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Nutrition of Rodents
HUSBANDRY AND NUTRITION
1094-9194/99 $8.00
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NUTRITION OF RODENTS Joseph J. Knapka, PhD
Nutrition is an environmental factor that influences the ability of animals to attain their genetic potential for growth, reproduction, and longevity or to respond to stimuli. The animal's nutritional status, therefore, has a profound effect on its health and well-being. The process of providing adequate nutrition to captive rodents involves establishing the requirements for approximately 50 essential nutrients, formulating and manufacturing diets with the required nutrient concentrations, and managing factors related to diet quality. These factors include the bioavailability of nutrients; diet palatability or acceptance by rodents; procedures involved in diet preparation, transport, and storage; and the concentration of chemical contaminants. The science of nutrition is a chemistry-based discipline that interacts to varying degrees with other physiologic and biological sciences. These aspects of nutrition are beyond the scope of this discussion, but they are adequately presented in general nutrition textbooks and in chapters of various biochemistry or physiology texts. In contrast, information regarding the practical aspects of rodent nutrition is not always readily available. Therefore, the practical aspects of providing rodents with nutritionally adequate diets is the emphasis of this presentation. NUTRIENT REQUIREMENTS
To provide a specific rodent species with a nutritionally adequate diet, it is essential to obtain an estimate of their nutrient requirements. Although many species of rodents are reared in zoos or in captivity as companion animals or pets, nutrient requirements have been well de-
From Laboratory Animal Nutrition, Brookeville, Maryland
VETERINARY CLINICS OF NORTH AMERICA. EXOTIC ANIMAL PRACTICE VOLUME 2. NUMBER 1. JANUARY 1999
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fined only for those species that are widely used in biomedical research. Estimates of nutrient requirements for rats, mice, guinea pigs, hamsters, gerbils, and voles have been published by the National Research Council (NRC).29 The most reliable estimates for the nutrient requirements for animal species are obtained from the results of feeding trials designed to measure the performance of animals consuming diets in which the nutrient of interest is the only dietary variable. Studies of this nature, involving many of the rodent species currently held in captivity, have not been conducted. To provide these species with nutritionally adequate diets, estimates of their quantitative nutrient requirements must be obtained from other sources. These include (1)the results from studies that were not designed to establish nutrient requirements but had a significant nutritional component, (2) the extrapolation of nutrient requirement estimates from other rodent species on the assumption that different rodent species would have similar nutrient requirements, and (3) the nutrient composition of diets that have resulted in acceptable animal performance. FACTORS POTENTIALLY INFLUENCING NUTRIENT REQUIREMENTS
When a diet for a specific rodent species or colony is being selected, it is essential to identify and consider the potential factors that influence nutrient requirements. The nutrient requirements of rodents are dynamic in that they are influenced by both genetic and environmental factors. In addition, unspecific stress factors can cause changes in dietary consumption that may require adjustments in dietary nutrient concentrations to ensure adequate nutrition is provided. Although the biochemical and the physiologic factors that are associated with the influence of genetics on the nutrient requirements of rodents have not been studied in detail, data have been published indicating that the nutrient requirements of rodents vary among spec i e ~ At .~~ least in the case of mice, data also have been published indicating that the nutrient requirements of stocks and strains within a rodent species may differ. For instance, rodent growth tables30 that indicate a twofold difference in body weight between the smallest and the largest mouse strain at 56 days of age have peen published. This suggests a considerable difference in nutrient requirements among these strains. Data that indicate mouse strains differ in requirements for protein,'* riboflavin? pantothenic acid,lO,24 manganese; and zinc" have also been published. There are numerous environmental factors that can influence the nutrient requirements of rodents. Among the most important ones is the stage of life cycle. Changes in nutrient requirements associated with growth, reproduction, lactation, maintenance, or aging have been documented in most species of farm animals. Generally, rodent diets that are
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adequate for maximum growth have been assumed to be adequate for optimal reproduction or long-term maintenance. However, according to some published data, diets that resulted in rodent postweaning maximum growth do not support maximum reprodu~tion,~~ and diets that resulted in rodent maximum growth or reproduction were not the best diets to use in longevity studies.34,35 These results indicate that, as in farm animal species, the nutrient requirements of rodents change with stages " of the life cvcle. The rearing eivironment is an environmental factor that can have a profound influence on the nutrient requirements of rodents. The nutrient requirements of rodents reared in germ-free or specific pathogen-free (SPF) environments may differ from those reared in conventional environments. When a diet that was known to be marginal in several B complex vitamin concentrations was fed to germ-free and conventionally reared mice, the reproductive performance was lower in the germ-free mice.22It has been shown that intestinal microflora in rodents synthesize and utilize B complex vitaminsz7and that these vitamins become available to rodents by way of ~oprophagy.~ Data indicating that dietary calcium concentrations that were not excessive for conventional mice cause soft-tissue calcification in germ-free mice3*is evidence that environments devoid of microorganisms may influence the availability of some dietary constituents. The process of sterilizing diets to accommodate germ-free or SPF environments influences the concentration of dietary nutrients. During autoclaving for 25 minutes at 121°C the reported39loss of vitamins include vitamin A, 50% to 60%; thiamin, 75% to 90%; vitamin B,, 17% to 35%; pantothenic acid, 33% to 47%; and riboflavin, 5% to 12%. Diets manufactured for autoclaving must be fortified with these vitamins to compensate for these losses. Experimentally induced stress resulting from procedures such as surgery can have profound effects on dietary or nutrient requirements. In most cases it can cause anorexia, which may require the use of more palatable forms of diets or diets with higher nutrient concentrations. Diets with higher nutrient concentrations, particularly energy, should be considered to comvensate for the decrease in feed consumvtion. When surgery alters organs or organ systems, it is often necessary to make adjustments in dietary nutrient concentrations to compensate for changes in physiologic function. I
REQUIRED NUTRIENTS
The nutrients required by rodents do not differ from those required by other mammalian species. The quantitative nutrient requirements of rodents are different from other species because of their small body size and their greater metabolic rate. Published results on the nutrient requirements of various rodent species frequently contain considerable inconsistencies because the research was conducted under various envi-
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ronmental conditions and, in some cases, with different strains within a rodent species. Protein and Amino Acids
The protein concentration in rodent diets is expressed as the percentage of crude protein. This value is determined by measuring the total amount of nitrogen in the diet, usually by the Kjeldahl method,17 then calculating the crude protein content on the basis that the average protein contains 16% nitrogen. Errors associated with this procedure occur because all feed ingredients and diets contain some nonprotein nitrogen and because there is variation in the nitrogen content of proteins. Therefore, the crude protein concentration of diet is always higher than the true protein concentration. The crude protein concentrations in readily available commercial rodent diets range from 17% to 24%. Practical experience as well as experimental dataTsindicate that rodent performance is acceptable when dietary protein is within this range, even though experimental results indicate the actual dietary crude protein requirement for rodents is 12% to 14% for growth and 17% to 19% for reproduction. For example, published data have indicated an acceptable performance in rodent species at dietary protein concentrations of 13.6% from casein13 and 12.5% when the protein source was a combination of casein and free amino acids.16 Positive nitrogen balance has been reported in rats fed diets containing approximately 7% crude p r ~ t e i nResults, .~ such as these, showing a considerable amount of variation in the apparent crude protein requirements in rodents may account for the high and variable crude protein concentrations in commercially available rodent diets. The practice in establishing the crude protein concentration in a rodent diet is to start with the minimal requirements of the most demanding strain, to increase the percentage of protein to compensate for differences in protein quality, and to include a safety factor. Except for the added cost, moderate amounts of excess dietary crude protein is of little consequence in most rodent production colonies. Ingested protein in excess of required amounts is deaminated, and the nitrogen is excreted through the kidneys. The amino acid residues are used as energy. However, protein is an inefficient source of energy because the deamination process requires energy. Although crude protein values are used to express! dietary protein concentrations and to estimate protein requirements, crude protein is not required by animals. Rather, amino acids, which are commonly referred to as the building blocks of proteins, are the nitrogen-bearing compounds that are required and utilized by animals. There are approximately 20 amino acids required for protein synthesis; these are classified as dietary essential and nonessential. The essential amino acids arginine, histidine, tyrosine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, and valine must be provided in the diet because
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of the inability of rodents to synthesize the amounts required for normal body functions. Dietary concentrations of dietary nonessential amino acids are not a concern because adequate amounts can be synthesized by rodents. The concentrations and the ratios of the essential amino acids determine protein quality. That is, the more consistent the concentration of each essential amino acid in the dietary protein is with the actual rodent requirement, the higher the protein quality. The required protein concentration in a specific diet is dependent on the amino acid profile of the protein involved. Even though dietary amino acid concentrations are important to the well-being of rodents, only a limited amount of data have been published regarding the quantitative requirements for these species. However, estimated amino acid requirements have been published for rodent species used in biomedical research.29
Fat and Energy
The fat content of commercial rodent diets is expressed in terms of crude fat, or ether extract, which is the ether-soluble fraction of the diet.2 In addition to fat, this fraction also contains other ether-soluble compounds, such as fat-soluble vitamins and plant pigments. Therefore, the percentage of dietary crude fat is always higher than the percentage of true fat. Since a unit weight of fat contains approximately 2.5 times as much energy as an equal unit weight of carbohydrate, the substitution of dietary fat for carbohydrate provides a means to increase dietary caloric density. The dietary caloric density requirements for the various stages of the life cycle of rodent species have not been determined experimentally. However, as in other mammalian species, the metabolic energy requirements for rodent maintenance can be expressed based on metabolic body size, kilocalorie = body weight in kilograms to the 3/4 power. The crude fat concentrations in commercial rodent diets range from 4% to 11%.In theory, a high-energy concentration is required for rodents that are gestating and lactating at the same time. However, because of obesity, high dietary concentrations of crude fat could have a more drastic negative effect on the reproductive performance of less prolific rodent species. In general, the reproductive performance in most rodent species is acceptable when the dietary crude fat concentration is from 4% to 6%. The weaning weight of rodent pups increases as the dietary crude fat concentration increases.19 Fat is not only a dietary energy source but also the source of the essential fatty acids, linoleic acid, and arachidonic acid. Vegetable fats such as soybean or corn oil are good sources of these essential fatty acids, in comparison to essential fatty concentrations in animal fats. Generally, a dietary concentration of 2% soybean or corn oil provides sufficient amounts of the essential fatty acids to meet the requirements
of most species of rodents.2yThe detailed histologic changes associated with an essential fatty acid deficiency in mice have been d e s ~ r i b e d . ~ ~ , ~ ~ Crude Fiber and Carbohydrates
The crude fiber fraction of a rodent diet contains complex carbohydrates such as cellulose and hemicellulose. The assay for crude fiber2 involves boiling diet samples in a dilute base and a dilute acid to digest protein, fat, vitamins, and simple carbohydrates and then ashing to separate the crude fiber from the minerals. Crude fiber is not digested by rodents. However, it has been described2yas a potentially beneficial dietary constituent in rodent diets. Crude fiber has been implicated as affecting diet palatability, digestion, lactation, intestinal microbial biosynthesis, and the consumption of other n ~ t r i e n t sCommercial .~ rodent diets contain between 2.5% and 4% crude fiber, but the optimal dietary concentrations have not been established experimentally. The fraction of the diet containing the simple carbohydrates such as sugars and starches is referred to as nitrogen free extract (NFE). This fraction is calculated as the difference between 100% and the total percentages of dietary moisture, ash, crude protein, crude fat, and crude fiber. The NFE content of commercial rodent diets ranges from 45% to 60%, depending on the concentration of the other nutrients. The simple carbohydrates are a source of energy for the body, but estimated quantitative requirements for simple carbohydrates in rodent diets are not available. Minerals
The total mineral content of a diet is expressed as ash and consists of the residue remaining after a diet sample has been subjected to complete oxidati~n.~ The concentrations of individual minerals are not identified in the ash fraction of the diet, but ash content is an indicator of diet quality. A good quality rodent diet contains from 7% to 8.5% ash. Higher ash concentrations may indicate marginal diet quality. There are approximately 14 minerals that have been shown to be required by rodents: calcium, chloride, magnesium, phosphorus, potassium, sodium and sulfur concentrations (generally expressed as a percentage of the diet), copper, iron, manganese, zinc,'.iodine, molybdenum and, selenium concentrations (generally expressed as milligrams per kilogram). The function and signs of deficiency for many of these minerals are summarized in Table 1. More detailed information in this regard and the estimated mineral requirements for rodent species used in biomedical research have been published by the NRCZ9In addition, there is a group of minerals that rodents may require in microgram per kilogram concentrations. Since the experimental data needed to estimate a quantitative requirement for minerals such as silicon, chromium, fluo-
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Table 1. FUNCTION AND DEFICIENCY SIGNS OF SELECTED MINERALS Mineral
Calcium Phosphorus Potassium Magnesium zinc Sodium Manganese
Function
Bone formation and bone growth. Bone and teeth formation, body fluids. Cellular constituent, blood pressure regulation. Similar to Ca and P in function, 'a phosphate activator.' Role in C02eliminating enzyme, an activator of various enzymes. Formation and retention of body fluids. Bone formation and enzyme activity.
Iron
Hemoglobin synthesis.
Iodine
Thyroxine formation, growth and development, new tissue formation. Cystine and methionine synthesis.
Sulphur
Deficiency Signs
Growth retardation, decreased food consumption, rear leg paralysis. Similar to calcium, poor reproduction. Reduced appetite and growth, diarrhea, distended abdomens. Irregular heart action, kidney damage, temperamental irritability. Retarded growth and hair development. Poor condition, appetite depression, death. Poor growth, irregular ovulation, and weak or dead young in females; testicular degeneration in males. Nutritional anemia, red cells reduced in size. Nutritional goiter. Hair loss, lameness, food consumption decrease, starvation.
ride, nickel, boron, lithium, and vanadium are not available, they are considered as potentially beneficial mineral elements.33 In comparison with estimated requirements, high concentrations of minerals are included in commercial rodent diets to compensate for the low bioavalibility of the chemical forms of minerals found in feed ingredients or due to the phytates associated with plant products. Phytates frequently influence mineral bioavailability. The ratios of various mineral concentrations to each other may be more important to the well-being of rodents than absolute mineral concentrations because an excessive amount of one mineral inhibits the normal absorption of others. For instance, it has been reportedz6that 1.25% dietary calcium induces bone resorption in aging mice when the dietary phosphorus concentration was at 1.2% but not at 0.6%. At high dietary concentrations, many of the minerals can have toxic effects on rodents. The toxicity signs for each of the minerals have been described.29 Vitamins
Organic compounds that have been shown to be essential to life but that do not meet the criterion for classification in any other of the
nutrient classes are referred to as vitamins. The vitamins are classified based on their solubility in fat and water. The fat-soluble vitamins are A, D, E, and K, whereas the water-soluble vitamins are B complex and vitamin C. The B complex vitamins include biotin, choline, cyanocobalamin (vitamin B,,), folic acid, niacin, pantothenic acid, pyridoxine, riboflavin, and thiamine. There is a considerable amount of variation in the chemical structure and function of vitamins. The function and the sims " of deficiency for many of the vitamins are summarized in Table 2. The fat-soluble vitamins are essential for various critical body functions. For instance, vitamin A is combined with a protein in visual purple to prevent night blindness; vitamin D is involved in calcium transport; vitamin E is an important natural antioxidanP6;and vitamin K is essential for normal blood clotting. It is essential that rodent diets provide adequate amounts of vitamins-A, D, and E; however, it appears that adequate amounts of vitamin K to meet body requirements are synthesized by the gut flora of conventionally reared rodents.38 The primary functions of the B complex vitamins are as coenzyme or cofactors in various metabolic processes in energy metabolism and tissue synthesis. Information regarding rodent requirements for the B comvlex vitamins is limited. and there is little consistencv in the data that'have been published. This may be attributed to the cokplex nature of the procedures associated with establishing the water-soluble vitamin requirements for rodent species. As indicated above, the gut flora of rodents synthesize many of the B complex vitamins, and through the practice of coprophagy they obtain at least a portion of their B complex Table 2. FUNCTION AND DEFICIENCY SIGNS OF SELECTED VITAMINS Vitamin
Vitamin A Vitamin D Vitamin E
Function
Deficiency Signs
Development of normal, healthy skin and bone. Essential for utilization of Ca and P in normal bone development. Essential for normal reproduction.
High infant mortality, malformation of bones, night blindness. Rickets, decreased bone ash.
Vitamin K
Essential for blood coagulation.
Vitamin C
Normal metabolism of Ca in man, monkey, and guinea. Essential for normal carbohydrate metabolism. Normal embryo development, amino acid metabolism. Fat metabolism, transport, synthesis of unsaturated fatty acids. Aids in tissue respiration.
Thiamin (Vitamin B,) Riboflavin (Vitamin B,) Vitamin B, Niacin
Abnormal gestation in females, sterility in males, muscular weakness and paralysis. Longer clotting time, hemorrhages in skin and tissue. Scurvy. ., Loss of appetite, muscular weakness, polyneuritis. Poor reproduction, deformed offspring. Convulsions, acrodynia in rats. Redness of skin, sores in mouth and intestinal tract.
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vitamin requirements from this s ~ u r c e Studies .~ designed to establish requirements for these vitamins must be conducted in environments in which intestinal microorganism populations are controlled or coprophagy is prevented. With the exception of the guinea pig, rodent species do not have a dietary requirement for vitamin C. However, the NRC reportz9 lists vitamin C as a potentially beneficial dietary constituent, and there is a considerable amount of documented discussion indicating that, at least for the rat, dietary vitamin C may be effective under specific conditions. Estimated vita& requirements for rodent species used in biomedical research have been published.29Although these estimates are based on the best available information, the estimates for the B complex vitamins should be considered guidelines for adequate rodent nutrition rather than absolute requirements. The vitamin concentrations in diets that have produced acceptable performance in the rodent species of interest should also be considered when dietary vitamin concentrations are selected. Water
Rodents require an ad libitum source of fresh clean water. Although the quantitative water requirements for rodent species have been established, environmental temperature is the primary factor influencing water requirement^.^ For instance, mice fed a dry diet and maintained at temperatures of 75" to 80°F (24" to 2TC) may die if deprived of water for 24 hours.28 It has also been shown that restricted water intake resulted in decreased voluntary food cons~mption.~ Water can contain sufficient concentrations of minerals to prevent clinical signs of deficiency when rodents are fed diets otherwise devoid of specific minerals. Water is also a potential source of pathogenic microorganisms and chemical contaminants. Unknown Nutritional Factors
There may be a concern by individuals engaged in the maintenance and production of some exotic rodent species that these animals may require unidentified or unknown nutrients. Although there is no scientific evidence to indicate the existence of any unknown nutrients, efforts are made to provide such nutrients by supplementing nutritionally balanced concentrated diets by feeding various succulent foodstuffs. Generally, these foodstuffs contain high-moisture and low-energy concentrations such as fresh fruits and vegetables, a combination of bread and milk, and so forth. Unless there is a substantial amount of scientific data showing the use of such foodstuffs is beneficial, their use should be curtailed or be very closely monitored. The concern is that these rodent species may
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vitamin requirements from this s ~ u r c e Studies .~ designed to establish requirements for these vitamins must be conducted in environments in which intestinal microorganism populations are controlled or coprophagy is prevented. With the exception of the guinea pig, rodent species do not have a dietary requirement for vitamin C. However, the NRC reportz9 lists vitamin C as a potentially beneficial dietary constituent, and there is a considerable amount of documented discussion indicating that, at least for the rat, dietary vitamin C may be effective under specific conditions. Estimated vitanfin requirements for rodent species used in biomedical research have been published.29Although these estimates are based on the best available information, the estimates for the B complex vitamins should be considered guidelines for adequate rodent nutrition rather than absolute requirements. The vitamin concentrations in diets that have produced acceptable performance in the rodent species of interest should also be considered when dietary vitamin concentrations are selected. Water
Rodents require an ad libitum source of fresh clean water. Although the quantitative water requirements for rodent species have been established, environmental temperature is the primary factor influencing water requirement^.^ For instance, mice fed a dry diet and maintained at temperatures of 75" to 80°F (24" to 27°C) may die if deprived of water for 24 hours.28 It has also been shown that restricted water intake resulted in decreased voluntary food cons~mption.~ Water can contain sufficient concentrations of minerals to prevent clinical signs of deficiency when rodents are fed diets otherwise devoid of specific minerals. Water is also a potential source of pathogenic microorganisms and chemical contaminants. Unknown Nutritional Factors
There may be a concern by individuals engaged in the maintenance and production of some exotic rodent species that these animals may require unidentified or unknown nutrients. Although there is no scientific evidence to indicate the existence of any unknown nutrients, efforts are made to provide such nutrients by supplementing nutritionally balanced concentrated diets by feeding various succulent foodstuffs. Generally, these foodstuffs contain high-moisture and low-energy concentrations such as fresh fruits and vegetables, a combination of bread and milk, and so forth. Unless there is a substantial amount of scientific data showing the use of such foodstuffs is beneficial, their use should be curtailed or be very closely monitored. The concern is that these rodent species may
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have a preference for the succulent foodstuff instead of a more concentrated diet. In these cases the succulent foodstuff becomes a substitution rather than a supplementation, and there is the potential of energy or nutrient deficiencies in the rodents involved. In this era when the technology is available to measure dietary constituents in the microgram per kilogram range, it is very unlikely that there are any unidentified nutrients that are essential for life. It is more likely that the quantitative requirements for the known nutrients have not been determined for many of the rodent species currently held in captivity.
RODENT DIETS
Diets used to maintain rodent colonies used in biomedical research are classified according to the basis of the degree of purification of the ingredients: natural-ingredient and purified diet and chemically defined diets.l Natural-ingredient diets are the most readily available and the most widely used diets. Natural-ingredient diets formulated to provide adequate nutrition for many of the rodent species used in biomedical research and some of the captive exotic species are commercially available. Although these are nutritionally well-balanced diets for the attended species, the quantitative ingredient composition, or formulation, of diets manufactured and marketed by commercial institutions is privileged information. Therefore, it is not possible to alter these formulations to accommodate the nutrient requirements of a rodent species that may have even slightly different nutrient requirements or to accommodate the requirements of a specific research project. A nutritionally balanced diet that has been formulated specifically for the species of interest should always be available. Although rodent species may survive when they are fed a diet consisting of a single cereal grain, this practice is discouraged because the nutrient composition of any of the cereal grains does not meet the requirements of any animal species. Natural-ingredient diet formulations in which the quantitative ingredient composition is readily available are presented in Table 3. These formulations can readily be altered to make required changes in their dietary nutrient compositions. Instructions for the f~rmulationof complete rodent diets have been published,17 and most animal feed diet manufacturers have access to computer programs for formulating diets.
DIET STORAGE
Nutrient stability in complete natural-ingredient rodent diets generally increases as environmental temperature and humidity in storage areas decrease. Diets stored at high temperatures and humidity may
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Table 3. EXAMPLES OF RODENT DIET FORMULATIONS Ingredient*
Ground wheat, g Ground corn, g Ground oats, g Wheat middlings, g Fish meal, g Soybean meal, g Alfalfa meal, g Corn gluten nieal, g Brewer's dried yeast, g Dried skim milk, g Soybean oil, g Dried molasses, g Salt, g Dicalcium phosphate, g Ground limestone, g Cobalt, mg Copper, mg Iodine, mg Iron, mg Magnesium, mg Manganese, mg Zinc, mg Vitamin A, IU Vitamin D, IU Vitamin E, mg Vitamin K, mg Biotin, mg Choline, mg Folic acid, mg Niacin, mg Pantothenate, mg Riboflavin, mg Thiamin, mg Pyridoxine, mg Vitamin B,, pg
Conventionalt
230 245 100 100 120 40 30 20 50 25 15 5 12.5 5 0.44 4.4 1.5 132 66 17.6 6060 5070 22 2.9 0.15 570 2.4 33 19.8 3.7 11 1.9 4.4
Autoclavable*
355 210 100 100 90 50 20 20 10 15 5 15 5 0.44 4.4 1.65 66 440 110 11 24,200 4,180 16.5 22 0.13 770 1.1 22 27.5 5.5 71.5 2.2 15.4
*Amount per kilogram of diet. tDiet designated as National Institutes of Health NIH-07.18 *National Institutes of Health, NIH-31 diet specifications NIH-11-137h, 1993.
deteriorate within several weeks, whereas the same diet stored in a freezer may be usable after several years of storage. The nutrients in natural-ingredient rodent diets can be expected to be stable and adequate for more than 168 days when such diets are stored in a cool (< 25°C [V°F]), dry (relative humidity approximately 50%) environment.12 With the exception of vitamin C, the most labile nutrients during diet storage are thiamine and vitamin A. Diets stored for long periods or under unusual environmental conditions should be assayed for at least these vitamins. For species such as the guinea pig, whose diet contains vitamin C that may not be protected by microencapsulation or other processes that protect this vitamin, this food may have to be used within 90 days
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of manufacture owing to the gradual oxidation of vitamin C during storage. Diets formulated with high fat (>lo%) concentrations may have to be stored at temperatures of 4°C (39°F) to retard rancidity as a result of peroxide formation. The refined ingredients used to manufacture purified diets generally do not contain antioxidants. As a result, the nutrients in this type of diet are subject to more rapid oxidation rates than those in natural-ingredient diets. It is recommended that purified diets be stored in 4°C environments when they cannot be used within 45 days of manufacture. DIETARY CONTAMINANTS
Dietary biological and chemical contaminants may have a negative influence on the well-being of captive rodent colonies. It is essential that managers of captive rodent colonies are aware of the potential influence of dietary contaminants. Biological Contaminants
Diets are a potential source of biological agents that may be pathogenic to captive rodents. The kind and concentration of these pathogens depend on such factors as the type of ingredients in the diet, sanitation programs in feed manufacturing facilities, and the protection of the finished product between manufacture and use. For the most part, animal feed users have very limited control over these factors, except in the selection of feed manufacturers or suppliers that implement rigid sanitation programs in facilities where feed ingredients are stored and diets are manufactured and warehoused. However, these efforts may be only partially effective in controlling dietary biological contaminants. The most reliable method to ensure against biological contaminations in rodent diets is to decontaminate diets before use. Steam autoclavingll, 37 and ionizing irradiationz1are widely used methods for decontaminating laboratory animal feeds of biological contaminants. Chemical Contaminants
Potential sources of chemical contaminants in,dietary natural ingredients include the soils where plants producing the ingredients are grown; the use of pesticides and the environmental conditions during the growing and the harvesting season of the ingredients; and the accidental contaminations that occur during processing, storage, and transportation of feed ingredients or complete diets. There are in excess of 50 dietary chemical constituents that are considered potential contaminants. These contaminants include heavy metals, pesticide residues, hormones, nitrosamines, or mycotoxins. Their dietary concentrations are
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generally low and variable,15,31, 32 depending on the ingredients used in a specific diet formulation. Dietary chemical contaminants are of the most concern to scientists conducting toxicology or bioassay studies in which even low concentrations have an influence on experimental results. Chemical contaminant concentrations in rodent diets can be managed when dietary ingredients are used that have a low-contaminant potential and when ingredients for contaminant concentrations are assayed before being used in diet formulation^.^^ SUMMARY
Access to a diet that provides adequate nutrition is one of the most important environmental factors influencing the well-being of rodent colonies. The dietary ingredient and nutrient composition, as well as the potential biological and chemical contaminant concentrations, are factors for consideration in selecting diets for a specific rodent colony. Estimated nutrient requirements have been published for the rodent species that are commonly used in biomedical research. The nutrient concentrations in adequate diets for other captive rodent species that are not used in biomedical research are more difficult to obtain. However, reasonable estimates of their nutrient requirements can be obtained by extrapolation of data from rodent species of a similar metabolic weight and size or from nutrient concentrations of diets that have a history of acceptable performance in the species of interest. Captive rodent colonies should be provided with nutritionally balanced diets with only limited amounts of succulent foodstuffs. The practice of feeding rodent colonies specific cereal grains is discouraged, since no single grain provides a balanced rodent diet. References 1. American Institute of Nutrition (AIN): Ad Hoc committee on standards for nutritional standards. Report of the committee. J Nutr 107:1340-1348, 1977 2. Association of Official Analytical Chemist (AOAC): Official Methods of Analysis of the Association of Official Analytical Chemist, ed 16. Washington, DC, 1995 3. Bell JM: A comparison of fibrous feedstuffs in nonruminant rations. Effects of growth responses, digestibility, rate of passage of ingesta volume. Can J Anim Sci 40:71, 1960 4. Bricker ML, Mitchell HH: The protein requirements of the adult rat in terms of protein contained in egg, milk, and soy flour. J Nutr 34491, 1947 5. Chew RM, Hinegardner RT: Effects of chronic insufficiency of drinking water in white mice. J Mammal 38:361, 1957 6. Daft FS, McDaniel EG, Herman LG, et al: Role of coprophagy in utilization of B vitamins synthesized by intestinal bacteria. FASEB J 22:129, 1963 7. Dalton DC: Effect of dilution of the diet with a filler on feed intake in the mouse. Nature 197:909, 1963 8. Erway L, Hurley LS, Fraser A: Congenital ataxia and otolith defects due to manganese deficiency in mice. J Nutr 100:643, 1970 9. Fenton PF, Cowgill GR: The nutrition of the mouse. I. A difference in the riboflavin requirements of two highly inbred strains. J Nutr 34:273, 1947
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10. Fenton PF, Cowgill GR, Stone MA, et al: Nutrition of the mouse. VIII. Studies on pantothenic acid, biotin, inositol, and p-aminobenzoic acid. J Nutr 42:257, 1950 11. Foster HL, Black CL, Pfau ES: A pasteurization process for pelleted diets. Lab Anim Care 14:373, 1964 12. Fullerton FR, Greenman DL, Kendall DC: Effects of storage conditions on nutritional qualities of semipurified (AIN-76) and natural ingredient (NIH-07) diets. J Nutr 112:567-573, 1982 13. Goettsch M: Comparative protein requirement of the rat and mouse for growth, reproduction and lactation using casein diets. J Nutr 70:307, 1960 14. Goodrick CL: The effects of dietary protein upon growth of inbred and hybrid mice. Growth 37:355, 1973 15. Greenman DL, Oller WL, Littlefield NA, et al: Commercial laboratory animal diets: Toxicant and nutrient variability. J Toxicol Environ Health 6:235-246, 1980 16. John AM, Bell JM: Amino acid requirements for the growing mouse. J Nutr 106:1361, 1976 . 17. Knapka JJ: Formulation of diets. In Beare-Rogers J (ed): Methods for Nutritional Assessment of Fats. American Oil Chemists' Society, 1985, pp 45-59 18. Knapka JJ, Smith KP, Judge FJ: Effect of open and closed formula rations on the performance of three strains of laboratory mice. Lab Anim Sci 24:480, 1974 19. Knapka JJ, Smith KP, Judge FJ: The effect of crude fat and crude protein on reproduction and weanling growth in four strains of inbred mice. J Nutr 107:61, 1977 20. Krishnarao GVG, Draper HH: Influence of dietary phosphate on bone resorption in senescent mce. J Nutr 102:1143, 1972 21. Ley FJ: The use of irradiation for the treatment of various animal feed products. Food Irradiation Information 1%-22, 1975 22. Luckey TD, Bengson MH, Kaplan H: Effect of bioisolation and the intestinal flora of mice upon evaluation of an Apollo diet. Aerosp Med 45:509, 1974 23. Luecke RW, Fraker PJ: The effect of varying dietary zinc levels on growth and antibody mediated response in two strains of mice. J Nutr 109:1373, 1979 24. Marsh JM, Fenton PF: Studies on some hormonal and genetic factors regulating nitrogen release from the isolated diaphragm. Endocrinology 65:916, 1959 25. Menton DN: The effects of essential fatty acid deficiency on the skin of the mouse. Am J Anat 122:137, 1968 26. Menton DN: The effects of essential fatty acid deficiency on the fine structure of mouse skin. J Morphol 132:181, 1970 27. Mickelsen 0 : Intestinal synthesis of vitamins in the non-ruminant. Vitam Horm 14:1, 1956 28. National Research Council, Board on Agriculture, Committee on Animal Nutrition, Subcommittee on Laboratory Animal Nutrition: Nutrient Requirements of Laboratory Animals, revised ed 3. Nutrient Requirements of Domestic Animals Series. Washington, DC, National Academy Press, 1978 29. National Research Council, Board on Agriculture, Committee on Animal Nutrition, Subcommittee on Laboratory Animal Nutrition: Nutrient Requirements of Laboratory Animals, revised ed 4. Nutrient Requirements of Domestic Animals Series. Washington, DC, National Academy Press, 1995 30. Poiley SM: Growth tables for 66 strains and stocks of laboratory animals. Lab Anim Sci 22:759, 1972 31. Rao GN: Significance of dietary contaminants in diet restriction studies. In Fishbein L (ed): Biological Effects of Dietary Restriction. New York, Springer-Verlag, 1991, pp 16-24 32. Rao GN, Knapka JJ: Contaminant and nutrient concentrations of natural ingredient rat and mouse diet used in chemical toxicology studies. Fundam Appl Toxicol 9:329-338, 1987 33. Reeves PG, Nielsen FH, Fahey GC: AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A diet. J Nutr 123:1939, 1993 34. Ross MH, Bras G: Food preference and length of life. Science 190:165, 1975
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35. Ross MH, Lustbader E, Bras G: Dietary practices and growth responses as predictors of longevity. Nature 262548, 1976 36. Scott ML: Vitamin E. In DeLuca HF (ed): Handbook of Lipid Research: Vol 2, The Fat Soluble Vitamins. New York, Plenum Press, 1978, pp 133-210 37. Williams FP, Christie RJ, Johnson DJ, et al: A new autoclave system for sterilizing vitamin fortified commercial rodent diets with lower nutrient loss. Lab Anim Care 18:195, 1968 38. Wostmann BS: Nutrition and metabolism of the germfree mammal. World Rev Nutr Diet 22:40, 1975 39. Zimmerman DR, Wostmann BS: Vitamin stability in diets sterilized for germfree mice. J Nutr 79:318, 1963 ' .
Address reprint requests to Joseph J. Knapka, PhD 19725 Olney Mill Road Brookeville, MD 20833
1094-9194/99 $8.00
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NUTRITION OF RODENTS Joseph J. Knapka, PhD
Nutrition is an environmental factor that influences the ability of animals to attain their genetic potential for growth, reproduction, and longevity or to respond to stimuli. The animal's nutritional status, therefore, has a profound effect on its health and well-being. The process of providing adequate nutrition to captive rodents involves establishing the requirements for approximately 50 essential nutrients, formulating and manufacturing diets with the required nutrient concentrations, and managing factors related to diet quality. These factors include the bioavailability of nutrients; diet palatability or acceptance by rodents; procedures involved in diet preparation, transport, and storage; and the concentration of chemical contaminants. The science of nutrition is a chemistry-based discipline that interacts to varying degrees with other physiologic and biological sciences. These aspects of nutrition are beyond the scope of this discussion, but they are adequately presented in general nutrition textbooks and in chapters of various biochemistry or physiology texts. In contrast, information regarding the practical aspects of rodent nutrition is not always readily available. Therefore, the practical aspects of providing rodents with nutritionally adequate diets is the emphasis of this presentation. NUTRIENT REQUIREMENTS
To provide a specific rodent species with a nutritionally adequate diet, it is essential to obtain an estimate of their nutrient requirements. Although many species of rodents are reared in zoos or in captivity as companion animals or pets, nutrient requirements have been well de-
From Laboratory Animal Nutrition, Brookeville, Maryland
VETERINARY CLINICS OF NORTH AMERICA. EXOTIC ANIMAL PRACTICE VOLUME 2. NUMBER 1. JANUARY 1999
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fined only for those species that are widely used in biomedical research. Estimates of nutrient requirements for rats, mice, guinea pigs, hamsters, gerbils, and voles have been published by the National Research Council (NRC).29 The most reliable estimates for the nutrient requirements for animal species are obtained from the results of feeding trials designed to measure the performance of animals consuming diets in which the nutrient of interest is the only dietary variable. Studies of this nature, involving many of the rodent species currently held in captivity, have not been conducted. To provide these species with nutritionally adequate diets, estimates of their quantitative nutrient requirements must be obtained from other sources. These include (1)the results from studies that were not designed to establish nutrient requirements but had a significant nutritional component, (2) the extrapolation of nutrient requirement estimates from other rodent species on the assumption that different rodent species would have similar nutrient requirements, and (3) the nutrient composition of diets that have resulted in acceptable animal performance. FACTORS POTENTIALLY INFLUENCING NUTRIENT REQUIREMENTS
When a diet for a specific rodent species or colony is being selected, it is essential to identify and consider the potential factors that influence nutrient requirements. The nutrient requirements of rodents are dynamic in that they are influenced by both genetic and environmental factors. In addition, unspecific stress factors can cause changes in dietary consumption that may require adjustments in dietary nutrient concentrations to ensure adequate nutrition is provided. Although the biochemical and the physiologic factors that are associated with the influence of genetics on the nutrient requirements of rodents have not been studied in detail, data have been published indicating that the nutrient requirements of rodents vary among spec i e ~ At .~~ least in the case of mice, data also have been published indicating that the nutrient requirements of stocks and strains within a rodent species may differ. For instance, rodent growth tables30 that indicate a twofold difference in body weight between the smallest and the largest mouse strain at 56 days of age have peen published. This suggests a considerable difference in nutrient requirements among these strains. Data that indicate mouse strains differ in requirements for protein,'* riboflavin? pantothenic acid,lO,24 manganese; and zinc" have also been published. There are numerous environmental factors that can influence the nutrient requirements of rodents. Among the most important ones is the stage of life cycle. Changes in nutrient requirements associated with growth, reproduction, lactation, maintenance, or aging have been documented in most species of farm animals. Generally, rodent diets that are
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adequate for maximum growth have been assumed to be adequate for optimal reproduction or long-term maintenance. However, according to some published data, diets that resulted in rodent postweaning maximum growth do not support maximum reprodu~tion,~~ and diets that resulted in rodent maximum growth or reproduction were not the best diets to use in longevity studies.34,35 These results indicate that, as in farm animal species, the nutrient requirements of rodents change with stages " of the life cvcle. The rearing eivironment is an environmental factor that can have a profound influence on the nutrient requirements of rodents. The nutrient requirements of rodents reared in germ-free or specific pathogen-free (SPF) environments may differ from those reared in conventional environments. When a diet that was known to be marginal in several B complex vitamin concentrations was fed to germ-free and conventionally reared mice, the reproductive performance was lower in the germ-free mice.22It has been shown that intestinal microflora in rodents synthesize and utilize B complex vitaminsz7and that these vitamins become available to rodents by way of ~oprophagy.~ Data indicating that dietary calcium concentrations that were not excessive for conventional mice cause soft-tissue calcification in germ-free mice3*is evidence that environments devoid of microorganisms may influence the availability of some dietary constituents. The process of sterilizing diets to accommodate germ-free or SPF environments influences the concentration of dietary nutrients. During autoclaving for 25 minutes at 121°C the reported39loss of vitamins include vitamin A, 50% to 60%; thiamin, 75% to 90%; vitamin B,, 17% to 35%; pantothenic acid, 33% to 47%; and riboflavin, 5% to 12%. Diets manufactured for autoclaving must be fortified with these vitamins to compensate for these losses. Experimentally induced stress resulting from procedures such as surgery can have profound effects on dietary or nutrient requirements. In most cases it can cause anorexia, which may require the use of more palatable forms of diets or diets with higher nutrient concentrations. Diets with higher nutrient concentrations, particularly energy, should be considered to comvensate for the decrease in feed consumvtion. When surgery alters organs or organ systems, it is often necessary to make adjustments in dietary nutrient concentrations to compensate for changes in physiologic function. I
REQUIRED NUTRIENTS
The nutrients required by rodents do not differ from those required by other mammalian species. The quantitative nutrient requirements of rodents are different from other species because of their small body size and their greater metabolic rate. Published results on the nutrient requirements of various rodent species frequently contain considerable inconsistencies because the research was conducted under various envi-
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ronmental conditions and, in some cases, with different strains within a rodent species. Protein and Amino Acids
The protein concentration in rodent diets is expressed as the percentage of crude protein. This value is determined by measuring the total amount of nitrogen in the diet, usually by the Kjeldahl method,17 then calculating the crude protein content on the basis that the average protein contains 16% nitrogen. Errors associated with this procedure occur because all feed ingredients and diets contain some nonprotein nitrogen and because there is variation in the nitrogen content of proteins. Therefore, the crude protein concentration of diet is always higher than the true protein concentration. The crude protein concentrations in readily available commercial rodent diets range from 17% to 24%. Practical experience as well as experimental dataTsindicate that rodent performance is acceptable when dietary protein is within this range, even though experimental results indicate the actual dietary crude protein requirement for rodents is 12% to 14% for growth and 17% to 19% for reproduction. For example, published data have indicated an acceptable performance in rodent species at dietary protein concentrations of 13.6% from casein13 and 12.5% when the protein source was a combination of casein and free amino acids.16 Positive nitrogen balance has been reported in rats fed diets containing approximately 7% crude p r ~ t e i nResults, .~ such as these, showing a considerable amount of variation in the apparent crude protein requirements in rodents may account for the high and variable crude protein concentrations in commercially available rodent diets. The practice in establishing the crude protein concentration in a rodent diet is to start with the minimal requirements of the most demanding strain, to increase the percentage of protein to compensate for differences in protein quality, and to include a safety factor. Except for the added cost, moderate amounts of excess dietary crude protein is of little consequence in most rodent production colonies. Ingested protein in excess of required amounts is deaminated, and the nitrogen is excreted through the kidneys. The amino acid residues are used as energy. However, protein is an inefficient source of energy because the deamination process requires energy. Although crude protein values are used to express! dietary protein concentrations and to estimate protein requirements, crude protein is not required by animals. Rather, amino acids, which are commonly referred to as the building blocks of proteins, are the nitrogen-bearing compounds that are required and utilized by animals. There are approximately 20 amino acids required for protein synthesis; these are classified as dietary essential and nonessential. The essential amino acids arginine, histidine, tyrosine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, and valine must be provided in the diet because
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of the inability of rodents to synthesize the amounts required for normal body functions. Dietary concentrations of dietary nonessential amino acids are not a concern because adequate amounts can be synthesized by rodents. The concentrations and the ratios of the essential amino acids determine protein quality. That is, the more consistent the concentration of each essential amino acid in the dietary protein is with the actual rodent requirement, the higher the protein quality. The required protein concentration in a specific diet is dependent on the amino acid profile of the protein involved. Even though dietary amino acid concentrations are important to the well-being of rodents, only a limited amount of data have been published regarding the quantitative requirements for these species. However, estimated amino acid requirements have been published for rodent species used in biomedical research.29
Fat and Energy
The fat content of commercial rodent diets is expressed in terms of crude fat, or ether extract, which is the ether-soluble fraction of the diet.2 In addition to fat, this fraction also contains other ether-soluble compounds, such as fat-soluble vitamins and plant pigments. Therefore, the percentage of dietary crude fat is always higher than the percentage of true fat. Since a unit weight of fat contains approximately 2.5 times as much energy as an equal unit weight of carbohydrate, the substitution of dietary fat for carbohydrate provides a means to increase dietary caloric density. The dietary caloric density requirements for the various stages of the life cycle of rodent species have not been determined experimentally. However, as in other mammalian species, the metabolic energy requirements for rodent maintenance can be expressed based on metabolic body size, kilocalorie = body weight in kilograms to the 3/4 power. The crude fat concentrations in commercial rodent diets range from 4% to 11%.In theory, a high-energy concentration is required for rodents that are gestating and lactating at the same time. However, because of obesity, high dietary concentrations of crude fat could have a more drastic negative effect on the reproductive performance of less prolific rodent species. In general, the reproductive performance in most rodent species is acceptable when the dietary crude fat concentration is from 4% to 6%. The weaning weight of rodent pups increases as the dietary crude fat concentration increases.19 Fat is not only a dietary energy source but also the source of the essential fatty acids, linoleic acid, and arachidonic acid. Vegetable fats such as soybean or corn oil are good sources of these essential fatty acids, in comparison to essential fatty concentrations in animal fats. Generally, a dietary concentration of 2% soybean or corn oil provides sufficient amounts of the essential fatty acids to meet the requirements
of most species of rodents.2yThe detailed histologic changes associated with an essential fatty acid deficiency in mice have been d e s ~ r i b e d . ~ ~ , ~ ~ Crude Fiber and Carbohydrates
The crude fiber fraction of a rodent diet contains complex carbohydrates such as cellulose and hemicellulose. The assay for crude fiber2 involves boiling diet samples in a dilute base and a dilute acid to digest protein, fat, vitamins, and simple carbohydrates and then ashing to separate the crude fiber from the minerals. Crude fiber is not digested by rodents. However, it has been described2yas a potentially beneficial dietary constituent in rodent diets. Crude fiber has been implicated as affecting diet palatability, digestion, lactation, intestinal microbial biosynthesis, and the consumption of other n ~ t r i e n t sCommercial .~ rodent diets contain between 2.5% and 4% crude fiber, but the optimal dietary concentrations have not been established experimentally. The fraction of the diet containing the simple carbohydrates such as sugars and starches is referred to as nitrogen free extract (NFE). This fraction is calculated as the difference between 100% and the total percentages of dietary moisture, ash, crude protein, crude fat, and crude fiber. The NFE content of commercial rodent diets ranges from 45% to 60%, depending on the concentration of the other nutrients. The simple carbohydrates are a source of energy for the body, but estimated quantitative requirements for simple carbohydrates in rodent diets are not available. Minerals
The total mineral content of a diet is expressed as ash and consists of the residue remaining after a diet sample has been subjected to complete oxidati~n.~ The concentrations of individual minerals are not identified in the ash fraction of the diet, but ash content is an indicator of diet quality. A good quality rodent diet contains from 7% to 8.5% ash. Higher ash concentrations may indicate marginal diet quality. There are approximately 14 minerals that have been shown to be required by rodents: calcium, chloride, magnesium, phosphorus, potassium, sodium and sulfur concentrations (generally expressed as a percentage of the diet), copper, iron, manganese, zinc,'.iodine, molybdenum and, selenium concentrations (generally expressed as milligrams per kilogram). The function and signs of deficiency for many of these minerals are summarized in Table 1. More detailed information in this regard and the estimated mineral requirements for rodent species used in biomedical research have been published by the NRCZ9In addition, there is a group of minerals that rodents may require in microgram per kilogram concentrations. Since the experimental data needed to estimate a quantitative requirement for minerals such as silicon, chromium, fluo-
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Table 1. FUNCTION AND DEFICIENCY SIGNS OF SELECTED MINERALS Mineral
Calcium Phosphorus Potassium Magnesium zinc Sodium Manganese
Function
Bone formation and bone growth. Bone and teeth formation, body fluids. Cellular constituent, blood pressure regulation. Similar to Ca and P in function, 'a phosphate activator.' Role in C02eliminating enzyme, an activator of various enzymes. Formation and retention of body fluids. Bone formation and enzyme activity.
Iron
Hemoglobin synthesis.
Iodine
Thyroxine formation, growth and development, new tissue formation. Cystine and methionine synthesis.
Sulphur
Deficiency Signs
Growth retardation, decreased food consumption, rear leg paralysis. Similar to calcium, poor reproduction. Reduced appetite and growth, diarrhea, distended abdomens. Irregular heart action, kidney damage, temperamental irritability. Retarded growth and hair development. Poor condition, appetite depression, death. Poor growth, irregular ovulation, and weak or dead young in females; testicular degeneration in males. Nutritional anemia, red cells reduced in size. Nutritional goiter. Hair loss, lameness, food consumption decrease, starvation.
ride, nickel, boron, lithium, and vanadium are not available, they are considered as potentially beneficial mineral elements.33 In comparison with estimated requirements, high concentrations of minerals are included in commercial rodent diets to compensate for the low bioavalibility of the chemical forms of minerals found in feed ingredients or due to the phytates associated with plant products. Phytates frequently influence mineral bioavailability. The ratios of various mineral concentrations to each other may be more important to the well-being of rodents than absolute mineral concentrations because an excessive amount of one mineral inhibits the normal absorption of others. For instance, it has been reportedz6that 1.25% dietary calcium induces bone resorption in aging mice when the dietary phosphorus concentration was at 1.2% but not at 0.6%. At high dietary concentrations, many of the minerals can have toxic effects on rodents. The toxicity signs for each of the minerals have been described.29 Vitamins
Organic compounds that have been shown to be essential to life but that do not meet the criterion for classification in any other of the
nutrient classes are referred to as vitamins. The vitamins are classified based on their solubility in fat and water. The fat-soluble vitamins are A, D, E, and K, whereas the water-soluble vitamins are B complex and vitamin C. The B complex vitamins include biotin, choline, cyanocobalamin (vitamin B,,), folic acid, niacin, pantothenic acid, pyridoxine, riboflavin, and thiamine. There is a considerable amount of variation in the chemical structure and function of vitamins. The function and the sims " of deficiency for many of the vitamins are summarized in Table 2. The fat-soluble vitamins are essential for various critical body functions. For instance, vitamin A is combined with a protein in visual purple to prevent night blindness; vitamin D is involved in calcium transport; vitamin E is an important natural antioxidanP6;and vitamin K is essential for normal blood clotting. It is essential that rodent diets provide adequate amounts of vitamins-A, D, and E; however, it appears that adequate amounts of vitamin K to meet body requirements are synthesized by the gut flora of conventionally reared rodents.38 The primary functions of the B complex vitamins are as coenzyme or cofactors in various metabolic processes in energy metabolism and tissue synthesis. Information regarding rodent requirements for the B comvlex vitamins is limited. and there is little consistencv in the data that'have been published. This may be attributed to the cokplex nature of the procedures associated with establishing the water-soluble vitamin requirements for rodent species. As indicated above, the gut flora of rodents synthesize many of the B complex vitamins, and through the practice of coprophagy they obtain at least a portion of their B complex Table 2. FUNCTION AND DEFICIENCY SIGNS OF SELECTED VITAMINS Vitamin
Vitamin A Vitamin D Vitamin E
Function
Deficiency Signs
Development of normal, healthy skin and bone. Essential for utilization of Ca and P in normal bone development. Essential for normal reproduction.
High infant mortality, malformation of bones, night blindness. Rickets, decreased bone ash.
Vitamin K
Essential for blood coagulation.
Vitamin C
Normal metabolism of Ca in man, monkey, and guinea. Essential for normal carbohydrate metabolism. Normal embryo development, amino acid metabolism. Fat metabolism, transport, synthesis of unsaturated fatty acids. Aids in tissue respiration.
Thiamin (Vitamin B,) Riboflavin (Vitamin B,) Vitamin B, Niacin
Abnormal gestation in females, sterility in males, muscular weakness and paralysis. Longer clotting time, hemorrhages in skin and tissue. Scurvy. ., Loss of appetite, muscular weakness, polyneuritis. Poor reproduction, deformed offspring. Convulsions, acrodynia in rats. Redness of skin, sores in mouth and intestinal tract.
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vitamin requirements from this s ~ u r c e Studies .~ designed to establish requirements for these vitamins must be conducted in environments in which intestinal microorganism populations are controlled or coprophagy is prevented. With the exception of the guinea pig, rodent species do not have a dietary requirement for vitamin C. However, the NRC reportz9 lists vitamin C as a potentially beneficial dietary constituent, and there is a considerable amount of documented discussion indicating that, at least for the rat, dietary vitamin C may be effective under specific conditions. Estimated vita& requirements for rodent species used in biomedical research have been published.29Although these estimates are based on the best available information, the estimates for the B complex vitamins should be considered guidelines for adequate rodent nutrition rather than absolute requirements. The vitamin concentrations in diets that have produced acceptable performance in the rodent species of interest should also be considered when dietary vitamin concentrations are selected. Water
Rodents require an ad libitum source of fresh clean water. Although the quantitative water requirements for rodent species have been established, environmental temperature is the primary factor influencing water requirement^.^ For instance, mice fed a dry diet and maintained at temperatures of 75" to 80°F (24" to 2TC) may die if deprived of water for 24 hours.28 It has also been shown that restricted water intake resulted in decreased voluntary food cons~mption.~ Water can contain sufficient concentrations of minerals to prevent clinical signs of deficiency when rodents are fed diets otherwise devoid of specific minerals. Water is also a potential source of pathogenic microorganisms and chemical contaminants. Unknown Nutritional Factors
There may be a concern by individuals engaged in the maintenance and production of some exotic rodent species that these animals may require unidentified or unknown nutrients. Although there is no scientific evidence to indicate the existence of any unknown nutrients, efforts are made to provide such nutrients by supplementing nutritionally balanced concentrated diets by feeding various succulent foodstuffs. Generally, these foodstuffs contain high-moisture and low-energy concentrations such as fresh fruits and vegetables, a combination of bread and milk, and so forth. Unless there is a substantial amount of scientific data showing the use of such foodstuffs is beneficial, their use should be curtailed or be very closely monitored. The concern is that these rodent species may
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vitamin requirements from this s ~ u r c e Studies .~ designed to establish requirements for these vitamins must be conducted in environments in which intestinal microorganism populations are controlled or coprophagy is prevented. With the exception of the guinea pig, rodent species do not have a dietary requirement for vitamin C. However, the NRC reportz9 lists vitamin C as a potentially beneficial dietary constituent, and there is a considerable amount of documented discussion indicating that, at least for the rat, dietary vitamin C may be effective under specific conditions. Estimated vitanfin requirements for rodent species used in biomedical research have been published.29Although these estimates are based on the best available information, the estimates for the B complex vitamins should be considered guidelines for adequate rodent nutrition rather than absolute requirements. The vitamin concentrations in diets that have produced acceptable performance in the rodent species of interest should also be considered when dietary vitamin concentrations are selected. Water
Rodents require an ad libitum source of fresh clean water. Although the quantitative water requirements for rodent species have been established, environmental temperature is the primary factor influencing water requirement^.^ For instance, mice fed a dry diet and maintained at temperatures of 75" to 80°F (24" to 27°C) may die if deprived of water for 24 hours.28 It has also been shown that restricted water intake resulted in decreased voluntary food cons~mption.~ Water can contain sufficient concentrations of minerals to prevent clinical signs of deficiency when rodents are fed diets otherwise devoid of specific minerals. Water is also a potential source of pathogenic microorganisms and chemical contaminants. Unknown Nutritional Factors
There may be a concern by individuals engaged in the maintenance and production of some exotic rodent species that these animals may require unidentified or unknown nutrients. Although there is no scientific evidence to indicate the existence of any unknown nutrients, efforts are made to provide such nutrients by supplementing nutritionally balanced concentrated diets by feeding various succulent foodstuffs. Generally, these foodstuffs contain high-moisture and low-energy concentrations such as fresh fruits and vegetables, a combination of bread and milk, and so forth. Unless there is a substantial amount of scientific data showing the use of such foodstuffs is beneficial, their use should be curtailed or be very closely monitored. The concern is that these rodent species may
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have a preference for the succulent foodstuff instead of a more concentrated diet. In these cases the succulent foodstuff becomes a substitution rather than a supplementation, and there is the potential of energy or nutrient deficiencies in the rodents involved. In this era when the technology is available to measure dietary constituents in the microgram per kilogram range, it is very unlikely that there are any unidentified nutrients that are essential for life. It is more likely that the quantitative requirements for the known nutrients have not been determined for many of the rodent species currently held in captivity.
RODENT DIETS
Diets used to maintain rodent colonies used in biomedical research are classified according to the basis of the degree of purification of the ingredients: natural-ingredient and purified diet and chemically defined diets.l Natural-ingredient diets are the most readily available and the most widely used diets. Natural-ingredient diets formulated to provide adequate nutrition for many of the rodent species used in biomedical research and some of the captive exotic species are commercially available. Although these are nutritionally well-balanced diets for the attended species, the quantitative ingredient composition, or formulation, of diets manufactured and marketed by commercial institutions is privileged information. Therefore, it is not possible to alter these formulations to accommodate the nutrient requirements of a rodent species that may have even slightly different nutrient requirements or to accommodate the requirements of a specific research project. A nutritionally balanced diet that has been formulated specifically for the species of interest should always be available. Although rodent species may survive when they are fed a diet consisting of a single cereal grain, this practice is discouraged because the nutrient composition of any of the cereal grains does not meet the requirements of any animal species. Natural-ingredient diet formulations in which the quantitative ingredient composition is readily available are presented in Table 3. These formulations can readily be altered to make required changes in their dietary nutrient compositions. Instructions for the f~rmulationof complete rodent diets have been published,17 and most animal feed diet manufacturers have access to computer programs for formulating diets.
DIET STORAGE
Nutrient stability in complete natural-ingredient rodent diets generally increases as environmental temperature and humidity in storage areas decrease. Diets stored at high temperatures and humidity may
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Table 3. EXAMPLES OF RODENT DIET FORMULATIONS Ingredient*
Ground wheat, g Ground corn, g Ground oats, g Wheat middlings, g Fish meal, g Soybean meal, g Alfalfa meal, g Corn gluten nieal, g Brewer's dried yeast, g Dried skim milk, g Soybean oil, g Dried molasses, g Salt, g Dicalcium phosphate, g Ground limestone, g Cobalt, mg Copper, mg Iodine, mg Iron, mg Magnesium, mg Manganese, mg Zinc, mg Vitamin A, IU Vitamin D, IU Vitamin E, mg Vitamin K, mg Biotin, mg Choline, mg Folic acid, mg Niacin, mg Pantothenate, mg Riboflavin, mg Thiamin, mg Pyridoxine, mg Vitamin B,, pg
Conventionalt
230 245 100 100 120 40 30 20 50 25 15 5 12.5 5 0.44 4.4 1.5 132 66 17.6 6060 5070 22 2.9 0.15 570 2.4 33 19.8 3.7 11 1.9 4.4
Autoclavable*
355 210 100 100 90 50 20 20 10 15 5 15 5 0.44 4.4 1.65 66 440 110 11 24,200 4,180 16.5 22 0.13 770 1.1 22 27.5 5.5 71.5 2.2 15.4
*Amount per kilogram of diet. tDiet designated as National Institutes of Health NIH-07.18 *National Institutes of Health, NIH-31 diet specifications NIH-11-137h, 1993.
deteriorate within several weeks, whereas the same diet stored in a freezer may be usable after several years of storage. The nutrients in natural-ingredient rodent diets can be expected to be stable and adequate for more than 168 days when such diets are stored in a cool (< 25°C [V°F]), dry (relative humidity approximately 50%) environment.12 With the exception of vitamin C, the most labile nutrients during diet storage are thiamine and vitamin A. Diets stored for long periods or under unusual environmental conditions should be assayed for at least these vitamins. For species such as the guinea pig, whose diet contains vitamin C that may not be protected by microencapsulation or other processes that protect this vitamin, this food may have to be used within 90 days
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of manufacture owing to the gradual oxidation of vitamin C during storage. Diets formulated with high fat (>lo%) concentrations may have to be stored at temperatures of 4°C (39°F) to retard rancidity as a result of peroxide formation. The refined ingredients used to manufacture purified diets generally do not contain antioxidants. As a result, the nutrients in this type of diet are subject to more rapid oxidation rates than those in natural-ingredient diets. It is recommended that purified diets be stored in 4°C environments when they cannot be used within 45 days of manufacture. DIETARY CONTAMINANTS
Dietary biological and chemical contaminants may have a negative influence on the well-being of captive rodent colonies. It is essential that managers of captive rodent colonies are aware of the potential influence of dietary contaminants. Biological Contaminants
Diets are a potential source of biological agents that may be pathogenic to captive rodents. The kind and concentration of these pathogens depend on such factors as the type of ingredients in the diet, sanitation programs in feed manufacturing facilities, and the protection of the finished product between manufacture and use. For the most part, animal feed users have very limited control over these factors, except in the selection of feed manufacturers or suppliers that implement rigid sanitation programs in facilities where feed ingredients are stored and diets are manufactured and warehoused. However, these efforts may be only partially effective in controlling dietary biological contaminants. The most reliable method to ensure against biological contaminations in rodent diets is to decontaminate diets before use. Steam autoclavingll, 37 and ionizing irradiationz1are widely used methods for decontaminating laboratory animal feeds of biological contaminants. Chemical Contaminants
Potential sources of chemical contaminants in,dietary natural ingredients include the soils where plants producing the ingredients are grown; the use of pesticides and the environmental conditions during the growing and the harvesting season of the ingredients; and the accidental contaminations that occur during processing, storage, and transportation of feed ingredients or complete diets. There are in excess of 50 dietary chemical constituents that are considered potential contaminants. These contaminants include heavy metals, pesticide residues, hormones, nitrosamines, or mycotoxins. Their dietary concentrations are
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generally low and variable,15,31, 32 depending on the ingredients used in a specific diet formulation. Dietary chemical contaminants are of the most concern to scientists conducting toxicology or bioassay studies in which even low concentrations have an influence on experimental results. Chemical contaminant concentrations in rodent diets can be managed when dietary ingredients are used that have a low-contaminant potential and when ingredients for contaminant concentrations are assayed before being used in diet formulation^.^^ SUMMARY
Access to a diet that provides adequate nutrition is one of the most important environmental factors influencing the well-being of rodent colonies. The dietary ingredient and nutrient composition, as well as the potential biological and chemical contaminant concentrations, are factors for consideration in selecting diets for a specific rodent colony. Estimated nutrient requirements have been published for the rodent species that are commonly used in biomedical research. The nutrient concentrations in adequate diets for other captive rodent species that are not used in biomedical research are more difficult to obtain. However, reasonable estimates of their nutrient requirements can be obtained by extrapolation of data from rodent species of a similar metabolic weight and size or from nutrient concentrations of diets that have a history of acceptable performance in the species of interest. Captive rodent colonies should be provided with nutritionally balanced diets with only limited amounts of succulent foodstuffs. The practice of feeding rodent colonies specific cereal grains is discouraged, since no single grain provides a balanced rodent diet. References 1. American Institute of Nutrition (AIN): Ad Hoc committee on standards for nutritional standards. Report of the committee. J Nutr 107:1340-1348, 1977 2. Association of Official Analytical Chemist (AOAC): Official Methods of Analysis of the Association of Official Analytical Chemist, ed 16. Washington, DC, 1995 3. Bell JM: A comparison of fibrous feedstuffs in nonruminant rations. Effects of growth responses, digestibility, rate of passage of ingesta volume. Can J Anim Sci 40:71, 1960 4. Bricker ML, Mitchell HH: The protein requirements of the adult rat in terms of protein contained in egg, milk, and soy flour. J Nutr 34491, 1947 5. Chew RM, Hinegardner RT: Effects of chronic insufficiency of drinking water in white mice. J Mammal 38:361, 1957 6. Daft FS, McDaniel EG, Herman LG, et al: Role of coprophagy in utilization of B vitamins synthesized by intestinal bacteria. FASEB J 22:129, 1963 7. Dalton DC: Effect of dilution of the diet with a filler on feed intake in the mouse. Nature 197:909, 1963 8. Erway L, Hurley LS, Fraser A: Congenital ataxia and otolith defects due to manganese deficiency in mice. J Nutr 100:643, 1970 9. Fenton PF, Cowgill GR: The nutrition of the mouse. I. A difference in the riboflavin requirements of two highly inbred strains. J Nutr 34:273, 1947
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10. Fenton PF, Cowgill GR, Stone MA, et al: Nutrition of the mouse. VIII. Studies on pantothenic acid, biotin, inositol, and p-aminobenzoic acid. J Nutr 42:257, 1950 11. Foster HL, Black CL, Pfau ES: A pasteurization process for pelleted diets. Lab Anim Care 14:373, 1964 12. Fullerton FR, Greenman DL, Kendall DC: Effects of storage conditions on nutritional qualities of semipurified (AIN-76) and natural ingredient (NIH-07) diets. J Nutr 112:567-573, 1982 13. Goettsch M: Comparative protein requirement of the rat and mouse for growth, reproduction and lactation using casein diets. J Nutr 70:307, 1960 14. Goodrick CL: The effects of dietary protein upon growth of inbred and hybrid mice. Growth 37:355, 1973 15. Greenman DL, Oller WL, Littlefield NA, et al: Commercial laboratory animal diets: Toxicant and nutrient variability. J Toxicol Environ Health 6:235-246, 1980 16. John AM, Bell JM: Amino acid requirements for the growing mouse. J Nutr 106:1361, 1976 . 17. Knapka JJ: Formulation of diets. In Beare-Rogers J (ed): Methods for Nutritional Assessment of Fats. American Oil Chemists' Society, 1985, pp 45-59 18. Knapka JJ, Smith KP, Judge FJ: Effect of open and closed formula rations on the performance of three strains of laboratory mice. Lab Anim Sci 24:480, 1974 19. Knapka JJ, Smith KP, Judge FJ: The effect of crude fat and crude protein on reproduction and weanling growth in four strains of inbred mice. J Nutr 107:61, 1977 20. Krishnarao GVG, Draper HH: Influence of dietary phosphate on bone resorption in senescent mce. J Nutr 102:1143, 1972 21. Ley FJ: The use of irradiation for the treatment of various animal feed products. Food Irradiation Information 1%-22, 1975 22. Luckey TD, Bengson MH, Kaplan H: Effect of bioisolation and the intestinal flora of mice upon evaluation of an Apollo diet. Aerosp Med 45:509, 1974 23. Luecke RW, Fraker PJ: The effect of varying dietary zinc levels on growth and antibody mediated response in two strains of mice. J Nutr 109:1373, 1979 24. Marsh JM, Fenton PF: Studies on some hormonal and genetic factors regulating nitrogen release from the isolated diaphragm. Endocrinology 65:916, 1959 25. Menton DN: The effects of essential fatty acid deficiency on the skin of the mouse. Am J Anat 122:137, 1968 26. Menton DN: The effects of essential fatty acid deficiency on the fine structure of mouse skin. J Morphol 132:181, 1970 27. Mickelsen 0 : Intestinal synthesis of vitamins in the non-ruminant. Vitam Horm 14:1, 1956 28. National Research Council, Board on Agriculture, Committee on Animal Nutrition, Subcommittee on Laboratory Animal Nutrition: Nutrient Requirements of Laboratory Animals, revised ed 3. Nutrient Requirements of Domestic Animals Series. Washington, DC, National Academy Press, 1978 29. National Research Council, Board on Agriculture, Committee on Animal Nutrition, Subcommittee on Laboratory Animal Nutrition: Nutrient Requirements of Laboratory Animals, revised ed 4. Nutrient Requirements of Domestic Animals Series. Washington, DC, National Academy Press, 1995 30. Poiley SM: Growth tables for 66 strains and stocks of laboratory animals. Lab Anim Sci 22:759, 1972 31. Rao GN: Significance of dietary contaminants in diet restriction studies. In Fishbein L (ed): Biological Effects of Dietary Restriction. New York, Springer-Verlag, 1991, pp 16-24 32. Rao GN, Knapka JJ: Contaminant and nutrient concentrations of natural ingredient rat and mouse diet used in chemical toxicology studies. Fundam Appl Toxicol 9:329-338, 1987 33. Reeves PG, Nielsen FH, Fahey GC: AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A diet. J Nutr 123:1939, 1993 34. Ross MH, Bras G: Food preference and length of life. Science 190:165, 1975
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35. Ross MH, Lustbader E, Bras G: Dietary practices and growth responses as predictors of longevity. Nature 262548, 1976 36. Scott ML: Vitamin E. In DeLuca HF (ed): Handbook of Lipid Research: Vol 2, The Fat Soluble Vitamins. New York, Plenum Press, 1978, pp 133-210 37. Williams FP, Christie RJ, Johnson DJ, et al: A new autoclave system for sterilizing vitamin fortified commercial rodent diets with lower nutrient loss. Lab Anim Care 18:195, 1968 38. Wostmann BS: Nutrition and metabolism of the germfree mammal. World Rev Nutr Diet 22:40, 1975 39. Zimmerman DR, Wostmann BS: Vitamin stability in diets sterilized for germfree mice. J Nutr 79:318, 1963 ' .
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