Beyond Comfort: A Review of the Critical Role of Bedding Selection in Rodent Welfare, Microenvironment, and Research Reproducibility

Abstract

This review addresses the critical but often underappreciated influence of bedding selection on rodent welfare, cage microenvironment, occupational health, and research outcomes in biomedical studies. Although contact bedding is widely preferred by mice and rats and is the current industry standard, there is considerable variation in bedding type, volume, sterility, and manufacturer practices, with no established universal protocols. We synthesize over 150 publications, evaluating corncob, wood, and cellulose-based beddings with respect to their physical attributes, absorbency, ammonia management, dust and contaminant control, and support of species-typical behaviors. The review highlights methodological challenges and inconsistent results across studies, with absorbency and ammonia accumulation particularly affected by bedding mass, volume, and microbial contamination, rather than material type alone. Occupational health risks, including ergonomic strain and allergen exposure, are impacted by bedding handling frequency and composition. Animal health is generally not strongly influenced by bedding type, although pine shavings and certain corncob products present toxicity and reproductive risks in some studies. Bedding also has documented impacts on hepatic enzyme induction, neurobehavioral development, pain sensitivity assays, and gut microbiome composition, posing substantial risks for experimental confounding and compromised reproducibility. Based on the evidence, cellulose bedding is recommended for minimizing negative outcomes, but the complexity and variability inherent in bedding selection necessitate careful documentation and transparent reporting. We advocate for standardized detail in publications to ensure comparability and rigor across rodent-based research.

Introduction

Housing mice and rats in solid-bottom, shoebox-style cages with contact bedding represents the current standard of care in biomedical research programs.¹ The Guide for the Care and Use of Laboratory Animals specifies that “animals should have adequate bedding substrate and/or structures for resting and sleeping” and that bedding should “dilute and limit animals’ contact with excreta”.² Empirical studies clearly and consistently demonstrate that rodents prefer contact bedding over wire-mesh flooring,^3–6 with evidence of positive affective responses, such as rewarding vocalizations, associated with bedding presence.⁷ Bedding also serves to enhance animal comfort, improves in-cage microenvironmental parameters, and supports species-typical behaviors.^2,8 Conversely, insufficient bedding induces physiologic and behavioral stress responses, including elevated basal corticosterone levels and impaired maternal care, and limited bedding supply is used experimentally to model early-life stress in rodents.^9,10

Although the critical role of bedding in promoting and maintaining rodent welfare is widely acknowledged, no standardized practices currently exist within the research community. Considerable variation occurs across institutions and studies with respect to bedding type, particle size, source, vendor, sterility, and depth; consequently, bedding may be a significant experimental variable. Selection of appropriate bedding material can be complex and may differ depending on the specific research objectives. Although over 150 publications address the influence of bedding on rodent husbandry practices, animal health, and research outcomes, synthesizing this information remains a substantial challenge for the research community. Existing discussions of bedding within textbooks and broader reviews of housing conditions are often outdated, narrow in scope, and lack the level of detail necessary to support data-driven decision-making by institutions and researchers.^6,8,11–20

This review provides a comprehensive synthesis of the available literature with an emphasis on the effects of the most common mouse and rat bedding materials (wood, corncob, and cellulose) on microenvironmental conditions, occupational health, animal welfare, and research outcomes. It also evaluates desirable bedding characteristics, species-specific preferences, and their implications for experimental rigor and reproducibility. The objective of this review is to serve as an inclusive reference for researchers, veterinarians, animal care personnel, and IACUC members, emphasizing the influence of bedding selection on research integrity and the critical importance of accurate documentation in scientific publications.

Considerations in Rodent Bedding Selection

The ideal bedding material should safeguard the health and physical well-being of rodents while supporting the expression of species-typical behaviors such as foraging, digging, burrowing, and nest building.^2,21,22 Ideally, bedding should simultaneously meet the needs of occupational safety, daily husbandry operations, and animal welfare without compromising experimental outcomes.^23–26 In practice, however, no single bedding type satisfies all of these criteria across diverse research applications. Figure 1 summarizes the key attributes of optimal bedding materials from the perspectives of multiple stakeholders.^{15,19,21–25,27}

Figure 1.

Characteristics of Optimal Rodent Bedding Based on Impact.

Synthetic bedding materials have been explored in an effort to develop a product that satisfies the diverse requirements of all stakeholders; however, no synthetic option has yet proven to be uniformly successful, leaving natural resources as the primary materials used for rodent bedding.^28,29 Several natural materials have been deemed unsuitable for research use due to undesirable properties. For instance, hay and straw are edible, dusty, and poorly absorbent; peat moss and newspaper tend to stain; and granulated clay litter has sharp edges and an unfavorable volume-to-weight ratio.^22,25 Additional materials that are rarely used or inappropriate for rodent research applications are summarized in Table 1.^30–47

Table 1.

Characteristics of Nonstandard Rodent Bedding Materials Reported in the Literature

Bedding Material	Reported observations		References
	Benefits	Concerns
Plant-derived bedding
Forage and forage-like materials
Alfalfa	—	High ammonia Presence of phytoestrogens Poor learning outcomes in rats Underperformed in mouse preference tests	30,31,47
Hay (pelleted)	Low levels of volatile organic compounds before and after autoclaving	—	32
Wheat straw	—	Significantly decreased ALT and AST compared with wood bedding in mice	32
Rice straw	—	Significantly decreased ALT compared with wood bedding in rats Significantly increased ALT and AST compared with wood bedding in mice	32
Agricultural byproducts
Banana midrib	No physiologic changes to mice	Ammonia levels exceeded 25 ppm within 3 d in static cages	34
Rice hulls (aka paddy husk)	—	High levels of bacteria, yeast, and mold Low absorbency Increased hepatic enzymes Decreased hepatic glutathione levels	35–37
Spelt	Sustainable, soft, cost-effective	Ingestion led to decreased food consumption by rats	38
Water hyacinth	No physiologic changes in mice	Ammonia exceeded 25 ppm in static cages within 3 d	34
Processed corn cob (Pure Lite; Harlan, Indianapolis, IN)	Less microbial contamination compared with standard corncob Similar ammonia control as autoclaved corncob	Autoclaving decreases ammonia control and increases microbial contamination	23
Wood-derived (uncommon hardwood or processed wood)
Uncommon wood products (walnut, pistachio, almond, apricot, neem)	Rats preferred resting on walnut and almond bedding Improved learning and memory on walnut and almond bedding Enhanced murine local immunity with neem	—	30,37
Recycled spruce (soft hydrothermal processing)	Reduced intra-cage particulates, hepatic enzyme induction, and ammonia levels compared with fresh spruce bedding	—	39
Eucalyptus pulp (unbleached, sterilized)	High water absorption; low dust content	—	40
Processed or manufactured fiber bedding
Compressed cotton fiber	Reduced dextran sodium sulfate sensitivity Improved survival in Notch1/Notch2 mutant mice	—	41
Cotton cloth (Agrebe, Globe Japan, Tokyo, Japan)	Preferred material for nest building (increased dwell time)	Significantly decreased breeding performance when used as sole bedding/nesting material; difficult to observe mice on health examination	42–44
Deodorized cellulose	—	Reduced litter size, lactation, and survival to weaning	45
Mineral/inorganic bedding
Bentonite clay	—	Underperformed in mouse preference tests	31
Perlite (Kaleblokbims, Erzurum, Turkey)	Decreased intra-cage microbial load	Increased oxidative stress; reduced intra-cage moisture leading to severe inflammation and degeneration of epithelial cells	46
Plastic (polycarbonate granules, Zoonlab, Castrop-Rauxel, Germany and polysulfone granules, Tecniplast, Buguggiate, Italy)	Reusable and recyclable; no adverse health concerns reported in mice	Less ammonia control compared with wood chip bedding; less preferred compared with wood chip bedding	29
Synthetic (polyethylene beads; Rio Ammunition, McEwen, TN)	—	Significant health concerns: mice were unkempt, lethargic, wet, and had decreased body weight	28
Vermiculite	—	High dust levels	40

The remainder of this review focuses on the 3 bedding types most used in contemporary laboratory animal facilities: corncob, wood, and cellulose-based substrates.^{13,15,20,26,38,48,49} Each bedding type is available from multiple vendors in various particle sizes, shapes, and blends as summarized in Table 2. All 3 bedding types are manufactured using multistep processes intended to control particle size, reduce dust, and limit contaminants. Although raw materials differ, production typically includes cleaning or debarking, drying, mechanical size reduction, and sieving or aspiration to remove fine particulates and minimize microbial load. These 3 bedding types are widely used due to their broad availability, relatively low cost, high absorbency, and effective odor control.^13,25,38 In addition, they are autoclavable, biodegradable, incinerable, compostable, and derived from renewable resources. However, they also present certain drawbacks: all are edible, may vary in composition depending on source, and can harbor microbial or chemical contaminants.^6,50–52 Softwood beddings such as pine and cedar are less commonly used because of their aromatic compounds, which have been shown to induce hepatic enzyme activity.⁸ The advantages and limitations of each bedding type are outlined in Table 2 and discussed in greater detail in the following sections.

Table 2.

Characteristics of the Most Common Rodent Bedding Materials

Type	Source	Sizes/formats	Benefits	Concerns	Impact on research
Cellulose	Reclaimed wood pulp Pure cellulose Virgin paper Food-grade or recycled paper	Compressed paper chipsa Irregular flakes Pellets	Outperforms others on preference tests Better nesting capabilities Lower endotoxin levels	—	Impacts on T-water maze outcomes
Corn cob	Lignocellulosic coreof maize cobs	¼ in. ⅛ in. Ground	Increased absorbency by volume Increased maternal grooming	Underperforms on preference tests Higher endotoxin levels Variable carbohydrate levels based on harvesting and storage processes	Impacts slow-wave sleep Disrupts reproductive cycles and reduces fertility in rats Attenuates stress response on behavioral tests Increases fasting blood glucose and calorie restricted weight loss Reduces hepatic microsomal protein
Wood	Hardwood (aspen, maple, birch) Softwood (pine, cedar, spruce) Mixed	Sawdust Chips Shavings Flakes	Increased absorbency by mass	Higher endotoxin levels Wood dust is potentially carcinogenic Less ammonia control (with unsterile products) Variation in physical properties by harvest location and storage	Immunostimulatory Impacts on mechanical threshold testing Increases severity of Dahl-sensitive rat phenotype Impacts hepatic enzyme function, drug metabolism, and anesthesia durationb

^a Can be mixed with corncob.

^b Softwood.

Microenvironmental Effects

The type and condition of contact bedding used has a significant effect on a rodent’s microenvironment, making bedding a central factor used in determining cage change frequency.^2,53 The following sections summarize the current literature regarding how bedding affects key microenvironmental parameters, including absorbency, ammonia accumulation, temperature and humidity, airborne dust, and contaminants.

Absorbency.

According to the Guide for the Care and Use of Laboratory Animals, sufficient bedding should be provided “to keep animals dry between cage changes.”² Adequate use of absorbent bedding helps maintain animal health,⁵⁴ stabilizes microenvironmental temperature and humidity,³⁴ and minimizes volatile pheromones that can influence rodent communication and behavior.⁵⁵

Despite its importance, there is no clear consensus in the literature regarding which bedding type exhibits the highest absorbency. Absorbency is influenced by several factors, including the product’s mass, the quantity used,^36,56 and the sterilization methods applied.^2,23

Reclaimed wood pulp (cellulose) bedding has been reported to be less absorbent than virgin cellulose bedding¹⁷; however, this difference is likely attributable to variations in mass and density rather than to the source material itself.⁵⁶ Bedding absorbency is positively correlated with volume but negatively correlated with mass.^36,56,57 When compared on a volume-to-volume basis, corncob bedding tends to exhibit higher absorbency than cellulose or wood chip bedding. In contrast, when comparisons are made on a mass-equivalent basis, cellulose and wood-chip beddings outperform corncob in absorbency.^36,56–59 One study reported that wood shavings were more absorbent than corncob on a volume-equivalent basis.³⁴ This may reflect differences in material density between wood shavings and wood chips; however, this variable has not been systematically documented.

Comparisons of absorption rate across bedding types have also produced inconsistent findings. While one study demonstrated faster absorption with corncob than with cellulose, another reported the opposite, and a third found no difference between corncob and wood shavings.^34,36,59 Direct comparison among these studies is challenging due to important differences in methodology and bedding source.

Selecting bedding with greater absorbency may extend cage-change intervals, especially when visual urine-spotting is used as the trigger.^21,59,60 However, absorbency metrics alone do not reliably predict ammonia accumulation.^34,61–63 Microbial activity appears to have a stronger influence.²³ While autoclaving reduces bacterial load, it also increases moisture, diminishing its absorbency^23,57; post-autoclave drying and cooling mitigate this effect.²

Ammonia.

Ammonia, a colorless gas produced by bacterial urease conversion of urinary urea, can irritate mucous membranes and compromise animal welfare.² Although no specific limits exist for rodents, human exposure limits (25-50 ppm over 8 hours)⁶⁴ often serve as reference values.

Ammonia levels are shaped by husbandry and environmental factors including humidity, cage ventilation, density, cage size, and bedding-change frequency.^1,2,65–73 Bedding sterilization reduces microbial load and therefore urease activity, decreasing ammonia production even in bedding with lower absorbency.²³ When bedding is sterile and bacterial effects are minimized, absorbency becomes the primary determinant of ammonia buildup.^40,59,62

Many studies have investigated the effects of wood, cellulose, and corncob bedding on in-cage ammonia concentrations,^{27,34–36,59,61–63,71,74–81} but results remain inconsistent due to important differences in experimental designs, environmental conditions, and ammonia measurement methodologies.

Increased air exchange in IVCs (individually ventilated cages) is generally associated with lower ammonia concentrations and extended cage-change intervals compared with static cages; however, some studies have reported unexpectedly low ammonia levels in static cages across multiple bedding types.^{40,74,76,82,83} In contrast, other investigations have shown that static cages containing aspen or reclaimed wood pulp bedding exhibit higher ammonia levels than do those containing other cellulose, wood, or corncob based beddings.^34,76,79,84 The relatively slow surface absorption rate of reclaimed wood pulp bedding may contribute to increased ammonia accumulation in static cages.³⁶ Nevertheless, when used in IVC systems, the ammonia control performance of aspen and reclaimed wood pulp beddings is comparable to that of other commonly used bedding materials.^27,71,76

Studies comparing corncob and other cellulose beddings yield mixed results: 1 reported no significant differences, 3 found superior ammonia control with corncob, while 4 other studies indicate that cellulose bedding is more effective under IVC conditions.^{59,61–63,78,81,84} Comparisons of corncob and wood bedding indicate that corncob can reduce ammonia levels when nonsterile sources are used.^34,35 However, when bedding is autoclaved or irradiated, bedding type has minimal influence on ammonia accumulation, highlighting the critical role of bacterial contamination in ammonia production.^27,36,76,79

Accurate ammonia measurement depends strongly on instrumentation. Multi-gas detectors yield the most precise readings, although their use in bedding studies remains limited.⁶⁷ Variations in sampling location, especially in relationship to the distance to latrines, can lead to over- or underestimation, reinforcing the need for multiple sampling points.^27,60 The unexplainably low ammonia readings reported in some studies^{40,74,76,82,83} may be attributable to unaccounted factors such as the location of the measurement point relative to latrines.

Bedding volume and particle size have also been evaluated, but only in a limited number of studies, primarily using aspen or corncob substrates. While one study found bedding volume to be a significant predictor of ammonia levels, all recorded values remained low (<5 ppm), likely due to high air exchange rates in IVC.⁸² Overall, evidence suggests that bedding volume and particle size do not substantially affect ammonia accumulation.^{82,83,85–87}

Temperature and humidity.

Bedding type has negligible impacts on in-cage temperature and humidity levels, with only slight increases in temperature and humidity noted in cages containing cellulose bedding.^{29,35,61,79,84} Bedding quantity, however, can affect humidity due to reduced absorbency with smaller bedding volumes. Cages with 250 g of aspen bedding showed higher humidity levels than did those with 400-500 g.^82,83

Dust.

Only a limited number of studies have compared dust generation across bedding types, and among them, only half assessed in-cage dust levels in the presence of animals. Most found that corncob, cellulose, and processed wood bedding generate similar levels of airborne particulates, both in and out of the cage.^88,89 One study reported higher dust production from corncob compared with wood bedding when measured outside the cage⁵⁷; however, these results may be unreliable due to deviations from recommended measurement techniques.⁹⁰ Bedding volume, rather than material, appears to drive in-cage dust levels, with larger volumes correlating with increased dust accumulation.⁸³

Contaminants.

Nonsterile bedding of all 3 common types may harbor microbial, fungal, and chemical contaminants. Environmental pathogens such as Cladosporium spp., Aspergillus spp., Fusarium spp., Penicillium spp., and aerobic yeasts and bacteria are most commonly detected.^91–95 Less frequently, viral agents and parasitic organisms have also been associated with bedding contamination.^96,97 In addition, natural bedding materials may contain chemical contaminants, including phthalates, volatile organic compounds, and pesticides.^32,98–100

The type and load of microbial contaminants vary widely across bedding types, and findings are inconsistent across studies.^35,88,92,94 One study evaluated bedding from 20 different vendors and observed substantial variability even among similar products.⁸⁸ This variability is influenced by multiple factors, including the source of the bedding, particle size and shape, vendor, processing methods, and storage conditions.^22,88,91,93 Modern bedding sanitation practices, such as gamma irradiation, autoclaving, and high-temperature drying (up to 1,200 °F/648.9 °C), substantially reduce microbial and volatile organic compound contamination.^22,62,88 However, some fungal species may persist despite autoclaving.^{32,88,94,95,101}

Endotoxin, a heat-stable LPS from the outer membrane of Gram-negative bacteria, has also been detected at high levels in wood and corncob bedding,^88,91,92 and at lower levels in cellulose bedding, even after autoclaving.^88,91 While dry-heat sterilization is the preferred method for endotoxin elimination in the pharmaceutical industry,¹⁰² the slow heat transfer and extended sterilization times required render it impractical for sterilizing bulk bedding supplies.¹⁰³

Although sterilization technologies mitigate many risks, regardless of the bedding type, residual microbial and chemical contaminants in the bedding material remain a concern for animal health, research reproducibility, and occupational exposure.^{93–95,98,99}

Occupational Health Implications

Bedding selection affects not only rodent microenvironment and cage-change frequency, but also the safety and ergonomics of animal care staff. More frequent cage changes may increase the risk of repetitive motion injuries, while larger bedding volumes within cages can contribute to ergonomic strain for animal care personnel.²⁶ Back injuries and muscle strains are also reported among animal procurement and cagewash staff responsible for lifting and handling heavy bags of bedding.¹⁰⁴ Manufacturer-reported weights of bedding bags range from 4.5 to 18 kg, with cellulose- and wood-based beddings typically weighing less than corncob bedding.

Procedures that aerosolize bedding (such as cage changes, bedding addition, or disposal) contribute to respiratory and dermal exposures among staff. These exposures may include rodent allergens, dust, zoonotic pathogens, and endotoxins, some of which also carry carcinogenic risk.^{21,88,91–93,104–106} Some wood-based beddings are also labeled with cancer warnings due to wood dust exposure risk.¹⁰⁷

Although relatively few studies have directly assessed the effects of bedding type on occupational health outcomes, several investigations identified bedding composition as a significant determinant of rodent allergen exposure. Cages using absorbent cellulose-based pads or corncob bedding have shown significantly reduced airborne allergen concentrations compared with wood-based beddings, with woodchip bedding showing reduced allergen concentrations compared with wood shavings, although the underlying mechanism remains unclear.^108–110 Allergen concentrations have not been consistently correlated with bedding dust levels, but larger bedding volumes may contribute to increased exposure.^83,92

Rodent Health Monitoring

Rodent colony health monitoring has begun transitioning from traditional live sentinel animal testing to environmental health monitoring approaches, including sentinel-free soiled bedding and exhaust duct testing.¹¹¹ These methods rely on PCR-based detection of nucleic acids obtained from swabs of soiled bedding or dust collected on filter media placed within IVC rack exhaust ducts. Bedding characteristics, such as absorbency and dust generation, may influence nucleic acid stability, pathogen distribution, and ultimately the sensitivity of environmental health monitoring methods. However, to our knowledge, no studies have systematically evaluated the effects of commonly used rodent bedding materials on environmental health monitoring performance. The limited studies that have assessed bedding effects on pathogen detection when live sentinel animals or nonstandard bedding materials were used have reported no significant differences in detection outcomes.^29,112

Rodent Bedding Preferences

When comparing wood, cellulose, and corncob bedding, both mice and rats consistently demonstrate a preference for cellulose-based products. This preference suggests that cellulose provides superior nesting characteristics and a more comfortable resting surface.^{4,5,31,58,113–115}

Most bedding preference studies have been conducted without the provision of separate nesting materials. Under these conditions, rodents tend to select the bedding, or combination of beddings, that best facilitates nest construction, often favoring larger, fibrous substrates.^{4,5,31,48,113–119} However, when nesting material is available, these bedding preferences often diminish or disappear.¹¹⁶ For example, in studies lacking nesting material, multiple mouse strains have shown a consistent preference for cellulose bedding over aspen and corncob alternatives, as indicated by longer lengths of stay in cages with cellulose bedding.^4,31,58,114

In one study that provided nesting material, large-flake cellulose bedding from reclaimed wood pulp sources was preferred and more effectively incorporated into nests compared with wood chip or small-flake cellulose bedding.¹¹⁴ In addition, species-specific behaviors such as digging and grooming are more frequently observed in mice housed on cellulose (shredded paper) bedding.⁴ Although cloth bedding has been shown to be preferred over cellulose and wood-based substrates in the absence of nesting material, its use is associated with poor breeding performance and is impractical for routine husbandry.^43,44

Most studies assessing bedding preference measure periodic or continuous dwell time (length of stay) in choice-test paradigms. Bedding preferences based on dwell time are typically most pronounced during the light phase, when rodents are at rest,^4,115 whereas during the dark phase they tend to explore the entire enclosure. Thus, dwell time during the light phase may reflect the comfort and suitability of the bedding as a resting surface. Consistent with this interpretation, corncob is less preferred compared with wood-based beddings across multiple studies.^{31,58,115,116} Moreover, rats exhibit reduced slow-wave sleep when housed on corncob bedding compared with aspen.^114,120 Because rodents typically defecate away from their resting area, fecal boli accumulation is an unreliable indicator of dwell time or bedding preference.^4,115

Rodents may exhibit a partial preference for the bedding type they were reared on,¹¹⁵ although this finding is not consistent across studies.⁵⁸ Preferences may also vary by sex and by the spatial position of bedding types within experimental cages.^4,38 While strain differences do not appear to substantially influence bedding preference,^4,48 one study reported that rodents preferred bedding that is visually similar to their coat color.⁴⁴

Bedding depth can also influence preference. One investigation has shown that mice prefer deeper bedding (6 L), which better allows for natural burrowing behavior.¹²¹ However, providing such excessively high volumes is often impractical due to increased cost and ergonomic challenges for animal care staff.²⁶ Because nesting material was not provided in this study, it remains unclear how access to nesting material might mitigate the desire for deeper bedding. The preferences for deep bedding were strongest during the light phase, suggesting that nesting material providing shelter may partially substitute for the protective and insulating benefits of burrowing.

Animal Health and Welfare Effects

Several studies have compared the effect of different bedding types, sizes, and depth on rodent health, behavior, and reproductive outcomes. The findings from these studies are discussed below.

General health.

Bedding type generally does not exert a significant influence on rodent food and water intake or on hematologic and clinical chemistry parameters.^{29,34,39,77,82,122,123} Although most studies report minimal physiologic impact, certain wood-based beddings, particularly pine shavings, have been linked to elevated oxidative stress and ALT in rats, and in vitro cytotoxicity.^33,124–126 Wood-based materials have also been historically implicated in the development of various health conditions such as periodontal and respiratory disease, blepharitis, pup mortality, infertility, and neoplasia.^{31,74,127–130} However, many of these associations have since been refuted or remain inconclusive due to potential confounding variables such as bedding contamination or preexisting disease conditions.^{77,82,131–133} While wood-based beddings have been found to increase footpad damage in hamsters and rabbits, to our knowledge, comparison of bedding type effect on pododermatitis in mice and rats has not been evaluated.^134,135

Corncob bedding can contain leukotoxin diols (bioactive substances capable of promoting human cancer cell xenograft growth in mice),^136,137 raising potential toxicological concerns, although their practical significance in rodent health remains uncertain.

While cotton nesting materials have been associated with an increased incidence of blepharitis, particularly in athymic nude mice, at this time, cellulose-based beddings have not been reported to significantly affect rodent health outcomes.¹³⁸

Growth and thermoregulation.

Across multiple studies, bedding type has not been shown to significantly influence body weight gain in healthy rodents.^{21,27,38,39,46,77,82,116,123,139–143} Nonetheless, bedding modifications that facilitate nesting or burrowing can impact thermoregulation, which in turn may influence growth, weight, and physical features.^{14,74,86,144–146}

Infection, toxicity, and respiratory risks.

As previously discussed, bedding contamination with infectious agents or toxins poses a direct threat to animal health and research reproducibility; therefore, bedding materials should be sourced from reputable vendors providing autoclaved or irradiated products with routine quality assurance testing. Although airborne particulate matter from bedding is known to accumulate in microisolation cages and may theoretically affect rodent respiratory health, no direct evidence currently links inhaled bedding dust to adverse health outcomes in rodents.⁸⁹

Respiratory tract irritants, acetic acid and sulfur dioxide, have been found to accumulate in sterile microisolation cages containing corncob bedding, which may impact the health and research outcomes of rodents housed on this substrate.⁸⁴

Ammonia toxicity.

Elevated in-cage ammonia concentrations are strongly associated with an increased risk of nasal epithelial damage and bacterial colonization.^147,148 Multiple studies investigating the impact of bedding on nasal pathology have reported that higher ammonia levels correlate with greater severity of nasal lesions. However, these studies were unable to fully control for other contributing variables such as endotoxin exposure, bedding dust, bacterial contamination, ambient temperature and humidity, or the presence of other volatile organic compounds.^{20,27,34,76,79,149} As a result, the independent effect of bedding type on nasal pathology remains uncertain. Furthermore, comparisons between ¼-in. and ⅛-in. corncob bedding revealed no significant differences in ammonia or nasal pathology scores, suggesting that particle size within the same bedding source may not substantially influence nasal health outcomes.⁸⁷

Species-specific behavior.

Most studies indicate that bedding type does not significantly influence rodent fecal cortisol levels, daily activity, or species-specific behaviors such as grooming, feeding, drinking, climbing, digging, and huddling.^{27,38,77,82,122} When alterations in blood cortisol concentrations were observed, these effects were attributed to differences in the nest-building properties of the bedding materials, particularly in the absence of supplemental nesting material.¹⁵⁰

Bedding volume may modulate species-specific behaviors. For example, increased bedding depth (80 cm) has been shown to reduce stereotypic behaviors, such as wire-gnawing, in hamsters.¹⁴⁴ In contrast, similar effects were not observed in mice housed with 250, 400, or 550 mL of aspen bedding,⁸² suggesting that larger bedding volumes or additional enrichment items may be required to elicit behavioral changes in this species. A higher incidence of fighting in mice housed in IVCs with corncob bedding was reported compared with those in static cages with wood chip bedding; however, the study could not conclusively determine whether the observed effect was due to bedding type or cage design.¹⁵¹

Reproduction and development.

While provision of bedding enhances rodent breeding performance, the specific bedding type can significantly influence reproductive outcomes.^142,152 Despite its widespread use, corncob bedding has been associated with disrupted estrous cycles, delayed puberty onset, and reduced fertility in rats.^141,143,153 The mechanisms underlying these reproductive alterations are likely multifactorial and remain incompletely understood.

Evidence shows that corncob bedding suppresses the expression of estrogen receptor-α cells in rats, a change that may influence reproductive behavior, although a direct causal link to reproductive decline has not been established.¹⁵⁴ Isomeric mixtures of tetrahydrofuran diols and leukotoxin diols, identified as non-estrogenic compounds in corncob bedding, have been demonstrated to alter reproductive behavior when administered orally. These compounds can prolong metestrus in female rats and reduce sexual behaviors, including lordosis, mounting, and intromission.^{136,137,155–157} Reduced female sexual receptivity in rats may be mitigated if females are cohoused with sexually experienced males.¹⁵⁸ Rats housed on corncob bedding have also exhibited increased maternal grooming behavior toward their offspring compared with those housed on cellulose-based bedding.¹⁵⁹

Zearalenone, an estrogenic mycotoxin commonly detected in corncob bedding, has been associated with early vaginal opening in mice, although this effect has not been replicated in rats.^49,141 Most studies comparing corncob, wood, and cellulose bedding types have found no adverse effects on mouse reproductive performance.^{31,123,154,160,161} In one study, mice housed on cellulose (compressed paper) bedding exhibited improved breeding performance relative to those on corncob bedding, although this benefit may have been due to superior nest construction when the compressed paper was incorporated into nesting material.¹⁶² As discussed previously, bedding with longer or more fibrous structures can enhance nesting and burrowing behaviors, which, in the absence of adequate supplemental nesting material, may positively influence both welfare and reproductive success.²⁴ Multiple bedding manufacturers offer the option to incorporate paper-based nesting materials in the bedding substrate, possibly eliminating the need for additional nesting material. While this may be cost effective and benefit animal welfare through foraging and nest building, to our knowledge, the impact of these combination bedding substrates on rodent reproduction has not been studied.¹¹⁹

Research Impact

The regulation on Good Laboratory Practice for Nonclinical Laboratory Studies states that “bedding used in animal cages or pens shall not interfere with the purpose or conduct of the study.”¹⁶³ However, bedding selection has emerged as a core environmental variable affecting experimental outcomes and reproducibility.¹⁵ Below is a summary of published findings on bedding-related research effects.

Physiology and immunology research.

Softwood beddings contain aromatic hydrocarbons that induce hepatic enzyme activity in rodents, leading to altered drug metabolism, including reduced barbiturate sleep times and changes in hepatic conjugation processes.^164–169 Induction of cytochrome P450 enzymes and associated physiologic changes are most pronounced with cedar bedding, which has been linked to increased liver-to-body weight ratios, increased seizure thresholds, and significantly shorter anesthesia recovery times compared with other softwood beddings.^{36,130,164,166,170,171} These effects are independent of bedding sterility or the quantity of softwood bedding used,^86,170 and are reversible. Anesthesia recovery times normalize within ∼48 hours after removal from softwood bedding,^167–169 with hepatic enzyme activity stabilizing within 6 weeks of removal.¹⁶⁶ Extraction or hydrothermal treatment of softwood bedding to remove essential oils and volatile aromatic hydrocarbons markedly reduces cytochrome induction.^39,169,172

In contrast, corncob bedding does not induce hepatic enzymes but has been associated with reduced hepatic microsomal protein production in mice and rats.^153,164 Hardwood-based beddings may contain immunostimulatory compounds that modulate gut-associated lymphoid tissue responses. Mice housed on hardwood bedding have demonstrated hypertrophy of Peyer’s patches and increased production of virus-specific intestinal IgA following rotavirus inoculation, although serum antibody levels remain unaffected by bedding type.^88,122,173

Behavior research.

Bedding type has been shown to significantly influence neurobehavioral development in rodents, with effects varying by sex and being most pronounced during early-life exposure.^{38,123,142,143,159,174} Mice raised on cellulose bedding demonstrated reduced performance in multiple T-water maze testing compared with those housed on wood-based bedding, although the underlying mechanisms remain unclear.¹²³

Rodents raised on corncob bedding tend to exhibit attenuated stress responses in behavioral assays such as the open field, light/dark box, resident–intruder, and social defeat tests when compared with animals reared on cellulose or wood-based beddings, independent of maternal care differences.^{123,154,159,174} The anxiolytic-like effects of corncob bedding are hypothesized to be mediated through altered estrogen signaling and changes in phosphorylated extracellular signal–regulated kinase expression in the hypothalamus and amygdala.^154,174

Conversely, some studies report increased anxiety-like behaviors in rats raised on corncob bedding in elevated plus maze, open field, and novel object recognition tests.¹⁴³ However, confounding factors such as cage type and diet were not fully controlled. Transitioning rats from pine to cedar bedding has also been associated with reduced anxiety responses, although it remains unclear whether the effect is due to bedding type or environmental novelty.¹⁷⁵

Bedding size and depth have not been shown to significantly affect measures of anxiety, depression, learning, or memory.^24,86,87

Pain research.

Contact bedding can influence the integrity of rodent footpad skin, potentially confounding studies that assess acute or chronic pain using paw sensitivity assays. The extent of this impact depends on multiple factors, including the animal model, pain assay employed, and the bedding material and particle size. In a rat model of peripheral nerve injury, fine sawdust bedding increased responsiveness to pinprick and acetone tests compared with coarse sawdust, suggesting that bedding texture may alter stimulus-evoked nociceptive thresholds.¹⁷⁶ In addition, rodents housed on aspen bedding exhibited greater sensitivity to mechanical and thermal stimuli than did those on cellulose bedding; however, these differences disappeared following inflammation induced by complete Freund’s adjuvant.¹¹⁴ The mechanisms underlying the influence of bedding type and particle size on paw sensitivity remain unclear. Histopathologic evaluation of mouse footpads housed on aspen shavings compared with corncob bedding revealed no discernible differences in tissue integrity.³⁴

Metabolism and microbiome research.

Because rodents routinely ingest their bedding, both the presence and type of bedding can influence key physiologic and metabolic outcomes, including fasting blood glucose levels, feed efficiency, and gut microbiota composition.^177,178 Mice housed with contact bedding exhibit significantly higher fasting blood glucose levels and body weights compared with those without bedding.¹⁷⁹ Although mice have been reported to consume wood bedding at the highest rate, followed by corncob, then cellulose, the greatest increase in fasting blood glucose levels have been observed in animals housed on corncob bedding compared with wood or cellulose materials.^21,179,180 Despite these differences in fasting states, bedding type does not appear to affect glucose tolerance, adipose tissue composition, or feed intake in rodents fed ad libitum.^{21,34,38,77,122,123,140,179,181} Under calorie restriction, mice experience increased intestinal taurine levels with wooden bedding and greater weight loss and higher bile acid deconjugation with corncob bedding.^178,179 Greater weight loss with corncob bedding and calorie restriction is suspected to be related to the higher insoluble fiber content and lower digestible energy yield of ingested corncob particles.¹⁷⁹

Relative to wire-grid flooring, bedding presence alters fecal and cecal bacterial composition and increases fermentable substrate availability, resulting in modified large-bowel fermentation profiles.^177,178 Several studies have reported that bedding type influences fecal and cecal microbial composition, richness, and diversity; however, findings are inconsistent across experiments.^{140,179,182–184} Variability in results may stem from uncontrolled differences in animal source, diet, water supply, sampling interval, and housing systems, all of which independently affect microbiome composition.^185,186

The type of diet provided also modulates the magnitude of bedding effects on the microbiome and feed conversion efficiency. Bedding-related differences in cecal and fecal bacterial composition are less pronounced in rodents fed a high-fat diet.^140,177 Feeding a high-fat diet has also been associated with reduced cecal digesta mass and decreased feed efficiency in animals housed on corncob bedding compared with those on wire-grid or cellulose substrates (effects not observed under low-fat dietary conditions).^177,181 While some studies suggest that a high-fat diet may interact with bedding type to influence body weight, these findings are not consistent across investigations.^140,181

Unrecognized bedding-related shifts in the microbiome can substantially alter experimental phenotypes and compromise reproducibility. For example, substitution of corncob bedding with wood bedding in Dahl salt-sensitive rats resulted in the development of hypertension, increased renal damage, and impaired renal function.¹⁸⁷ The authors attributed these phenotypic changes to altered bedding consumption and its downstream effects on gut microbiota.

Conclusion

Contact bedding is preferred by rodents and supports stable microenvironmental conditions. However, if not carefully selected, characterized, and documented, bedding can act as an uncontrolled extrinsic variable, affecting research outcomes and reproducibility.^14,188,189 Literature indicates a general preference for cellulose-based bedding as a resting substrate compared with wood or corncob alternatives, although distinguishing bedding preference from nesting behavior remains challenging.

Microenvironmental parameters (absorbency, ammonia levels, temperature, humidity, dust generation, and microbial contamination) are not consistently affected by bedding type across studies. Inconsistencies are further complicated by variable methods and incomplete reporting. Future studies should consider the use of AI-powered smart IVCs, which enable continuous measurement of cage microenvironmental parameters, including bedding moisture content.^190,191 When combined with the capacity to track and record multiple behavioral outcomes, AI-powered technology may also facilitate the identification of bedding-associated phenotypic changes.

Bedding sterility, density, and volume are key determinants of the cage microenvironment and should be carefully considered at the institutional level. Bedding that supports extended cage change intervals may further enhance animal welfare and research outcomes by improving breeding performance and reducing anxiety and aggression.^74,192–194 Accordingly, the use of irradiated or autoclaved bedding, along with routine quality control assessments of vendor practices, is recommended.²⁵ Although mice demonstrate a preference for bedding volumes exceeding 0.5 L,¹²¹ an optimal amount remains undefined and is likely influenced by bedding density. Increased bedding depth can promote species-specific behaviors and greater environmental stability; however, these benefits must be balanced against occupational health concerns.

The Guide for the Care and Use of Laboratory Animals recommends bedding that facilitates burrowing behavior. Although the effects of bedding type and depth have been evaluated in mice and hamsters,^86,121,144 data for rats remain limited. AAALAC International has noted that rat husbandry practices have not advanced at the same pace as for other rodent species and emphasizes the importance of providing opportunities to dig, explore, and chew.¹⁹⁵ In our experience, IVCs for rats frequently lack sufficient bedding to support burrowing or nesting, necessitating the use of supplemental enrichment.

While bedding type often exerts minimal influence on general health or overt behavior, some materials have demonstrated significant experimental impacts, leading to their reduced use. Softwood products, for example, have largely been replaced by hardwood alternatives because of aromatic hydrocarbon impacts on hepatic enzymes. More recently, one commercial supplier discontinued corncob bedding production, citing variability concerns.¹⁹⁶ Compared with wood and cellulose, corncob bedding is less preferred by rodents and has significant impacts to rodent reproduction and research. Among available materials, cellulose-based bedding has shown the least disruption to rodent health and research outcomes.

Given the influence of bedding on research results and the lack of standardization in the field, detailed reporting of bedding characteristics is essential for ensuring research transparency and reproducibility. Underreporting of bedding and other extrinsic factors may stem from journal space limitations or inconsistent adherence to reporting standards.^49,197–200 The ARRIVE guidelines, ACLAM position statements, and NIH reproducibility workshops all highlight the importance of disclosing bedding details, although specific reporting requirements remain undefined.^201–204 Recent literature recommends that publications include bedding type and amount, manufacturer, catalog and lot numbers, sterilization status, and contamination data.^{14,49,87,154,180,188,205,206} Incorporating these details will strengthen cross-study comparability and help identify conditions under which bedding selection may influence experimental outcomes.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This work was internally funded.