What Do We Need Our Livestock to Do in Regards to Feed Primary Colors

Introduction

Providing for an animal's nutritional needs, as well as an appropriate environment to reside, is the cornerstone of animal husbandry. Feed and bedding can directly influence experimental outcomes, as their chemical and physical characteristics can affect the animal's physiology (Baker and Lipman 2015). The production and subsequent handling of feed and bedding, whether by the manufacturer or distributor, and continuing within the animal facility, can also affect the quality of the product in use. The selection, management, and provision of high-quality feed and bedding is essential to ensure the health and welfare of the animals and the integrity of the results obtained from them.

In this chapter, feed and bedding are reviewed separately. The importance of, and the methods by which, feed and bedding are selected and their impact on research are discussed. Processing, handling, and storage of feed and bedding is also reviewed. The reader is provided an overview of the concepts relevant to selecting and managing feed and bedding in the laboratory animal setting. As the scope of this topic is broad, the reader should consult the references for additional information.

Feed

The provision of high-quality feed to animals is essential to meet their physiologic needs, specifically growth, maintenance, and reproduction. There are numerous products, which differ in nutrient content, available in various formulations from a variety of feed manufacturers (Fox and Newberne 1980). A primary directive when formulating feed is to ensure sufficient content of the six classes of nutrients: water, carbohydrates, fats (lipids), proteins, minerals, and vitamins. While all animals require each of the six classes of nutrients, some species may require higher levels of specific nutrients than others. Additionally, nutrients are considered either essential or nonessential. Essential nutrients are those that an animal cannot synthesize or cannot synthesize in sufficient quantities to maintain health and must be obtained from an external source, that is, in the diet. It is important to recognize, when selecting a diet, that a particular nutrient may be essential (i.e., necessary in the diet or otherwise provided) for some species but not for others. Nonessential nutrients are those nutrients that can be produced by the animal or its microbial flora and are not a dietary requirement. Examples of nutrient categories are provided in the following paragraphs, with select examples of species that have species-specific nutrient requirements.

Fiber, a complex carbohydrate, provides bulk for the contents of the digestive tract and is important for efficient and effective digestion in most laboratory animal species, particularly rabbits, guinea pigs, some species of nonhuman primates, and ruminants. These species should be fed a high-fiber diet to ensure proper gastrointestinal (GI) physiology and to avoid GI obstruction. Vitamins are needed in small amounts to maintain health. Some vitamins may be produced in the body, but not in sufficient quantities. These vitamins must be supplied in the diet. For example, vitamin C cannot be synthesized by guinea pigs and some nonhuman primate species. These species are dependent on receiving their vitamin C requirement in their feed or, less commonly, in their water, or through the provision of dietary supplements, for example, fruit; therefore, their diets are specially formulated to include vitamin C, without which they may develop scurvy. Vitamin D is supplied to laboratory animals, in the form of cholecalciferol (D3), in their feed, although the absolute requirement for vitamin D is unknown for many species. Some nonhuman primate species, if not exposed to ultraviolet (sun) light, require vitamin D3 in their diet to help prevent rickets, as they cannot utilize vitamin D2 to meet their needs. Amino acids, the building blocks of proteins, are needed to carry out many important bodily functions. The amino acid taurine is essential for cats but not for other species. For this reason, cat foods are supplemented with taurine to avoid a deficiency that may lead to blindness and tooth decay. The primary functions of fats are to supply and store energy. High-fat diets (up to 11%) are at times used as a response to increased neonatal morbidity and mortality, or to support breeding in rodents, because their higher energy content can help the animal meet the energy demands of gestation and lactation. Minerals, in the appropriate ratios, are necessary to maintain normal physiology. A mineral deficiency or the provision of minerals in an inappropriate ratio can lead to disease. For example, a diet deficient in calcium can lead to rickets in young animals of many species, or in mature animals to osteoporosis, a disease in which the bones are fragile, making them susceptible to fracture.

Subcommittees of the National Research Council Committee on Animal Nutrition have prepared comprehensive reports for the nutrient requirements of commonly used laboratory animal species (NRC 1977, 1982, 1993, 1994, 1995, 1998, 2000, 2001, 2003a, 2003b, 2006a, 2006b, 2007); these publications also include information on quality assurance, contaminants and toxicants, bioavailability, and palatability.

Formulas and Types

Most laboratory diets are natural ingredients and nutritionally complete. Natural-ingredient diets are formulated from agricultural- and/or animal-based ingredients, such as processed whole grains and fish meal, and commodities subjected to limited refinement. Formulations of natural-ingredient diets differ depending on the species to which they are fed. Even within a species, there may be different formulations depending on the animals' use. For example, there are many formulations of rodent diets, differing principally with respect to protein and fat concentration for breeding versus nonbreeding animals and for growth and maintenance.

Most natural-ingredient diets are "closed" formula, in which the individual components of the diet are proprietary and are not specified by the manufacturer, although a guaranteed analysis is provided, including a list of ingredients and nutrients with their calculated values. The diet's ingredients may change in association with commodity prices or availability. Closed-formula diets typically contain ingredients such as ground corn, ground oats, alfalfa meal, soybean meal, and ground wheat. Vitamins, minerals, and fat are added to ensure nutritional adequacy. Categories of closed-formula diets include least-cost, fixed, and variable formula diets.

Least-cost formula is the practice of substituting one ingredient in a diet with a lower-cost ingredient at any given time, to be cost-effective. Of the various types of closed-formula diets, least cost introduces the most variables due to the possibility of constant and broad change in the diet's ingredients.

Fixed formula ensures the ingredient formulation does not change, unless the formula itself is updated by the manufacturer to change nutritional content. It is important to note that since the nutritional content of the dietary constituent can naturally vary with harvest location and across growing seasons, there will be some variability from batch to batch in a fixed formula diet.

The goal of a variable formula is to maintain the concentrations of known nutrients in the diet in response to the naturally changing nutritional content of the ingredients. The formula is altered as ingredients are assayed to ensure the diet remains nutritionally stable across batches (Knapka 1997). Variable formula diets, although likely to be more stable with regard to nutritional content than least-cost and fixed formula diets, may still vary over time, as the ingredients used do differ from place to place and season to season.

Although less commonly utilized, "open"-formula diets are manufactured in accordance with an established known ingredient formulation that is known to the purchaser. Open-formula diets were generated to reduce diet as a variable and may be natural ingredient, but most commonly are purified diets. For example, in the 1970s, the American Institute of Nutrition (AIN) initiated a program to standardize laboratory animal diets, formed a committee, and designed the open-formula AIN-76 rodent diet to be used as a standard reference diet in an effort to reduce some variability. AIN-76A was revised and improved in 1993, and two new formulations were derived: AIN-93G for growth, pregnancy, and lactation, and AIN-93M for adult maintenance. Open-formula diets offer certain advantages, including, but not limited to, the following: their quantitative ingredient formulations are available to the user, they facilitate control of potential research variables, they allow for repeatability in research, and the diets can be purchased from multiple vendors, encouraging competitive and quality incentives (Barnard et al. 2009).

Purified diets, also referred to as semisynthetic diets, are open-formula diets produced from purified components, such as carbohydrates (e.g., starch or sucrose), purified sources of protein (e.g., lactalbumin and casein), refined oils, and synthetic vitamins and minerals. Purified diets are typically used when altering the nutritional content of the diet or when compounding with additives. Purified diets allow for complete ingredient control. Specific ingredients can be added or excluded. The nutritional content of a purified diet is highly stable from batch to batch, as the specific ingredients and amounts of each are known. Purified diets are considerably more expensive than natural-ingredient diets.

Chemically defined diets are formulated from chemically pure nutrients, for example, specific carbohydrates, triglycerides, individual amino acids, essential fatty acids, vitamins, and minerals. Chemically defined diets are generally utilized when altering a specific nutritional dietary component. They are extremely expensive and highly labile.

It is important to note that natural-ingredient diets vary greatly from purified and chemically defined diets, and while each has its own advantages; it is not feasible to compare results when a natural-ingredient diet is used for a control study for cost savings, and a purified or chemically defined diet is used for the test diet.

Certified diets are natural-ingredient diets produced to meet the requirements of the governmental good laboratory practice (GLP) standards, requiring periodic feed analysis for environmental contaminants that may interfere with research studies (CFR 2004; EU 2004; OECD 2011). Feed samples are analyzed and certified to contain no more than the established maximum level of environmental contaminants, including heavy metals, chlorinated hydrocarbons, organophosphates, and aflatoxins. Many contaminants are found naturally in plant materials or are agricultural residues. Diets may also be contaminated during storage or formulation. A batch-specific certificate of analysis with a guarantee that the contaminants do not exceed the acceptable maximum limits is provided with the diet. For preclinical toxicology studies required to meet GLP standards, sufficient diet is commonly procured to ensure that the animals are fed diet from a single batch for the duration of the study.

Medicated and compounded diets have drugs or other chemicals added. They are routinely used in research facilities housing rodents. Many compounded diets are available "off the shelf" from commercial suppliers. Examples of medicated diets include feed containing the anthelmintic fenbendazole, as well as several antibiotics, including trimethoprim/sulfamethoxazole and doxycycline. Fenbendazole feed is commonly employed to treat mice for nematodes (e.g., Syphacia and Aspiculuris spp.), trimethoprim/sulfamethoxazole-compounded feed is used to control pneumocystosis in immunodeficient mouse strains, and doxycycline-containing feed is used to induce or inhibit transcription in conditional transgenic mice that contain components of the tetracycline transactivator system (Coghlan et al. 1993; Lewandoski 2001; Ryding et al. 2001). Compounded feed is manufactured in special manufacturing plants, distinct from those used to produce other diet types, to ensure there is no carryover of the added compound to noncompounded feed. Compounded feed can be manufactured using distinct dyes, permitting the diet to be differentiated from standard diets. Feed can also be pigmented with a small amount of a Food, Drug, and Cosmetic Act (FD&C) food color to distinguish different diets. Diets can also be compounded with various additives for specific needs. For example, at the authors' institution, feed compounded with vitamin E is used to treat ulcerative dermatitis in C57BL/6 (B6) mice and genetically engineered mice on a B6 background (Lawson et al. 2003). Medicated and compounded diets may have a shorter shelf life, as indicated by the manufacturer.

The majority of diets provided to rodents, and therefore used in animal research, are pelleted (Figure 27.1a and b), which provides the densest and highest energy content per unit weight, allowing efficient delivery. The effect of the pelleting process prevents ingredient separation during handling and feeding, minimizes waste, and decreases the need for storage space. Pelleting involves mixing, grinding, and exposing various dietary constituents, such as proteins, fibers, and minerals, to steam (typically 65°C–80°C); compressing the conditioned ingredients into a die through which the meal is forced, expelled, and cut to the desired pellet length; conveying the meal to a dryer, which reduces moisture content to levels that impede microbial growth, permitting a relatively long shelf life; and cooling and sieving before packaging (Tobin et al. 2007). Since pelleting subjects feed constituents to heat and pressure and then rapid cooling, the bacterial and fungal loads that may be found in unprocessed ingredients are reduced (Halls and Tallentire 1978). Pelleting ensures formulation stability, allows easy verification of animal access to feed, is dustless and prevents inhalation, and in rodents, provides a hard substrate to "wear" down incisors.

Figure 27.1

Various feed forms: (a) pellet (rodent), (b) pellet (rabbit), (c) extruded collet (nonhuman primate), (d) extruded collet (dog), (e) meal, and (f) powdered. (Courtesy of Envigo [a–d] and PMI LabDiet [f].)

Extruded diet (Figure 27.1c and d) is produced by extrusion, a process in which ingredients are exposed to high heat and pressure. Initially, the feed is prepared as it is for pelleting; however, the ingredients are more finely ground, increasing gelatinization of starch. Dietary constituents are conditioned at ~80°C–95°C before being exposed to a temperature of ~150°C under high pressure when forced through the die. During the process, the material moves from an area of high pressure to one of lower pressure, in which superheated water vapor, trapped in the feed, cools and begins to expand and pop, introducing and trapping air into the feed, producing a product less dense than a typical pellet. The resulting collet (extruded pellet) is cut, dried, cooled, and sieved to remove fines (Tobin et al. 2007). Because extruded diets are produced at higher temperatures than pelleted diets, the microbiologic burden is less. Extruded diet is typically used for larger species, such as nonhuman primates, canine, and swine. While extruded diets are commonly fed to rodents in Europe, extruded rodent diets are infrequently fed to rodents in the United States, principally because of their low density and cost. Many of the advantages of pelleting also apply to extruded feeds, such as formulation stability, easy verification of animal access, and being dustless. Additional advantages of extruded diets are the ability to add high levels of fat to the diet, the diet is more digestible, and it performs better following autoclaving, with little to no clumping or excessive hardening, which commonly occurs when a pelleted diet is subjected to steam sterilization. However, extruded feeds are more expensive to manufacture and, being less dense, need to be fed more often and require more space for storage.

Meal diets (Figure 27.1e) consist of ground ingredients that are mixed together with no further processing, that is, pelleting or extrusion. Microbiologic levels are much higher than those found in pelleted or extruded feed due to the lack of heat used during processing, making meal more susceptible to rancidity and insect infestation (Eva and Rickett 1983). If the ingredients in the meal are different-sized particles, segregation of ingredients can occur. Also, some species may be able to isolate the particles they prefer, leaving the others behind, thus negating a "balanced" diet. Feeding meal mixes generally require the use of a container, such as a jar, trough, or tub.

Powdered or ground diet (Figure 27.1f) is a pelleted or extruded diet that is ground to a powder after production. Powdered diets are commonly used when needing to provide additives after formulation. However, waste is high with powdered diets, and caking commonly occurs when exposed to the microenvironment. Powdered diet requires special feeders, which can easily tip, spilling the feed, which is subsequently soiled with urine, feces, or saliva. Due to its considerably greater surface area, powdered diets can spoil much easier than pellets and can separate. Some powdered diets are formulated to be suspended in water. These liquid diets are uncommon and are most often used for alcohol studies or postsurgical recovery. The diet has to be prepared more often and needs to be monitored. Once in solution, the stability of nutrients is significantly shorted.

Autoclavable diets, which are formulated to be sterilized prior to provision, are enriched with heat-labile nutrients, including thiamin, vitamins A, B12 and E, pantothenic acid, and pyridoxine, whose concentrations are reduced during the autoclaving process. Steam autoclaving at 121°C for 15–20 minutes is frequently recommended for diet sterilization. Some diet formulations may be adversely affected at this temperature, and pasteurization is used in lieu of sterilization. Pasteurization is typically achieved by exposure to a temperature of 107°C for 15–20 minutes in an autoclave (Caulfield et al. 2008). Slightly lower temperatures and durations can be used to pasteurize with decreasing effects on the nutritional and microbial content of the diet (Faith and Hessler 2006). Unlike sterilization, pasteurization does not kill all microorganisms in the diet, but instead achieves a "log reduction" in the number of viable organisms. Autoclavable pelleted diets may be coated with silicon dioxide or calcium bentonite to reduce the likelihood of clumping and adherence, which occurs as a result of pellet swelling during steam sterilization. To avoid clumping postautoclaving, feed may be decanted into bags with additional space to accommodate swelling, or it may be sterilized on trays at a depth of ~3 inches. Sterilization and pasteurization cycles must be developed and verified to ensure the sterility or microbial load reduction of the feed. Pulsed vacuum sterilization, which removes air from the autoclave chamber, is preferred to ensure adequate steam penetration when autoclaving feed in the manufacturer's original packing materials (bag). Feed subjected to excessive sterilization may be depleted of essential nutrients and the protein quality may be reduced, but more likely it may become too hard""a result of the polymerization of select feed constituents""for some rodent strains to eat (Ford 1987). Validation of autoclave cycles poststerilization for sterility or postpasteurization for desired microbial reduction is recommended on a regular basis. Additional postautoclaving monitoring should include assurance that appropriate levels of heat-labile constituents are provided at suitable levels (Lipman 2007). Access to mass spectroscopy and other technologies is available to evaluate feed for contaminants if contamination is expected.

Gamma-irradiated diets have become commonplace and have replaced the use of autoclaved diets in many settings, as they require less processing after receipt and are not subject to the effects of heat and temperature that result from autoclaving. Most feed producers subject irradiated diet to between 10 and 40 kGy (1 and 4 Mrad) by exposing the bags to a cobalt source or electron beam. As feed bags are palletized and then irradiated, irradiation exposure differs, depending on a specific bag's location within the load or even among pellets or collets in a single bag. The irradiation dose is stated as the minimal dose exposure. Some diets will be exposed to greater amounts of irradiation. Although irradiated feed is not purported to be sterile, bacterial (cells and spores) and fungal loads are markedly reduced, to less than 100 bacteria or fungi per gram of feed (Cover and Belcher 1992). This is in contrast to standard diets whose bacterial loads fluctuate seasonally and can reach levels as high as 500,000 total bacteria per gram of feed. Irradiation is purported to be ineffective against some viruses, for example, mouse parvovirus (MPV), as the exposure dose is insufficient (W. Shek, personal communication, 2004; Lipman 2007); however, some laboratory animal veterinarians affirm that sporadic parvovirus outbreaks can be eliminated by using irradiated diet. In contrast to steam sterilization, irradiation has much less of an effect on nutritional quality. Irradiated and fortified autoclavable diets are commonly used for axenic and microbiologically defined rodents, and immunodeficient animals (NRC 2011).

Various soft and/or moist diets are commercially available. These are commonly available for dogs and cats. In rodents, they are used at weaning to ease the transition from lactation to a solid diet; at postsurgery to hasten recovery; to improve the nutrition of animals subject to the effects of experimental manipulation; for select mutants, such as those with dental deformities, that have difficulty ingesting hard feed; for animals having difficulty ambulating; and during shipping.

Supplemental fiber is frequently provided to ruminants, as well as rabbits and guinea pigs, in addition to a balanced commercial diet. Common plants used include timothy and alfalfa hay. Hay should be of good quality, contain few thick tough stems and weeds, and have no mold or dust. Irradiated, cubed hay is commercially available to provide a product free of parasites and microorganisms.

Live feed may be used for certain species, such as fish, amphibians, and reptiles. Examples of live feed include brine shrimp larvae, rotifers, paramecia, algae, protozoa, drosophila, crickets, waxworms, and mealworms. Some reptiles need live feed to be supplemented with a balanced commercial diet or calcium powder, augmented with vitamin D3 to assist in absorbing the calcium, sprinkled onto live prey at feeding time. Live feed is highly perishable and requires on-site equipment and processes to maintain daily production of rations. Standardized timing for collection of live feed ensures that the highest nutritional value is provided. If sourcing a live product, it is imperative to have a reliable vendor for procurement to avoid inconsistencies (e.g., hatching rates). Additionally, the nutritional values of various types of live feed differ and must be taken into account when selecting the type to be used for a particular species.

Flaked or dry diets are available and used for certain aquatics species. Generally, flaked diets are used for convenience and efficiency in large colonies, for example, zebrafish. Nutritional superiority of flaked or dry over live feed for aquatics species is under debate, although many facilities use a mixture of both live and flaked diets to ensure a nutritionally complete diet for the various stages of development (Lawrence et al. 2012). One advantage of flaked or dry feed is the ability to prevent the introduction of unwanted pathogens by treating the product with gamma-irradiation.

Influence of Feed on Experimental Results

A fundamental goal in scientific research is to eliminate variables. There are important considerations in choosing the right diet for an experiment. When deciding on what type of diet to use, it is best to look in the literature to see what diet has been used in similar studies. Additionally, food provided as enrichment should be factored into the overall caloric intake to ensure adequate nutrition and prevent undesired health or experimental effects. Feed constituents and formulations serve as research variables and, in specific research areas, require the use of purified and chemically refined diets. Some contaminants, such as heavy metals, including arsenic, cadmium, lead, and mercury, and pesticides, may be introduced from the environment. Phytoestrogens, although not necessarily negative in all circumstances, are a well-known example of a naturally occurring compound in plants that have estrogenic activity and which can interfere with behavior, reproduction, bone development, and metabolic activity. Therefore, feed manufacturers are producing diets that avoid protein sources known to contain isoflavones (Allred et al. 2001; Ju et al. 2001, 2002; Thigpen et al. 2001, 2002, 2003). The main subclasses of phytoestrogens found in ingredients used in research diets are isoflavones (found in soybean meal), coumestans (found in alfalfa), and lignans (mainly associated with plant fibers) (Tobin et al. 2007). To date, numerous studies in animals leave little doubt that isoflavones affect a variety of experimental endpoints (Baker and Lipman 2015). For example, rodents consuming isoflavones have been noted to have fewer tumors and/or a delay in tumor development in mammary, liver, colon, and prostate cancer (Leiter 2009). Although experimental results may vary depending on many factors, the evidence for isoflavones' role in cancer is mounting and must not be dismissed as insignificant in cancer research (Leiter 2009). Phytoestrogens have been reported to cause reproductive problems, including impaired ovarian function and reduced fecundity, in sheep and cattle (Adams 1995). Diets containing alfalfa, which fluoresce naturally, can affect image quality when performing fluorescence optical imaging (Inoue et al. 2008). Therefore, alfalfa-free imaging diets are available for rodents. Many other studies describe the experimental impact of various dietary constituents. It is impossible to know which yet-to-be-discovered compounds may have such an effect. Additionally, as noted earlier, even closed-formula diets cannot be exactly replicated batch after batch to ensure complete nutritional consistency. At the very least, the concept of feed as a variable in most research should be taken into account when planning studies.

Bedding

Bedding is an integral component of the husbandry provided to most terrestrial species. It is used to absorb, dilute, and/or limit the animal's contact with its excreta, and is used for nest building; provides insulation and therefore allows the animal to thermoregulate; can serve to provide environmental enrichment; minimizes the growth of microorganisms; and in some cases, reduces the accumulation of intracage ammonia (Perkins and Lipman 1995; Smith et al. 2004). Importantly, bedding can also influence experimental data. A variety of materials are utilized as both contact and noncontact bedding. By definition, contact bedding that which the animals have direct contact. Noncontact bedding is typically provided as a sheet or on a roll, lining a pan or cage, and does not typically come into physical contact with the animal; rather, it sits below a rack or cage to collect and absorb urine and feces. Bedding selection should be based on a variety of factors, the most important of which are animal preference and materials that minimally interfere with the investigations for which the animals are used. For example, mice exhibit a preference for large fibrous materials that they can manipulate and use to build a nest (Blom et al. 1996; Van de Weerd et al. 1997). Bedding that enables burrowing is encouraged for some species, such as mice and hamsters. Pigs naturally forage and explore, even if there are no obvious stimuli (Wood-Gush et al. 1993), so if bedding is provided, it should be of a nature that encourages and satisfies that behavior (Bollen and Ritskes-Hoiinga 2007). No type of bedding is ideal for all species under all management and experimental conditions. For example, in nude or hairless mice that lack eyelashes, fibers from some forms of cellulose bedding can result in periorbital abscesses (White et al. 2008). Vendors' manufacturing, monitoring, and storage methods are important in bedding selection, as bedding may be contaminated with toxins and environmental pollutants, as well as bacteria, fungi, and vermin (NRC 2011). Other considerations for bedding selection are product cost; availability; absorbency; palatability, or lack thereof; ease of handling, transportation, and storage, including packaging and product weight (dry and wet); the ability to sterilize and/or obtain gamma-irradiated product; disposability; the ability to control ammonia accumulation; and the amount of associated dust.

Types of Bedding and the Potential to Affect Research

Bedding is generally manufactured from plant materials such as wood, cotton, and corncob, which are subject to varying degrees of processing. Minimally processed wood is the most commonly used contact bedding. Soft- or hardwoods, devoid of bark, are chopped, shredded, or shaved, and then heated at temperatures up to 1200°F to reduce the bacterial and moisture content before packaging. Hardwood bedding (Figure 27.2a) is manufactured from aspen, beech, maple, and/or birch. Softwoods, such as pine or cedar, are generally avoided, as the volatile aromatic amines that give these materials their pleasant aroma alter hepatic microsomal enzyme concentrations and therefore xenobiotic processing (Ferguson 1966; Vessel 1966). Most wood bedding, especially shredded or shaved products, has excellent nest-building characteristics (Blom et al. 1996). Larger species, such as pigs, may be provided with shaved wood products, which encourage natural foraging, assist in body heat retention, and absorb urine (Figure 27.2).

Figure 27.2

Various bedding types: (a) wood, (b) corncob, (c) cellulose, and (d) noncontact cage board. (Courtesy of P.J. Murphy [a], the Andersons, [b], and Shepherd Specialty Papers [c and d].)

Corncob (Figure 27.2b), produced from the woody-ring portion of the cob by processing with a hammer mill and roller mill and subsequently dried, is available in several pellet sizes. One-eighth inch, 1/4 inch, or a mixture of both are commonly used for rodents. Corncob has excellent characteristics with respect to inhibiting the accumulation of ammonia, and therefore is preferred when using static isolator caging (Perkins and Lipman 1995; Smith et al. 2004). The specific characteristics of corncob inhibiting ammonia accumulation are unknown, but they are unrelated to its absorbency. Corncob can be abrasive and has been associated with foot lesions in highly immunocompromised mouse strains (author's [N.S.L.] personal communication). Off-gassing of acetic acid has also been observed, presumably from the decay of residual organic matter (Perkins and Lipman 1995). The density of corncob limits nest building, and therefore it is frequently supplemented with nesting material or mixed with other bedding types. The authors recommend autoclaving or purchasing irradiated product, as the porosity of corncob leads to mold growth in unsterilized or nonirradiated product underneath the sipper tube, where spillover occurs. Corncob expands and adheres during autoclaving, requiring the need to dissociate the pellets after steam sterilization.

A variety of processed wood products, for example, cellulose (Figure 27.2c), both virgin and recycled, are available for use as both contact and noncontact bedding. Products differ in absorbency, color, shape, and size. They may also be blended with other products, such as corncob. Cellulose products are typically more expensive than wood and generally have good nest-building characteristics. In some countries, nontraditional bedding is used (e.g., in India, bedding composed of rice husks is commonly used); however, appropriate quality assurance practices should be in place.

Certified bedding, in which levels of specific toxic environmental contaminants are measured and determined to not exceed maximum concentrations, is available for use in studies that must meet GLP standards.

A variety of products, generally manufactured from cellulose, are available for use as noncontact bedding (Figure 27.2d). Noncontact bedding includes plastic-backed absorbent paper and cage board. Products are available in precut sheets, formed trays, and roll stock. Material used for noncontact bedding can be impregnated with antibiotics, for example, neomycin, to inhibit bacterial growth and the subsequent ammonia production.

Feed and Bedding Handling and Processing

The receipt, handling, storage, and distribution of feed and bedding are integral components of a biosecurity program, especially for barrier-maintained rodent species. These processes are heavily influenced by floor plan, the operational concepts employed in the facility, and the level of adventitious agent exclusion. Quality assurance programs for feed and bedding processes should include monitoring and validating sterilization or pasteurization and/or decontamination procedures.

Some facility designs provide distinct corridors to separate the distribution of feed and bedding (with or without associated caging) between the clean cage wash (CCW), the animal holding rooms (AHRs) and the soiled cage wash (SCW), in order to control cross-contamination between clean and soiled supplies. However, the use of dual-corridor facilities has become less common, as the space utilization is less efficient than single-corridor facilities, animal facility construction and operational costs have escalated, and the use of specific pathogen-free animals has become commonplace. Regardless, facility management must take into account the specific design of the facility to make important decisions in the interest of minimizing or eliminating contamination during the processing, storage, and handling of these materials.

The method of processing feed and/or bedding into CCW is of critical importance, as the bulk of the materials used in a vivarium originate from this area and are distributed throughout the facility. An operational failure in CCW, resulting in contamination, is likely to be widely spread. Feed may be autoclaved into the facility, but use of gamma-irradiated diet has become commonplace and has replaced the use of autoclaved diets in a variety of settings, as they require less processing after receipt and are not subject to the effects of heat and temperature that result from autoclaving. Validation of autoclave cycles poststerilization for sterility or postpasteurization for desired microbial reduction is recommended on a regular basis. Biological indicators are used to verify the sterility of bedding. Similar to bedding, additional postautoclaving monitoring should include assurance that appropriate levels of heat-labile constituents within feed are at suitable levels. Access to mass spectroscopy and other technologies is available to evaluate bedding for contaminants if contamination is expected.

Irradiated feed is available in a variety of packaging. To provide assurance that a particular bag or package of diet has been irradiated, the bags or packages are individually marked with indicator labels using polyvinyl chloride (PVC) impregnated with an acid-sensitive dye. Irradiation results in a color change caused by the release of hydrochloric acid within the PVC label (Tobin et al. 2007). The outer paper packaging of irradiated diets can be exposed to contaminants during shipping and storage and therefore should be sprayed with a disinfectant solution and/or carefully removed to reveal an inner plastic bag. The sealed inner plastic bag also permits decontamination with a liquid or spray disinfectant. This is generally done using a spray bottle, or alternatively, there are automated misting chambers available. Irradiated diet can also be obtained in small, watertight, vacuum-sealed plastic bags for use in isolators, change stations, or BSCs. Compromised packages are easily identified, as the vacuum seal is lost and the bag inflates. The use of vaporized hydrogen peroxide, as a method to decontaminate the external surface of feed bags, even those with an inner plastic bag, must be carefully considered, as the hydrogen peroxide gas may penetrate the feed (author's [N.S.L.] personal communication). Subjecting irradiated feed bags to an extremely short "flash" sterilization cycle has been employed as a method for surface decontamination prior to relocation of feed into a rodent barrier (Thurlow et al. 2007). Feed for nonrodent species is not typically irradiated or available in a formulation that permits autoclaving. These bags are generally surface decontaminated with a liquid disinfectant or sterilant prior to, or upon, entry into the facility. It may be necessary to ensure biosecurity that feed used for nonrodent species be stored and handled separately from feed used for barrier-maintained rodents.

Bedding comes in a variety of sizes and packaging and is generally packaged by cubic feet, as opposed to weight, and in autoclavable paper bags. Bulk totes of bedding can be procured for larger facilities to reduce handling by staff when used with a pneumatic dispensing system. Irradiated bedding products generally receive a minimum dose of 15 kGy, and each bag is marked with an indicator label confirming exposure. Smaller bags are vacuumed packaged and shipped in protective cartons. Certified bedding is packaged in lot-coded, certified stamped bags and arrives with a corresponding laboratory analysis report. Bedding is typically autoclaved into CCW or gamma-irradiated and surface decontaminated using a method similar to that used to process feed. During autoclaving, bedding can absorb moisture and, as a result, loose absorbency and support the growth of microorganisms. Therefore, if autoclaved, sufficient time should be provided for drying before stacking, or alternatively, gamma-irradiated bedding may be used.

Once treated, caging may be filled with bedding manually, for example, with a scoop, or using an automatic dispenser. Several types of automated dispensers are available. The most accurate is a dispenser into which cages are inserted and a metered amount of bedding is released, using a foot-activated or automated switch, into the cages (Figure 27.3). This dispenser type is frequently employed with articulated-arm or foundry robotic cage washing systems or in facilities processing limited numbers of rodent cages. In-line bedding dispensers are also available for tunnel washers (Figure 27.4). These dispensers continuously "rain" bedding from above. Cages are flipped as they exit the unload end of the washer and are subsequently filled as they pass under the dispenser. The amount of bedding dispensed is dependent on both the speed of the belt on which the cage moves and the volume of bedding continuously released from the dispenser. In-line dispensers are useful when processing large numbers of rodent cages. Dispensers can be filled manually, or automated delivery systems are available to fill the unit to reduce the labor-intensive task of handling the bedding. The latter is particularly valuable if the point of receipt, that is, loading dock, is a considerable distance away from the cage wash. Regardless of the method of distribution, given the significance of exposure to particulate, consideration must be given, in consultation with an occupational health expert, to the use of respiratory protection when handling loose bedding.

Figure 27.3

Automatic bedding dispenser. (Courtesy of Tecniplast.)

Figure 27.4

In-line bedding dispenser with integrated pneumatic delivery system. (Courtesy of MSKCC.)

The flow of feed and bedding from the CCW or another postprocessing distribution point facility to the AHR, whether in cages or other containers, is also of critical importance. Some facilities steam sterilize all materials that come in contact with laboratory rodents, including all cage components, before leaving the CCW for distribution in order to ensure unwanted infectious agents are excluded. Other facilities limit treatment to feed and bedding (such as sterilization), as they pose the more likely risk of introducing excluded agents. In single-corridor facilities, the use of reusable or disposable equipment covers during transport is highly recommended to reduce the likelihood of cross-contamination during the transport of both clean and soiled equipment containing feed and bedding.

The SCW is the repository of all returning and potentially contaminated material. Therefore, procedures that ensure adequate containment are necessary. The potential for contamination resulting from an infectious disease outbreak is omnipresent, and caging containing soiled bedding from contaminated animals is commonly processed along with those from "clean" colonies. Soiled material sterilization is generally limited to situations when a known contaminant is present. Given the significance of animal allergens, prevention or minimization of exposure should be of high priority during the handling and disposal of bedding (Harrison 2001). A variety of solutions are available for this purpose, including, but not limited to, dust masks, N95 particulate respirators, Class I biological safety cabinets, and pneumatic waste disposal systems with integrated downdraft stations.

There are several systems marketed to the laboratory animal industry for soiled bedding disposal. Systems differ based on whether the bedding is altered by adding water before transport and disposal. In dry systems, bedding is dumped into a collecting receptacle and transported by vacuum or auger to a waste receptacle, generally at a loading dock, for subsequent removal. The receptacles are commonly outfitted with a shredder or grinder to macerate larger materials that may be inadvertently introduced into the system. There are also several systems that convert bedding and cellulose materials, such as cage board, into a slurry by pulverizing them and adding water. The material can then be flushed into the sanitary waste system, if code permits, or the water can be extracted and the waste, which is reduced in volume, is containerized for disposal. The collecting receptacle used in all these systems is generally provided with downdraft, for personnel protection against aerosols. Foundry, or articulated-arm, robots can be used with a tunnel washer for waste disposal. On the soiled side of the cage wash, the robot's gripper picks up the uppermost row of soiled cages, which have been nested on specially designed pallets. The robot then dumps the cages and places them on the tunnel washer belt. After washing, the robot on the clean side collects the cages from the washer belt, inserts them into an automated bedding dispenser, and subsequently stacks the bedded cages onto pallets.

Feed and Bedding Receipt and Storage

Upon receipt, bags of feed and bedding should be checked for milling and expiration dates and examined to ensure that they are intact and unstained, demonstrating that their contents have not been damaged, potentially exposed to vermin, penetrated by liquids, or otherwise contaminated. Bags with defects should be rejected because of the possibility of contamination.

Inherent risks for feed and/or bedding storage are nutrient degradation, contamination, spoilage, and vermin infestation. It is important to know the manufacture date and other factors that affect the shelf life of a diet. Stale food or food transported or stored inappropriately can become deficient in nutrients. Stock should be rotated regularly upon receipt of each shipment to ensure that the oldest material is used first and to ensure that the freshest is always available. Feed and bedding should be stored off the floor on pallets, racks, or carts in a manner that facilitates sanitation and visual confirmation of the manufacture date. Placing materials a minimum of 6 inches away from the wall will help protect them against pests, permit air circulation, and allow for cleaning behind and under the stored material. Opened feed and bedding bags should be stored in vermin-proof containers, such as plastic lined cans with tight-fitting lids, to minimize contamination and avoid the potential spread of pathogens. Containers should be cleaned regularly and new liners placed before adding new material, with the expiration date of any materials marked on the container. Chemicals should not be stored in the same room as feed and bedding because of the possibility that bags could be contaminated by the chemicals.

Feed should be maintained under cool, dry conditions and in a well-ventilated area not exposed to direct sunlight. A constant cool temperature and low humidity in storage areas help to avoid spoilage. Daily minimum and maximum temperatures and humidity values should be closely monitored to ensure appropriate environmental conditions where feed and bedding are stored. Natural-ingredient diets should be stored at temperatures less than 70°F, and relative humidities of ~50% are ideal. Exposure of feed to elevated temperatures induces rancidity, in which unsaturated fats and lipids are oxidized and converted into hydroperoxides, which subsequently break down into volatile aldehydes, esters, alcohols, ketones, and hydrocarbons, giving the feed a disagreeable odor and taste. Most natural-ingredient, dry laboratory animal diets stored properly can be used up to 6 months, or longer in some cases, after manufacture. Specialty diets may have a shorter shelf life. Specialty diets, such as purified diets, containing high fat levels are subject to spoilage and must be kept in a 4°C cooler to extend the shelf life. Nonstabilized vitamin C in manufactured feeds generally has a shelf life of only 3 months, but commonly used stabilized forms can extend the shelf life of the diet to 6 months. Other perishable items, such as fruit and vegetables, used for larger species, should be stored in appropriate containers and kept at a lower temperature to avoid spoilage. Live food sources need to be maintained and managed to ensure a steady supply and the health and suitability of the organism as a food and should be stored in a type-appropriate manner to preserve nutritional content, minimize contamination, and prevent entry of pests. The shelf life for live food sources, such as brine shrimp eggs, or commercial (pellet or flake) diets for aquatics species should be determined based on manufacture recommendations or follow commonly accepted practices (NRC 2011). Unopened canned feed can typically be stored for up to 2 years or longer. General feed and bedding inventory quantities should be maintained such that they may be rotated into circulation and consumed before the set expiration date (for feed) but have sufficient on-hand stock in case of a delivery delay. It is important to recognize that some manufacturers ship directly to the end user, while others ship to distributors, who then send feed and bedding to the end user.

Pests such as cockroaches and wild rodents are potential carriers of disease-causing agents and should be prevented from any contact with materials used in the laboratory setting. Whether in the plant (vendor), distributor's warehouse, or the animal facility, a stringent pest control program should be in place with regular inspections forwarded for review and archive by the end user, for example, the facility manager or attending veterinarian. All storage locations should be vermin-proof. Walls and floors must be free of cracks and crevices. Pipelines, drains, and air filters should be well sealed and inspected frequently. Newly received supplies should be thoroughly inspected for evidence of pests. Inside a facility, pests seek food, water, and the protection of dark areas for shelter and breeding. In addition to using metal or plastic dunnage racks, carts or shelves should be used to keep materials off the ground and away from the walls, doors and entrances should have rodent guards or door sweeps to prevent entry of vermin, and insect screens may be used in specific locations. Movable equipment, cage racks, shelves, and drawers must be routinely moved and cleaned. Humane mechanical traps should be placed on the floor against walls, which is where rodents commonly travel, and insect pheromone traps at key locations throughout the area. Traps that catch pests live require frequent observation. Electrical insect trap lamps may be used to monitor insect activity, but should not be used as a primary method of control. Cleanliness and good housekeeping are the primary methods of vermin control for feed and bedding storage locations. If a facility is infested with insects, pesticides are undesirable, but may be necessary, in which case feed and bedding would need to be protected. Inappropriate use of pesticides in an animal facility can be dangerous to personnel and experimental animals. Exposure of research animals to pesticides can compromise research data. The introduction of pesticides makes controlling experimental variables more difficult""hence the importance of vermin-proofing storage areas and cleanliness prior to relying on traps or other means of ridding an area of pests.

Regular monitoring of the pest control program with inspections is a must for plant (vendor), distributor, and animal facility locations. Inspections should give assurance that the pest control program is being executed as designed and to monitor vermin activity levels, and action should be taken if deficiencies are found. Reports from the plant and distributor locations should be shared regularly with animal facility management.

Conclusion

Feed and bedding are the single most important commodities used in the maintenance of laboratory animals. They are also among the primary factors contributing to variables in the animals' environment and have the potential to impact the research studies in which the animals are utilized. Understanding the types of feed and bedding available and selecting those appropriate for the research to be conducted are critical decisions made by facility management and veterinarians, in concert with research staff. Ensuring that the procedures for receipt, storage, processing, and distribution of feed and bedding are well defined is an essential management responsibility to ensure animal health and welfare, and ultimately contribute to attaining high-quality research data.

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