26 Standard corncob bedding has a variable amount of bacteria, ranging from 23 to more than 1100 coliform CFU per gram of bedding.28,40 Bacteria present in bedding affect lung inflammation and other aspects of mouse health, contributing to stress of the animal.18,30,44 The choice of using PCC as promoted by the distributor can be assessed in terms of energy consumption, institutional cost, and animal stress.2 If bedding does not need to be autoclaved, the associated energy consumption and institutional cost will be eliminated. If data published by the manufacturer indicating that PCC and autoclaved corncob share similar initial bacterial loads are accurate,24-26 then there should be less initial bacteria for production of ammonia and longer intervals until the ammonia threshold is reached. We hypothesized that intracage ammonia levels would be comparable between PCC and autoclaved corncob beddings and that both housing materials would result in an increased cage-change interval. Compared with a standard schedule, a prolonged cage-change interval would expose mice to fewer stressful events, husbandry staff would be exposed to fewer allergens, and facilities would benefit from cost savings. This study was designed to compare the effectiveness of the corncob bedding currently used in our facilities with that of PCC. We determined the number of days required for cages on ventilated and static racks to reach aerosol concentrations of 25 ppm ammonia. Absorptive capacity and bacterial load and species were quantified to fully evaluate and compare these bedding materials. These studies primarily used 1/4-in. corncob bedding with mice because it is a common size used in research; however, 1/8-in. bedding was included in some aspects of the study design to determine whether and how the particle size of the same material affects absorption and bacterial content. These studies demonstrated that using autoclaved corncob or PCC bedding can extend the time required to reach maximal ammonia concentrations, as compared with nonsterile corncob or autoclaved PCC. In addition, corncob bedding is more absorptive than PCC and may therefore contribute to decreased humidity levels within cages.