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decline in hemoglobin concentration in a dog given a semipurified diet. Subcutaneous injections of 15 µg of folic acid per kilogram of body weight restored hemoglobin concentration.

Sheffy (1964) depleted 4- to 5-week-old Beagle puppies of folic acid by feeding a casein-based diet containing sulfasuxidine and 0.11 mg vitamin B12 per kilogram of diet. After 9 weeks of depletion, all dogs developed erratic appetites and weight gains decreased, but there were no changes in concentration of hemoglobin. All dogs were inoculated with distemper and hepatitis antigens, and half the dogs were also given 27.5 µg folic acid per kilogram of body weight. Depleted dogs had delayed antibody production responses against both distemper and infectious hepatitis antigens. Antibodies were detected in depleted dogs supplemented with folic acid 8 days after challenge with antigen, whereas depleted dogs without folic acid did not show antibodies until 17 days after being challenged. Folic acid supplementation allowed resumption of normal growth. Sheffy suggested a requirement for folic acid of less than 1.2 µg per kilogram of body weight.

The folic acid requirement of dogs fed an adequate diet of nonpurified ingredients that does not contain bacteriostatic agents is probably met by microbial synthesis in the intestine. Diets inadequate in choline, methionine, and vitamin B12 may induce deficiencies because of their interaction with folic acid.

It is suggested that the requirement given by the National Research Council (1974) for dogs of 4 µg folic acid per kilogram of body weight for adults and 8 µg folic acid per kilogram of body weight for growing dogs be maintained. On a dietary basis, these quantities are supplied in diets containing 54 µg folic acid per 1,000 kcal ME.

Signs Of Deficiency

Folacin deficiency results in erratic appetite, decreased weight gain, watery exudate from the eyes, glossitis leukopenia, hypochromic anemia with a tendency to microcytosis, and decreased antibody response to infectious canine hepatitis and canine distemper virus (Krehl and Elvehjem, 1945; Afonsky, 1954; Sheffy, 1964).

In the metabolism of histidine in the folate-replete animal, the formimino group from formiminoglutamate is transferred to tetrahydrofolate. When there is a metabolic deficiency of folate the urinary excretion of formiminoglutamate is elevated. A clinical test for folate deficiency is the administration of a load of histidine and the measurement of formiminoglutamate in urine (Tabor and Wyngarden, 1958).

Hypervitaminosis Folacin

Although oral toxicity of folacin has not been described in the dog, Vogel et al. (1964) demonstrated inhibition of hepatic alcohol dehydrogenase in the dog by intravenous administration of 80 mg folic acid per kilogram of body weight 4 hours after intravenous ethanol infusion.

Biotin

Requirements

Spontaneous biotin deficiency occurs rarely in animals because biotin is well distributed among foodstuffs, and a good part, if not all, of the requirement for the vitamin is met by microbial synthesis in the gut (Murthy and Mistry, 1977). The deficiency can, however, be induced by the inclusion of unheated (raw) egg white in the diet. Raw egg white contains the protein avidin, which forms a stable and biologically inactive complex with biotin. One molecule of avidin binds four molecules of biotin (Green, 1963) so firmly that 15 min of steaming at 100°C released only 0 to 10 percent of the bound biotin (Wei and Wright, 1964). Steaming for 2 h at 100°C released 55 to 65 percent of the biotin, while autoclaving for 15 min at 120°C produced complete dissociation. Uncombined avidin was found to be relatively heat-labile.

Shen et al. (1977) took 18-day-old Beagle puppies and divided them into two groups. One group was force-fed raw egg white along with a basal diet without biotin. The other group received an equal amount of heated egg white with the basal diet containing biotin. Within 10 days the group fed the raw egg white showed a significant reduction in activities of pyruvate and propionyl CoA carboxylases in liver and kidney homogenates. However, activities of these enzymes in heart and brain were less affected, which is consistent with data from rats and chicks.

Intestinally active antibiotics or sulfa drugs that inhibit microbial synthesis of biotin may also be expected to increase the need for biotin in the diet. Greve (1963) fed diets containing spray-dried egg white and sulfaguanidine to dogs and produced evidence of biotin deficiency. Assay of the urine of these dogs revealed less than 0.05 pg biotin per milliliter, as compared to "normal" dog urine that contained 7 to 13 pg biotin per milliliter. Unfortunately, the biotin concentration of the diet was not reported.

Incomplete availability of biotin to chicks has been reported by Wagstaff et al. (1961) and Anderson and Warnicke (1970). There have been reports of apparent