n-6 Fatty Acids

The mouse, like the rat, requires linoleic acid to avoid classical signs of EFA deficiency; however, the precise requirement for n-6 fatty acids has not been determined. Cerecedo et al. (1952) reported that 5 mg linoleate/day alleviated clinical signs of EFA deficiency in three mouse strains (C57, DBA, C3H) that had become EFA-deficient after eating a fat-free diet for more than 50 days postweaning. The n-6 stores of the mice were unknown; it is likely that the requirement is higher in the young growing mouse.

The rate of depletion of tissue linoleate is biphasic (Tove and Smith, 1959) with the most rapid loss occurring when linoleate comprised more than 20 percent of the depot fat. Also, female and immature male mice lose linoleate more quickly than mature males during the slower, second phase of depletion. The linoleate requirement for pregnant and lactating mice is unknown, although it should increase during lactation as in the rat.

The previous recommendation (National Research Council, 1978) was 0.3 percent dietary linoleate, based on the n-6 requirement of the rat. However, the present recommendation for a standard rat diet is 0.68 percent of dietary ME as linoleate; therefore, the current recommended amount of dietary linoleic acid for mice is 0.68 percent.

n-3 fatty acids

As in the rat and other mammals, n-3 fatty acids are sequestered in certain tissues in the mouse; thus, an essential function is likely. Two studies (Rivers and Davidson, 1974; Wainwright et al., 1991) have attempted to demonstrate a need for n-3 fatty acids. No attempt is made here to indicate a specific requirement. Gross depletion of tissue 22:6(n-3) in conjunction with abnormalities of the retina have been demonstrated in other species only when fed diets that contain oils with extremely high ratios (150:1) of n-6:n-3 fatty acids (as found in safflower, sunflower, and peanut oil) (Neuringer et al., 1988). Diets containing other common oils with more moderate ratios of n-6:n-3 fatty acids will not result in a depletion of n-3 fatty acids. (See Appendix Table 1 for fatty acid composition of common dietary fats and oils.)

Signs of EFA Deficiency EFA deficiency in the mouse was first described by White et al. (1943) and later by Decker et al. (1950) using weanling mice inbred in their respective laboratories (strain was not reported). Cercedo et al. (1952) produced EFA deficiency in three strains of weanling mice (C57, DBA, C3H), all of which were reported to have dermatitis of the thorax and extremities, scaliness of the ears, alopecia, and growth retardation. Mice fed a fat-free diet also developed lighter colored hair in the lower dorsal region. A "spectacle" eye condition was reported in two-thirds of the DBA mice but only noted occasionally in the C57 mice. The DBA mice developed the EFA deficiency syndrome more rapidly than did the C57 mice. Berkow and Campagnoni (1983) reported reduced myelination and abnormal myelin composition in C57BL/6J female mice fed EFA-deficient diets during the rapid growth phase.

Growth

Of the studies reviewed, none used natural-ingredient diets to estimate the protein requirements of growing mice. For male mice, purified diets will support growth rates up to 1.2 g/day for the 2-week period following weaning. The AIN-76 purified diet (American Institute of Nutrition, 1977), which contains 20 percent casein supplemented with 0.3 percent DL-methionine, will support gains of 0.98 to 1.2 g/day in Carworth Farms No. 1 X Swiss male mice (Bell and John, 1981; Keith and Bell, 1989) (Table 3-4). Others (Maddy and Elvehjem, 1949; Hirakawa et al., 1984; Toyomizu et al., 1988) using either Swiss or Y strain mice have reported growth rates of 0.8 g/day, 1 g/day, and 1 to 1.1 g/day when 19, 22.7, and 27 percent casein diets, respectively, were used. Improving the dietary amino acid pattern by supplementing casein with an equivalent amount of nitrogen as a mixture of indispensable amino acids lowers the protein required for CD-1 mice that gain 1.3 g/day to 15.5 or 16.5 percent of the diet (Bell and Keith, 1992). Females grow more slowly, and lower dietary protein concentrations are needed to maximize growth. Goettsch (1960) found that a diet containing 20 percent casein would support a growth rate of 0.73 g/day in male Swiss mice but that 14 percent would support the growth rate of 0.59 g/day in female Swiss mice. Thus, diets containing 18 percent crude protein, equivalent to 20 percent casein supplemented with methionine or casein alone at 23 to 27 percent of the diet, support growth rates of more than 1 g/day in male mice.

TABLE 3-4

Protein Requirements for Growth for Various Strains of Mice.

Reproduction

Both casein-based purified and natural-ingredient diets have been used in studies of the protein requirement for reproduction and lactation. In a study limited to the first gestation/lactation, a diet containing 16.7 percent casein resulted in the lowest age at first estrus (30.5 days versus 36.7 days) and largest litter size (7.5 versus 7.1), while a diet containing 20 percent casein was needed to support lactation, as demonstrated by the weight of the dam and litter at 21 days in Swiss-STM mice (Goettsch, 1960) (Table 3-5). Natural-ingredient diets varying in composition but containing 18 to 24 percent crude protein have been used to evaluate the protein requirement for mice over 6- to 9-month periods (Bruce and Parkes, 1949; Hoag and Dickie, 1962) or over four to seven litters for five strains [BALB/cAnN, C3H/HeN, C57BL/6N, N:NIH(S), and DBA/2N] (Knapka et al., 1974, 1977). Although strain differences were observed, a natural-ingredient diet containing 18 percent crude protein supported litter sizes of six to seven and a weaning percentage of 80 to 85 percent over four to five litters (Knapka et al. 1974, 1977). Thus natural-ingredient diets containing 18 percent crude protein from a mixture of animal and plant proteins will meet the protein needs of gestating/lactating mice through several pregnancies.

TABLE 3-5

Protein Requirements for Reproduction for Various Strains of Mice.

Growth

Differences in growth potential among strains have been observed (see Table 3-6). Mice of the C57BL/6 strain gained 0.44 g/day, while CD-1 mice gained 0.68 g/day (Olejer et al., 1982); and Swiss-Webster mice gained 0.4 g/day, while the Rockland strain gained 0.78 g/day (Maddy and Elvehjem, 1949). Few studies have focused on estimating amino acid requirements of mice (John and Bell, 1976; Bell and John, 1981). The requirement for L-arginine is suggested to be less than 0.1 percent for mice gaining 0.8 g/day (Bell and John, 1981) and less than 0.3 percent for mice gaining 0.9 g/day (John and Bell, 1976). Milner et al. (1975) report evidence of arginine deficiency in BFDSCH mice that gained 0.4 g/day when fed a diet devoid of L-arginine. Bauer and Berg (1943) found that arginine could be deleted from the DL-amino acid diet of mice gaining 0.11 g/day. A level of 0.3 percent arginine should meet the requirements of mice with a growth potential of 1 g/day. The requirement for L-histidine is 0.2 percent of the diet for mice gaining more than 1 g/day (John and Bell, 1976). Olejer et al. (1982) showed that 0.1 percent would meet the needs of C57BL/6 mice growing 0.4 g/day but that 0.2 percent was required for CD-1 mice, which grew 0.7 g/day. L-carnosine at 0.29 percent can replace 0.2 percent L-histidine (Olejer et al., 1982). Parker et al. (1985) showed that deletion of L-histidine from the amino acid mixture resulted in weight loss and that the single-test concentration of 0.33 percent L-histidine met the needs of Swiss-Webster mice gaining 0.3 g/day. The L-histidine requirement for growing mice seems to be met at 0.2 percent of the diet. The L-isoleucine requirement is 0.4 percent of diet for mice gaining 1.0 g/day (John and Bell, 1976). Diets containing 0.7 percent and 0.5 percent L-leucine and L-valine, respectively (John and Bell, 1976), meet the needs of mice growing 1 g/day; therefore, these concentrations are set as the requirements. The requirement for L-threonine is set at 0.4 percent of diet since it will support a gain of 1.1 g/day in Carworth Farms No. 1 X Swiss mice (John and Bell, 1976). The L-lysine requirement of 0.4 percent of diet for mice gaining 0.9 g/day (John and Bell, 1976) was confirmed (Bell and John, 1981). The L-methionine requirement is 0.5 percent of diet for mice gaining 1 g/day (John and Bell, 1976). L-cysteine may replace as much as one-half to two-thirds of methionine in diets of mice gaining 1 g/day, and D-cysteine does not spare L-methionine (Friedman and Gumbmann, 1984a). D-methionine may have a value as high as 60 percent that of L-methionine in mice gaining 1 g/day (Friedman and Gumbmann, 1984a). The L-phenylalanine requirement of 0.4 percent of diet is supported by the work of John and Bell (1976) and Bell and John (1981). In estimates of the requirement for L-phenylalanine, dietary L-tyrosine must be taken into account as it may replace as much as 50 percent of L-phenylalanine (Friedman and Gumbmann, 1984b). Growth rates of 1.2 g/day in Swiss-Webster mice required 0.76 percent of L-phenylalanine or 0.38 percent L-phenylalanine + 0.38 percent L-tyrosine (Friedman and Gumbmann, 1984b). D-phenylalanine has a growth promoting value that is one-third that of L-phenylalanine. Based on the above discussion, the requirement of L-phenylalanine + L-tyrosine is set at 0.76 percent of the diet (where L-tyrosine may replace 50 percent of L-phenylalanine). The requirement for L-tryptophan of 0.1 percent of diet for mice gaining 0.9 g/day (John and Bell, 1976) was confirmed (Bell and John, 1981). MacEwan and Carpenter (1980) showed that 0.05 percent niacin reduced the L-tryptophan requirement from 0.125 percent to 0.1 percent of the diet in C3HeJ mice gaining 0.7 g/day. The requirement for L-tryptophan is retained at 0.1 percent of the diet.

TABLE 3-6

Amino Acid Requirements for Growth for Various Strains for Mice.

Reproduction

No studies were found regarding estimated amino acid requirements for gestation and lactation. Concentrations similar to those listed for growth can be expected to meet the requirements for gestation. Higher concentrations may be required for lactation.

Calcium and Phosphorus

Reports regarding the quantitative calcium and phosphorus requirements of mice have not been published; therefore, the estimated requirements for these minerals are based on the dietary concentrations that have resulted in acceptable performance in mice. Purified diets that contain 4.0 g Ca/kg diet and 3 to 12 g P/kg diet (Morris and Lippincott, 1941; Mirone and Cerecedo, 1947), 5.0 g Ca/kg diet (Wolinsky and Guggenheim, 1974), and 8.0 g Ca/kg diet and 4.0 mg P/kg diet (Bell and Hurley, 1973) have been shown to support growth and reproduction in mice. Natural-ingredient diets containing 12 g Ca/kg and 8.6 g P/kg (Knapka et al., 1974, 1977) also have been reported to support growth and reproduction in BALB/cAnN, C57BL/6N, N:NIH(S), C3H/HeN, and DBA/2N mice.

Limiting dietary phosphorus to 3.0 g P/kg appears to promote bone calcification in mice. Bell et al. (1980) found that female B6D2F₁ mice had higher concentrations of calcium and phosphorus in their bones when fed 6.0 g Ca/kg diet with 3.0 g P/kg diet than when fed 6.0 to 24.0 g Ca/kg diet with 12.0 g P/kg. Further work demonstrated that female B6D2F₁ mice grew better when fed 15 percent casein with 6.0 g Ca/kg and 3.0 g P/kg diet (basal diet) than when fed 15 percent or 30 percent protein with 6.0 g Ca/kg and 12.0 g P/kg diet (Yuen and Draper, 1983). Moreover, bone calcium concentrations were higher when mice were fed the basal diet than when they were fed the diets containing elevated concentrations of phosphorus.

Little has been written about the problem of nephrocalcinosis in mice. However, Yuen and Draper (1983) observed that calcium concentrations in the kidneys of B6D2F₁ mice more than doubled when dietary phosphorus was increased from 3.0 g P/kg to 12.0 g P/kg and protein was held constant at 15 percent of the diet. This suggests that excess dietary phosphorus has the same negative effects in mice as in rats; and as regards calcium, there is no evidence to indicate that the requirements for calcium are greater for mice than rats. Therefore, the 5.0 g Ca/kg diet and 3.0 g P/kg diet estimated as the requirements for the rat are also the estimated requirements for the mouse.

Signs of Calcium and Phosphorus Deficiency Wolinsky and Guggenheim (1974) and Ornoy et al. (1974) reported that Swiss mice consuming a diet containing only 0.2 g Ca/kg experienced decreased weight gain, bone ash, and serum calcium. These effects were much less marked in mice than in rats, however. Mice increased the concentration of calcium-binding protein in the duodenal mucosa and reduced skeletal growth. Decreased growth rather than osteoporosis was the more prominent sign of deficiency.

Chloride

(See ''Sodium and Chloride" section.)

Magnesium

Magnesium has been shown to be a dietary essential for mice, but the optimal intake for this species has not been well established. Alcock and Shils (1974) reported that mice fed diets containing 20 mg Mg/kg diet developed signs of deficiency, but these signs did not develop when the diet contained 400 mg Mg/kg diet. Fahim et al. (1990) reported that mice fed a diet containing 111 mg Mg/kg diet grew more slowly and had lower concentrations of magnesium in their serum than mice fed a diet containing 335 mg Mg/kg diet. A purified diet containing 730 mg Mg/kg diet supported normal growth and development of mice (Bell and Hurley, 1973). Dubos et al. (1968) reported sudden death in lactating mice fed a diet containing 500 mg Mg/kg diet but not in mice fed a diet containing 700 mg Mg/kg. This parallels the finding for rats (Hurley et al., 1976) that the magnesium requirement for lactation is higher than for growth. Natural-ingredient diets containing 1,800 and 2,600 mg Mg/kg diet provided for good growth and reproduction in three mouse strains (Knapka et al., 1974). The magnesium concentration in the widely used AIN-76 (American Institute of Nutrition, 1977) purified diet is 500 mg/kg. Since the data regarding the quantitative magnesium requirements for mice are inconsistent and not definitive, 500 mg Mg/kg diet is the estimated requirement for this species, and the requirement for lactation may be as high as 700 mg/kg, at least for some strains.

Signs of Magnesium Deficiency Alcock and Shils (1974) reported magnesium-deficient mice developed rapid and usually fatal convulsions without previous hyperirritability. Soft tissue calcification resulting from magnesium deficiency has been reported in the hereditarily diabetic KK mouse strain (Hamuro et al., 1970).

Phosphorus

(See "Calcium and Phosphorus" section.)

Potassium

Bell and Erfle (1958) found that mice (Carworth Farms No. 1) fed purified diets with potassium concentrations of 2.0 g/kg diet did not exhibit signs of potassium deficiency, such as poor growth, inanition, lusterless eyes and hair, and dry scaly tails. A natural-ingredient diet containing 8.2 g K/kg diet (Knapka et al., 1974) and a purified diet containing 8.9 g K/kg (Bell and Hurley, 1973) supported good growth and reproduction in mice. Based on these results, the estimated required potassium concentration for mice is 2.0 g K/kg diet.

Sodium and Chloride

Sodium and chloride requirements of mice have not been studied. Two natural-ingredient diets containing 3.6 and 4.9 g Na/kg diet (Knapka et al., 1974) and a purified diet containing 3.9 g Na/kg diet (Bell and Hurley, 1973) are known to support good growth and reproduction. However, the estimated requirement for sodium and chloride is 0.5 g Na/kg diet and 0.5 g Cl/kg diet, which is identical to that estimated for rats. The actual requirements may be lower for mice. Rowland and Fregley (1988) observed that adrenalectomized mice are not as dependent as adrenalectomized rats on supplemental sodium. Mice are also less likely than rats to ingest saline solutions.

Copper

Specific studies to determine copper requirement of young growing mice have not been published. Knapka et al. (1974) reported satisfactory growth and reproduction by feeding mice a natural-ingredient diet containing 16 mg Cu/kg diet. Hurley and Bell (1974) reported adequate growth and development in young mice when they were fed a purified diet containing 4.5 mg Cu/kg diet. Mulhern and Koller (1988) showed that C57BL/6J weanling male and female mice fed an egg white-glucose-based purified diet containing 2 mg Cu/kg diet maintained values for serum ceruloplasmin activity and immune response for 8 weeks that were not significantly different from mice fed diets containing 6 mg Cu/kg diet; however, 1 mg/kg was not sufficient.

Reeves et al. (1994) used nonlinear modeling techniques to estimate the copper requirement of adult male Swiss-Webster mice fed the AIN-93M purified diet. By feeding mice a range of dietary copper, from 0.8 to 6.5 mg/kg for 12 weeks, they estimated that minimal dietary concentrations of 2.5 and 4 mg Cu/kg diet were required to maintain maximal concentrations of serum copper and serum ceruloplasmin activity, respectively. However, under other environmental and dietary conditions the copper requirement for adult male mice might be more than 4 mg/kg diet.

Based on these limited results, the estimated minimal requirement for both immature and adult mice is set at 6 mg Cu/kg diet. No information is available about the requirements for pregnancy and lactation. However, because of the similarity between the estimated requirement for copper in young rats (Johnson et al., 1993; Klevay and Saari, 1993) and adult mice (Reeves et al., 1994), the estimated requirement for pregnancy and lactation in mice is similar to that for rats; 8 mg Cu/kg diet. High dietary concentrations of zinc, cadmium, and ascorbic acid may increase the dietary requirement for copper (Davis and Mertz, 1987).

Signs of Copper Deficiency Copper-deficient mice have low plasma copper concentrations, low plasma ceruloplasmin activity, anemia, enlarged hearts, altered catecholamine metabolism, thymus and spleen atrophy, and low hepatic cytochrome P-450 concentrations (Prohaska and Lukasewycz, 1989a,b; Gross and Prohaska, 1990; Prohaska, 1990; Phillips et al., 1991; Arce and Keen, 1992).

Signs of Copper Toxicity Mice are relatively resistant to copper toxicosis. Pregnant mice fed diets containing 2,000 mg Cu/kg diet throughout gestation did not carry litters to term; when the high-copper diet was restricted to days 7 to 12 of gestation, the resorption frequency was higher than 50 percent and surviving fetuses were normal. The diet's toxicity to embryos was apparently caused by an indirect effect of reduced food intake rather than by a direct effect of excess copper on the fetus (Keen et al., 1982).

Iron

Sorbie and Valberg (1974) reported that iron concentrations of 25 to 100 mg Fe/kg diet supported normal growth and hematopoiesis in male C57BL/6J mice, although, liver iron storage in these animals was low compared to mice fed natural-ingredient diets containing between 220 to 240 mg Fe/kg diet. When the dietary iron concentration was increased to 120 mg Fe/kg diet, liver iron stores were similar to those obtained with the natural-ingredient diet. The 120 mg Fe/kg diet supported good reproduction for three generations. The requirement for iron is set at 35 mg/kg diet based on the concentration in the widely used purified diet, AIN-76 (American Institute of Nutrition, 1977). Higher concentrations may be necessary for reproduction. Two natural-ingredient diets known to provide good health and reproduction in three mouse strains contained between 198 and 255 mg Fe/kg diet (Knapka et al., 1974).

Signs of Iron Deficiency Compared to controls fed a diet containing 122 mg Fe/kg diet, male CD-1 mice fed a low-iron diet (2 mg Fe/kg) for 30 days were characterized by low body weights, anemia, and suppressed T-lymphocyte-dependent functions associated with antibody production and blastogenesis (Blakley and Hamilton, 1988). Kuvibidila et al. (1990) reported similar T-cell abnormalities in female C57BL/6 mice fed low-iron diets (10 mg Fe/kg) for 40 days; in addition, these investigators noted a reduction in mature B-cell populations.

Signs of Iron Toxicity NMR1 mice fed high-iron diets (ranging from 0.5 to 3.5 percent Fe-fumarate) for 4 weeks were characterized by iron concentration-dependent increases in liver and colon iron concentrations and tissue lipid peroxidation (Younes et al., 1990).

Manganese

Manganese concentrations of 3 mg/kg diet or less are clearly inadequate for optimal growth and development of several mouse strains, while diets containing 45 to 50 mg Mn/kg diet are adequate for all criteria tested (Hurley and Bell, 1974; Hurley and Keen, 1987). Consumption of diets containing 5 mg Mn/kg throughout gestation and lactation resulted in maternal and weanling tissue manganese concentrations and liver manganese superoxide dismutase and arginase activities that were similar to those observed for mice fed diets containing 45 mg Mn/kg diet (C. K. Keen and S. Zidenberg-Cherr, Dept. of Nutrition, University of California, Davis, 1990, unpublished data). Given the lack of data supporting a dietary requirement of manganese in excess of 5 mg/kg diet, the estimated requirement for manganese has been reduced to 10 mg/kg diet to account for possible differences among various strains. This is lower than the 1978 NRC recommendation of 45 mg/kg diet and reflects the absence of data supporting the need for such a high concentration of manganese coupled with the possible negative effects of excess manganese on iron metabolism (Hurley and Keen, 1987).

Signs of Manganese Deficiency A deficiency of manganese during prenatal development can result in congenital irreversible ataxia, which is characterized by lack of equilibrium and retraction of the head. The ataxia is caused by abnormal development of the otoliths (Erway et al., 1970; Hurley and Keen, 1987). Prenatal manganese deficiency can result in an increased frequency of early postnatal death, although birth weight and early postnatal body weight gain are not typically affected (Hurley and Bell, 1974). Offspring fed manganese-deficient diets into later life can show obesity and fatty livers and abnormalities in cellular ultrastructure including altered integrity of cell and mitochondrial membranes, which may be linked in part to alterations in the free radical defense system (Bell and Hurley, 1973; Zidenberg-Cherr et al., 1985).

Zinc

Using weight gain, tissue zinc concentration, and response to immunization as criteria, weanling and adult mice housed individually in wire-bottom stainless steel cages have a dietary zinc requirement on the order of 10 mg/kg diet when egg white or casein is used as the primary protein source (Luecke and Fraker, 1979; Morgan et al., 1988a,b). The requirement is higher when soybean protein is used (»20 mg/kg), presumably because of its high phytic acid content (Beach et al., 1980).

Precise requirements for pregnancy and lactation have not been established. A concentration of 5 mg Zn/kg diet has been reported to be inadequate (Beach et al., 1982, 1983), while satisfactory reproduction has been demonstrated using diets containing 30 mg Zn/kg diet or more (Bell and Hurley, 1973; Knapka et al., 1974; Beach et al., 1982, 1983; Keller and Fraker, 1986). Based on these results, the estimated dietary zinc requirement for growing and adult mice is 10 mg/kg diet and for pregnant and lactating dams is 30 mg/kg diet. The dietary requirement for zinc can be influenced by housing conditions; for example, mice maintained in galvanized cages, in cages with a solid bottom, or in groups have a lower requirement for "dietary" zinc because zinc is available from cage materials and feces.

Signs of Zinc Deficiency An inadequate intake of zinc is characterized by marked reductions in plasma zinc, which occur within a few days (Peters et al., 1991), followed by subsequent mild-to-severe anorexia (Beach et al., 1982, 1983). Prolonged consumption of a zinc-deficient diet can result in growth retardation/failure, alopecia, atrophy of lymphoid tissue, significant impairment of multiple components of the immune system, and alterations in lipid and protein metabolism (Nishimura, 1953; Beach et al., 1980; Hambidge et al., 1986; Morgan et al., 1988a,b; Keen and Gershwin, 1990). The introduction of a zinc-deficient diet during pregnancy can result in severe embryonic and fetal pathologies including prenatal death and a high incidence of central nervous system, soft tissue, and skeletal defects and postnatal behavioral abnormalities (Golub et al., 1986; Keen and Hurley, 1989).

Signs of Zinc Toxicity Mice are relatively resistant to zinc toxicosis; Aughey et al. (1977) reported no significant effects associated with giving 500 mg Zn/L water for up to 14 months.

Iodine, Molybdenum, and Selenium

Negative effects on the physiological or biochemical status of mammals have been shown if the diet is unsupplemented with iodine, selenium, and molybdenum. Cobalt is essential but only as a part of vitamin B₁₂. No systematic effort has been made, however, to establish the requirements of iodine, selenium, and molybdenum for the mouse.

Iodine deficiency was produced in mice by feeding them diets containing 20 µg I/kg diet for 8 weeks (Many et al., 1986). These mice experienced enlarged thyroid glands when compared to controls fed 200 µg I/kg diet. Marginal iodine deficiency was produced in mice consuming diets with 42 µg I/kg diet (Van Middlesworth, 1986). Mice were able to adapt to a low-iodine intake by maintaining normal concentrations of iodine in the thyroids. When they were challenged with a mycotoxin, however, the iodine concentration decreased. There was no effect of the toxin on thyroid iodine content in mice fed 150 µg I/kg diet.

There are a number of reports on producing selenium deficiency in mice. The amount of dietary selenium that caused a considerable reduction in the activity of liver glutathione peroxidase ranged from 10 to 16 µg/kg diet .

Control mice in these studies were fed selenium concentrations ranging from 330 to 500 µg Se/kg diet (Wendel and Otter, 1987; Otter et al., 1989; Toyoda et al., 1989; Weitzel et al., 1990; Peterson et al., 1992).

Based on these works, it seems reasonable to suggest that the minimal requirement of selenium and iodine for the mouse might be at least as much as for the rat. Data for molybdenum requirements of the mouse are even more sparse than those for selenium or iodine, and it is suggested that those values established for the rat are good estimates for the mouse (see Table 3-3).

Vitamin A

It has been shown by Wolfe and Salter (1931) that vitamin A is required by the mouse, and the mouse has been used extensively in studies of vitamin A metabolism and of the role of vitamin A in the prevention of cancer. Little work has been done to establish the vitamin A requirement of mice, however, and depleting the mouse of its vitamin A stores is difficult (McCarthy and Cerecedo, 1952). If rapid depletion is desired, it is necessary to use the pups from a pregnant female fed a vitamin A-deficient diet from about day 10 of gestation; this will produce very low vitamin A stores in the pups (Smith, 1990). Young mice weaned from dams fed a standard diet may require up to 1 year to show overt signs of deficiency. Santhanam et al. (1987) have explored methods to slowly produce vitamin A deficiency in mice. Both BALB/c and Swiss mice eventually developed deficiency signs when fed a cereal grain-based diet calculated to contain 1,200 IU vitamin A/kg (roughly equivalent to 1.2 µmol retinol/kg diet). BALB/c mice maintained good health, showed good growth, and stored modest liver reserves of retinyl esters when fed a diet calculated to contain 2,400 IU vitamin A/kg (2.5 µmol/kg). The AIN-76 diet was formulated to contain 4,000 IU vitamin A/kg (4.2 µmol retinyl esters/kg). This diet has been adequate for normal growth and reproduction in mice (American Institute of Nutrition, 1977).

When fed a natural-ingredient diet that contained 13,371 IU/kg (roughly 14 µmol/kg), A/J mice were found to accumulate vitamin A reserves in their livers as they increased in age from 9 to 216 days old (Sundboom and Olson, 1984). However, 644-day-old A/J mice were found to have about one-half the liver vitamin A stores observed in mice 216 days old.

Based on these limited data, the vitamin A (retinol) requirement of the mouse seems to be similar to the requirement of the rat. Therefore, a dietary concentration of 2,400 IU/kg diet (2.5 µmol/kg diet; 0.72 mg/kg diet) is adequate to meet the requirements of the mouse.

Ideally, retinyl esters should be added to animal diets in stabilized gelatin beadlets, which will protect the vitamin A from oxidation. An alternative procedure is to slowly dissolve the retinyl esters in the dietary lipid, which contains an antioxidant, before the lipid is mixed into the diet. If the second procedure is used, the diet should be freshly prepared at least every other week. Dissolving the retinyl esters in a solvent and adding them directly to the other dietary constituents without the protection afforded by the dietary oils or by gelatin beadlets will result in substantial oxidative destruction of the vitamin. The storage and treatment of the diet are also very important. Zimmerman and Wostmann (1963) reported that vitamin A activity was decreased by 20 percent as a result of steam sterilization.

Signs of Vitamin A Deficiency One of the early and significant consequences of vitamin A deficiency is impairment of the functional immune system (Smith et al., 1987). If conditions are sanitary the main overt sign observed early in deficiency is a decreased rate of weight gain. As the deficiency progresses many epithelial tissues become keratinized, including those of the seminal vesicles, testes (Van Pelt and De Rooij, 1990), bladder, kidney, trachea, esophagus, salivary glands, and lungs (McCarthy and Cerecedo, 1952). Xerophthalmia of the eye occurs if the mice are exposed to unsanitary conditions or are subjected to stress.

Signs of Vitamin A Toxicity The studies of vitamin A toxicity in mice focused on the teratological aspects of the toxicity. Kochhar et al. (1988) have found that a single dose of 349 µmol/kg BW on day 10.5 of gestation produced cleft palates and limb deformities in ICR mice. A lower dose of 175 µmol/kg BW did not produce the deformities. Giroud and Martinet (1962) reported that three doses of 6.5 µmol vitamin A/kg BW on days 8, 9, and 10 of gestation caused death or resorption of 63 percent of the fetuses and malformations in others.

Vitamin D

The AIN-76 diet was formulated to contain 0.025 mg cholecalciferol/kg (0.65 µmol or 1,000 IU/kg) (American Institute of Nutrition, 1977). This amount of vitamin D is adequate and may represent a considerable excess. However, a lesser amount cannot be recommended until the more sensitive criterion of vitamin D status has been evaluated at lower intakes.

Signs of Vitamin D Deficiency The mouse is quite resistant to the development of rickets—a disease caused by vitamin D deficiency. Beard and Pomerene (1929) found that mice fed vitamin D-deficient diets developed signs of rickets within 7 to 14 days. Rickets spontaneously healed between days 20 to 27 without vitamin D supplementation, but osteoporosis was present in many of the animals after healing. Delorme et al. (1983) found that both the 10,000 and 25,000 molecular weight kidney vitamin D-dependent calcium binding proteins were reduced to about one-third the normal concentrations in vitamin D-deficient Swiss mice. In contrast, milk production and the calcium content of the milk were normal in CD-1 mice fed a vitamin D-deficient diet (Allen, 1984).

Signs of Vitamin D Toxicity The LD₅₀ of a single intraperitoneal injection of cholecalciferol for CF₁ mice was found to be 355 µmol/kg BW (Hatch and Laflamme, 1989). No toxicity was found after the injection of 104 µmol/kg (1.7 × 10⁶ IU/kg). In contrast, only 5.5 nmol 1,25-dihydroxycholecalciferol/kg was required to produce toxicity in C57BL/6J mice (Crocker et al., 1985).

Vitamin E

Bryan and Mason (1940) observed fetal resorption in vitamin E-deficient female mice similar to that observed in rats but observed no evidence of testicular injury in vitamin E-deficient males. They reported that administration of 81 nmol all-rac-α-tocopherol daily for the first 10 days of gestation was adequate to maintain the first pregnancy. This corresponds to a dietary concentration of 10 IU RRR-α-tocopherol/kg diet (15.7 µmol/kg). Goettsch (1942) found that a single dose of vitamin E equivalent to 1.8 IU to 2.4 IU RRR-α-tocopherol (1.16 to 1.55 µmol) given at the start of the gestation period was adequate to maintain pregnancy in mice between 3 and 6 months old. Mice 7 to 12 months old required a larger dose equivalent to 5 IU RRR-α-tocopherol (7.78 µmol) to maintain pregnancy. Trostler et al. (1979) found that male C57BL/6J mice fed a diet containing the equivalent of 62 µmol RRR-α-tocopherol/kg diet (lower dose not used) had a growth rate equal to or greater than that observed with mice fed 124 µmol/kg diet. However, more than 124 µmol/kg diet was required to prevent the accumulation of malondialdehyde in liver and adipose tissue. Yasunaga et al. (1982) gave daily intraperitoneal injections of all-rac-α-tocopherol to BALB/c mice and measured their response to mitogens. The best responses were obtained in mice injected with amounts equivalent to 7.8 to 28 µmol RRR-α-tocopherol/kg BW. The lower dose is equivalent to 50 µmol RRR-α-tocopherol/kg diet. Based on these data the vitamin E requirement for mice is estimated to be 22 mg/kg or 32 IU/kg RRR -α-tocopherol/kg diet (50 µmol/kg diet) when lipids comprise less than 10 percent of the diet. When all-rac-α-tocopheryl acetate is used as the dietary source, the equivalent amount would be 32 mg/kg diet.

Signs of Vitamin E Deficiency Pappenheimer (1942) reported muscular dystrophy and hyaline degeneration in vitamin E-deficient mice but at a lower incidence than was observed in rats. No lesions were found in the central nervous system. Davies et al. (1987) did not find an accumulation of lipofuscin in neural tissues. The only tissue to show lipofuscin accumulation in vitamin E deficiency was the liver (Csallany et al., 1977). Spermatogenesis remained active in vitamin E-deficient mice for up to 439 days (Pappenheimer, 1942).

Signs of Vitamin E Toxicity Yasunaga et al. (1982) found that male C3H/He mice injected intraperitoneally daily with 212 µmol all-rac -α-tocopherol/kg BW showed a weight loss by day 7. Injections of 846 µmol/kg BW/day were lethal. α-Tocopheryl quinone, a major metabolite of α-tocopherol, has been found to interfere with the mouse's ability to metabolize vitamin K and resulted in bleeding (Woolley, 1945).

Vitamin K

Vitamin K has not been considered essential for mice reared under conventional conditions because of the substantial contribution from coprophagy. However, with the increased use of specific-pathogen-free animals for research, this is probably no longer true. Both specific-pathogen-free CF₁ mice (Fritz et al., 1968) and germ-free ICR/JCL mice (Komai et al., 1987) were reported to die quickly from hemorrhagic diathesis when fed vitamin K-free diets. Addition of 16 µmol menadione/kg to the diet prevented hemorrhaging problems in the specific-pathogen-free mice. Studies using the more sensitive criterion of vitamin K status have not been conducted with mice as they have been with rats (Kindberg and Suttie, 1989). Therefore, the estimated requirement of vitamin K for mice is 1 mg phylloquinone/kg diet (2.22 µmol/kg diet), based on the requirement of the rat.

Vitamin B12

Intestinal bacteria in mice synthesize undetermined amounts of vitamin B₁₂ that can be utilized by the host. The presence of endogenous vitamin B₁₂ generally confounds attempts to determine the quantitative requirements of this vitamin for mice. Jaffé (1952), however, reported a vitamin B₁₂ requirement in excess of 5 µg/kg diet for growth and between 4 and 5 µg/kg diet for reproduction and lactation. Lee et al. (1962) demonstrated that mice require vitamin B₁₂ for gestation. The widespread use of the AIN-76 (American Institute of Nutrition, 1977) purified diet containing 10 µg vitamin B₁₂/kg has not resulted in any reports of vitamin B₁₂ deficiency signs. This indicates that the vitamin B₁₂ concentration in the AIN-76 diets is adequate for mice. In the absence of more recent and definitive data regarding the vitamin B₁₂ requirements for mice, 10 µg vitamin B₁₂/kg diet is the estimated requirement for this species. However, it is noted that lower dietary concentrations may be adequate for mice with conventional intestinal flora, but higher concentrations may be required when the availability of endogenous B₁₂ is limited under conditions such as antibiotic feeding, germ-free environments, or coprophagy prevention.

Signs of Vitamin B₁₂ Deficiency Young mice deficient in vitamin B₁₂ show retarded growth and renal atrophy (Lee et al., 1962). Deficiency causes death both before and after birth.

Biotin

In contrast to other rodent species, mice fed purified casein-based diets appear to have a requirement for biotin that exceeds the amount obtained from coprophagy. Several investigators have observed signs of biotin deficiency or suboptimal weight gain when mice were fed biotin-deficient diets (Nielsen and Black, 1944; Fenton et al., 1950; Lakhanpal and Briggs, 1966). Fenton et al. (1950) found that 0.823 µmol biotin/kg diet was adequate for the mouse, but they did not use other concentrations. The AIN-76 diet was formulated to contain 0.2 mg biotin/kg diet (0.82 µmol biotin/kg). In the absence of more recent and definitive data regarding the dietary requirements of mice, the concentration of 0.2 mg biotin/kg diet is the estimated safe and adequate dietary concentration.

Signs of Biotin Deficiency Watanabe and Endo (1989, 1991) observed teratogenic effects of biotin deficiency in mice fed a spray-dried egg white diet (containing avidin). The teratogenic effects were much more severe in the fast-growing ICR and C57BL/6N strains than in the slower growing A/Jax mice. The other signs of deficiency include alopecia, achromotrichia, and growth failure, as well as decreased reproduction and lactation efficiency (Nielsen and Black, 1944).

Choline

Choline was first recognized as a dietary essential for the mouse by Best et al. (1932), who observed fatty livers in choline-deficient mice. Since choline can be synthesized from methionine (see Chapter 2), and its metabolism is influenced by folic acid and vitamin B₁₂ , a minimum requirement for choline is difficult to establish. Meader and Williams (1957) found that mice fed a diet containing 80 g casein/kg and 400 g lard/kg required 5 g choline chloride/kg diet (35,800 µmol/kg) to support growth and prevent lipid accumulation in the liver. However, Williams (1960) found this level of choline to be toxic in long-term studies. Therefore, caution should be exercised in adding high concentrations of choline to the diet. The widely used AIN-76 diet was formulated to contain 2 g choline bitartrate/kg (7,900 µmol/kg). This amount provides an adequate concentration of choline for diets containing optimal concentrations of methionine. Thus 2 g choline bitartrate/kg diet (7,900 µmol/kg) is the estimated safe and adequate dietary concentration.

Signs of Choline Deficiency Choline-deficient mice had fatty livers with modular parenchymal hyperplasia and lower conception rates with low viability of the young (Mirone, 1954; Buckley and Hartroft, 1955; Meader and Williams, 1957). In contrast to earlier descriptions of fibrosis, Rogers and MacDonald (1965) observed that C57BL mice, unlike rats, did not develop cirrhosis or fibrosis of the liver but only fatty livers. There were acute and chronic inflammations on necrosis of individual hepatic cells. Proliferation of parenchymal cells increased with fat deposition. There was increased thymidine uptake by endothelial, perivascular, and parenchymal cells. Fifty-four percent of the choline-deficient mice died during a 24-week period.

Signs of Choline Toxicity Choline is a very toxic nutrient with a narrow margin of safety. Williams (1960) observed that a dietary concentration of 5 g choline chloride/kg diet (35,800 µmol/kg) induced weight loss in BALB/c mice after they were fed that diet for 6 months, and there were no survivors after 9 months. After 15 weeks 52 percent of the mice had myocardial lesions and by 33 weeks 100 percent of the mice had myocardial lesions. The lesions were most frequently fibrosis with limited necrosis of muscle fibers and fibroid necrosis of coronary arteries (Thomas et al., 1968).

Folates

Weir et al. (1948) documented the essentiality of folic acid in growing mice. Fenton et al. (1950) obtained satisfactory growth in mice fed defined diets containing 0.5 mg folic acid/kg diet (1.1 µmol/kg). Heid et al. (1992) found that 0.4 to 0.5 mg/kg diet (0.9 to 1.1 µmol/kg) was necessary for successful pregnancy outcome in Swiss-Webster mice. Based on these results, 0.5 mg folic acid/kg diet (1.1 µmol/kg) is the estimated requirement for mice.

Signs of Folate Deficiency Weir et al. (1948) observed the following effects after feeding mice a folate-deficient diet for 50 days: a decrease in white cell count from 6,000 to 4,000/mm³, disappearance of megakaryocytes and nucleated cells from the spleen and hemosiderin accumulation, and disappearance of normal cell types from bone marrow. Other signs of deficiency reported include impaired antibody response (Rothenberg et al., 1973), decreased organ growth—particularly brain and liver (Shaw et al., 1973)—and decreased fetal implantations and increased resorption (Heid et al., 1992).

Niacin

Adequate data are not available to estimate the niacin requirement of mice. Male BK albino mice have been shown to convert [14C]tryptophan to N-methyl-nicotinamide, a urinary metabolite of niacin (Bender et al., 1990). The mouse may require increased niacin when tryptophan is fed at suboptimal concentrations. Based on the requirements of the rat, a dietary concentration of 15 mg nicotinic acid/kg diet (120 µmol/kg) is the estimated requirement for mice under the most adverse conditions (Hundley, 1949).

Pantothenic Acid

Sandza and Cerecedo (1941) found that subcutaneous injections of 63 nmol Ca-d-pantothenate 6 days each week would maintain an optimal growth rate in albino mice. Morris and Lippincott (1941) reported that a diet containing 21 µmol Ca-pantothenate/kg produced growth equivalent to a diet containing 168 µmol/kg in C3H mice. Fenton et al. (1950) obtained maximal growth in C57 mice with diets containing 13 µmol/kg diet, but more than 17 µmol/kg diet was required for optimal growth in the A and C3H strains of mice. Based on these limited data, 10 mg Ca-d-pantothenate/kg diet (21 µmol/kg) seems to be adequate for optimal growth in most strains of mice, but some strains may have higher requirements. Data on the requirement for pregnancy and lactation are not available, but the AIN-76 diet was formulated to contain 16 mg Ca-d-pantothenate/kg diet (33.6 µmol/kg). This diet has been shown to be adequate to support pregnancy and lactation in mice.

Signs of Pantothenic Acid Deficiency The following pantothenic acid-deficiency signs in growing mice were reported by Morris and Lippincott (1941): loss of weight; loss of hair, particularly of the ventral surface, flanks and legs; dermatosis; partial posterior paralysis; other neurological abnormalities; and achromotrichia.

Vitamin B6 (Pyridoxine, Pyridoxal, Pyridoxamine)

According to Miller and Baumann (1945) and Morris (1947), mice grew satisfactorily when fed diets containing 1 mg pyridoxine-HCl/kg diet. Pyridoxamine and pyridoxal were found to be less active than pyridoxine. Bell et al. (1971) found 0.2 mg pyridoxine-HCl/kg diet limited growth in two strains, whereas 8.2 mg/kg supported normal growth. A comparison of reproductive performance of C57BL and I strains found that concentrations of 1 to 6 mg/kg diet resulted in fewer productive matings, smaller litters, and a lower survival rate in the I strain. Increasing dietary pyridoxine concentrations to 8 to 12 mg/kg improved the reproductive performance, but further improvement was not obtained using concentrations of 410 or 1,230 mg/kg diet; however, these concentrations did improve the survival rate of C57BL mice over a concentration of 1 to 6 mg/kg (Hoover-Plow et al., 1988). The concentrations of pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate were determined in female mice fed purified diets containing 0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 mg/pyridoxine-HCl/kg diet for 5 weeks. Plasma, erythrocyte, whole blood, liver, and brain pyridoxal-5'-phosphate and liver and brain pyridoxamine-5'-phosphate concentrations correlated with dietary concentrations (r = 0.81 to 0.94) and did not plateau over the entire dietary ranges of values (Furth-Walker et al., 1990). During pregnancy mice fed open-formula diets containing 8.13 mg pyridoxine-HCl/kg had increased erythrocyte and whole blood (2.9- and 1.6-fold) and decreased plasma (50 percent) pyridoxal-5'-phosphate. Liver pyridoxal-5'-phosphate, and pyridoxamine-5'-phosphate decreased 25 percent, but brain concentration remained unchanged (Furth-Walker et al., 1989). The recommended concentration of vitamin B₆ for reproduction is set at 8 mg/kg diet. The concentration of 1 mg/kg set by Miller and Baumann (1945) and by Morris (1947) seems to be adequate for maintenance and growth.

Signs of Vitamin B₆ Deficiency Vitamin B₆ deficiency signs include poor growth, hyperirritability, posterior paralysis, necrotic degeneration of the tail, and alopecia (Beck et al., 1950). Investigators (Keyhani et al., 1974) observed in B6-deficient CF₁ mice a progressive hypochromic microcytic anemia with hypersideremia. It was accompanied by an increase in reticulocyte count not observed in vitamin B₆ deficiencies of other species.

Riboflavin

Based on data reported by Fenton and Cowgill (1947a,b) and Wynder and Kline (1965), mice require 4 mg riboflavin/kg diet for normal growth. However, diets resulting in normal reproduction in mouse colonies generally contain 6 or 7 mg riboflavin/kg diet (American Institute of Nutrition, 1977). In the absence of more definitive data regarding the requirements for reproduction, the estimated riboflavin requirements for this species is 7 mg/kg diet.

Signs of Riboflavin Deficiency Ariboflavinosis in the mouse was described by Lippincott and Morris (1942). They reported the development of either atrophic or hyperkeratotic epidermis with normal sebaceous glands, myelin degeneration in the spinal cord, and corneal vascularization with ulceration. Morris and Robertson (1943) found that adult mice lost weight and young mice grew poorly and died within 9 weeks when fed diets containing 0.4 to 0.6 mg riboflavin/kg diet. Kligler et al. (1944) showed that riboflavin-deficient mice had lowered resistance to Salmonella infection. Hoppel and Tandler (1975) reported striking increases in the size of hepatic mitochondria and a greatly decreased capacity for ADP-stimulated respiration in riboflavin-deficient mice. In some animals, livers were yellow and the cytoplasm of the cells was engorged with small lipid droplets. In other animals, the livers were redder than normal, and their hepatocytes contained few lipid droplets. Genetically diabetic (KK) mice had a higher riboflavin requirement than Swiss albino mice based on activity coefficients of erythrocyte glutathione reductase (Reddi, 1978). Riboflavin deficiency during gestation led to brain, orofacial, limb and gastrointestinal malformations in the offspring. The degree of severity and malformation pattern varied with the strain of mice studied (Kalter, 1990).

Thiamin

Hauschildt (1942) established the minimum requirement of thiamin for normal growth of mice at 10 µg/day. This would correspond to a concentration of approximately 3 mg/kg diet. Morris and Dubnik (1947) later found the growth requirement to be 4 to 6 µg/day for mice fed a diet containing 22 percent fat.

Results of studies on the specific requirements for reproduction and lactation have not been reported, but Mirone and Cerecedo (1947) found that 20 mg/kg diet were adequate. The purified diet (American Institute of Nutrition, 1977) containing 6 mg thiamin-HCl/kg diet has been used in numerous mouse colonies resulting in normal growth and reproduction. In the absence of more definitive data the concentration of 5 mg thiamin-HCl/kg diet that was the estimated requirement in the previous issue of this report (National Research Council, 1978) is being retained.

Signs of Thiamin Deficiency Morris (1947) and Jones et al. (1945) reported violent convulsions, especially when the animal was held a few seconds by the tail; cartwheel or circular movements; brain hemorrhages; decreased food intake; poor growth; early mortality; silvery-streaked muscle lesions; and testicular degeneration. The onset of ataxia in thiamin-deficient Swiss-Webster mice was preceded by a rapid rise in brain α-ketoglutarate (Seltzer and McDougal, 1974). Deficiency (4 to 21 days) led to increased activity in hepatic thiamin pyrophosphatase, alkaline phosphatase, and acid phosphatase (Tumanov and Trebukhina, 1983). Exposure of thiamin-deficient mice to ethanol resulted in brain damage that was more severe than either treatment alone (Phillips, 1987).

Ascorbic acid

The successful maintenance of mouse colonies fed diets devoid of ascorbic acid has confirmed the demonstration by Ball and Barnes (1941) that the mouse requires no dietary source of vitamin C. The plasma concentration of dehydroascorbate in mice fed graded concentrations of ascorbic acid (from 0 to 80 g/kg diet) increased with greater dietary concentrations; ³10 g/kg resulted in significantly greater dehydroascorbate concentration in heart, kidney, lung, and spleen. Concentrations in eyes, were only slightly increased; and brain, adrenal gland, and leukocyte concentrations were unchanged in mice consuming diets containing 80 g/kg (Tsao et al., 1987; Tsao and Leung, 1989).

During the first 8 days of pregnancy an inverse relationship was found between ascorbic acid intake and the concentration of peroxidase in the corpus luteum, blastocyst, and endometrium (Agrawal and Laloraya, 1979). A diet of 10 g ascorbic acid/kg increased average life span by 8.6 percent, decreased body weight by 6 to 7 percent, and increased the maximal life span 2.9 percent (from 965 to 993 days) in C57BL/6J male mice (Friedman et al., 1987).

Myo-inositol

Although Woolley (1941) reported that myo-inositol would alleviate a condition characterized by hair loss, other studies (Martin, 1941; Cerecedo and Vinson, 1944; Fenton et al., 1950; Shepherd and Taylor, 1974a) did not confirm the essentiality of myo-inositol for growth of mice. Studies with other rodent species indicate that they require myo-inositol under conditions of microbial suppression and physiological stress. Shepherd and Taylor (1974b) found that myo-inositol enhanced intestinal lipid transport in rats fed a 31 percent fat diet. Burton and Wells (1977) observed that rats fed 0.5 percent dietary phthalysulfathiazole required myo-inositol to prevent fatty liver during lactation; 500 mg myo-inositol/kg diet was sufficient. Anderson and Holub (1976) found that either tallow or the highly unsaturated canola oil caused liver fat accumulation in myo-inositol-deficient rats fed succinyl sulfathiazole, whereas corn oil or soybean oil did not; 0.5 percent myo-inositol was protective. Unlike rats (Bondy et al., 1990), the peripheral nerves of mice fed diets containing galactose (20 percent) were not depleted of myo-inositol (Calcutt et al., 1990).

If gnotobiotic, germ-free, or antibiotic-treated mice are fed diets containing tallow or the highly saturated rapeseed oil, then myo-inositol may be required in the diets. A purified diet fed to germ-free rats and mice contained 1,000 mg myo-inositol/kg diet (Wostmann and Kellogg, 1967). Chemically defined diets that supported growth and limited reproduction in germ-free CF_W or C3H mice contained 238 mg myo-inositol/kg diet (Pleasants et al., 1970, 1973). A concentration of 500 mg/kg diet was adequate for any combination of antibiotics, lactation, and unusual fat intake in rats and gerbils and seemed to be the upper limit of the myo-inositol requirement. However, conventionally reared mice fed ordinary diets have not been found to require dietary myo -inositol since the early studies of Woolley (1941, 1942).