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2—
Nutrient Requirements and Signs of Deficiency

Energy

When food is completely oxidized in a bomb calorimeter, the total combustible energy released as heat is known as gross energy (E). Gross energy values for mixed carbohydrates, fat, and protein average 4.15, 9.40, and 5.65 kcal/g, respectively. However, not all gross energy contained in food is available for metabolism. An undigested fraction is excreted in the feces. The difference between the gross energy consumed and the gross energy in the feces is referred to as apparent digestible energy (DE). Additional energy losses occur, namely, energy in excreted urine and combustible gases. For practical reasons, only energy in urine is subtracted from DE to determine metabolizable energy (ME) (NRC, 1981). The ME content of a food is a valid expression of the energy available to the dog and a basis for comparisons of the feeding value of various foodstuffs. ME values for most of the individual ingredients listed in Table 7 (see p. 48) have not been determined for dogs. Therefore, approximations of their ME content must be calculated. A method recommended for calculation is based on assumed apparent digestibilities of 80 percent for protein, 90 percent for ether extract, and 85 percent for nitrogen-free extract (NFE). The resulting digestion coefficients are then multiplied by gross energy values of 4.40 (5.65 – 1.25*), 9.40, and 4.15 kcal for protein, ether extract, and NFE, respectively. It is assumed that no ME was derived from crude fiber. The resulting values of 3.50, 8.46, and 3.50 kcal are reasonable estimates of the ME available to dogs from protein, fat, and carbohydrate (NFE) from feed ingredients commonly used in the manufacture of dog foods (see Chapter 4, section on Metabolizable Energy).

Expressing the energy requirements for all dogs is not a simple task. Adult size and growth rates of breeds vary greatly. Mature body weights range from 1 kg for the Chihuahua to 90 kg for the St. Bernard. From a physiological viewpoint, energy requirements of animals with widely differing weights are not directly related to body weight but are more closely related to body weight raised to some power, Wb, where W equals weight in kilograms and b is an exponent calculated from experimental data. Brody et al. (1934) found that the basal heat production of mature warm-blooded animals, ranging in size from mice to elephants, could be described by the expression Y = 70.5 W0.73, where Y equals kilocalories per 24 h and W equals body weight in kilograms. Kleiber (1961) argued that over such a range in body size, Brody's expression and Y = 70 W3/4 would not be significantly different and that the latter would be simpler to use.

More recently Heusner (1982) demonstrated that the use of the 0.75 interspecific mass exponent in Kleiber's equation is a statistical artifact. Many people assumed that the relationship between basal metabolic rate (BMR) and metabolic body weight was similar for mice and cows. Heusner argued that the theoretical exponent should be 0.67 to describe the intraspecies (e.g., dogs) relationship of energy to mass.

Requirements for Adult Maintenance

Energy requirements for maintenance of adult dogs have been studied by Cowgill (1928) and estimated by Arnold and Elvehjem (1939) from the prediction equations of Brody et al. (1934). Abrams (1962) also published estimates of maintenance energy requirements of adult male dogs that conform closely to previous values, although it is not clear how these data were derived.

* Gross energy of protein corrected for nitrogen energy loss in metabolic products.



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Page 2 2— Nutrient Requirements and Signs of Deficiency Energy When food is completely oxidized in a bomb calorimeter, the total combustible energy released as heat is known as gross energy (E). Gross energy values for mixed carbohydrates, fat, and protein average 4.15, 9.40, and 5.65 kcal/g, respectively. However, not all gross energy contained in food is available for metabolism. An undigested fraction is excreted in the feces. The difference between the gross energy consumed and the gross energy in the feces is referred to as apparent digestible energy (DE). Additional energy losses occur, namely, energy in excreted urine and combustible gases. For practical reasons, only energy in urine is subtracted from DE to determine metabolizable energy (ME) (NRC, 1981). The ME content of a food is a valid expression of the energy available to the dog and a basis for comparisons of the feeding value of various foodstuffs. ME values for most of the individual ingredients listed in Table 7 (see p. 48) have not been determined for dogs. Therefore, approximations of their ME content must be calculated. A method recommended for calculation is based on assumed apparent digestibilities of 80 percent for protein, 90 percent for ether extract, and 85 percent for nitrogen-free extract (NFE). The resulting digestion coefficients are then multiplied by gross energy values of 4.40 (5.65 – 1.25*), 9.40, and 4.15 kcal for protein, ether extract, and NFE, respectively. It is assumed that no ME was derived from crude fiber. The resulting values of 3.50, 8.46, and 3.50 kcal are reasonable estimates of the ME available to dogs from protein, fat, and carbohydrate (NFE) from feed ingredients commonly used in the manufacture of dog foods (see Chapter 4, section on Metabolizable Energy). Expressing the energy requirements for all dogs is not a simple task. Adult size and growth rates of breeds vary greatly. Mature body weights range from 1 kg for the Chihuahua to 90 kg for the St. Bernard. From a physiological viewpoint, energy requirements of animals with widely differing weights are not directly related to body weight but are more closely related to body weight raised to some power, Wb, where W equals weight in kilograms and b is an exponent calculated from experimental data. Brody et al. (1934) found that the basal heat production of mature warm-blooded animals, ranging in size from mice to elephants, could be described by the expression Y = 70.5 W0.73, where Y equals kilocalories per 24 h and W equals body weight in kilograms. Kleiber (1961) argued that over such a range in body size, Brody's expression and Y = 70 W3/4 would not be significantly different and that the latter would be simpler to use. More recently Heusner (1982) demonstrated that the use of the 0.75 interspecific mass exponent in Kleiber's equation is a statistical artifact. Many people assumed that the relationship between basal metabolic rate (BMR) and metabolic body weight was similar for mice and cows. Heusner argued that the theoretical exponent should be 0.67 to describe the intraspecies (e.g., dogs) relationship of energy to mass. Requirements for Adult Maintenance Energy requirements for maintenance of adult dogs have been studied by Cowgill (1928) and estimated by Arnold and Elvehjem (1939) from the prediction equations of Brody et al. (1934). Abrams (1962) also published estimates of maintenance energy requirements of adult male dogs that conform closely to previous values, although it is not clear how these data were derived. * Gross energy of protein corrected for nitrogen energy loss in metabolic products.

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Page 3 Payne (1965), using Abrams's data, published estimates of metabolizable energy requirements for maintenance of growing, adult, pregnant, and lactating dogs. Abrams (1976) recalculated available data related to the determination of BMR in dogs. Estimates were made of energy requirements for maintenance of adult dogs in terms of DE and expressed as joules (J) needed per day or per kilogram of body weight. Constants were determined by regression analysis and an equation for calculating kJ energy requirement was derived. Of significance is that estimates for males exceeded those for females. Abrams (1976) reported greater metabolizable energy requirements for dogs weighing less than 20 kg than those calculated by the method of the 1974 NRC report (132 × BW), but his values did not differ greatly for heavier dogs. More recently, Blaza (1981) measured ME requirements for maintenance of medium and giant breeds of dogs. Great Dane and Newfoundland dogs required 1.5 and 1.3 times, respectively, the calculated ME from the equations of NRC (1974). Requirements of Labrador Retrievers were comparable to requirements predicted from NRC (1974). The Subcommittee on Dog Nutrition decided to seek available data for maintenance based on controlled feeding conditions and to develop a prediction equation using the approach advocated by Thonney et al. (1976). Limited data were available for individual dogs weighing from 4 to 36 kg. Seven breeds (Beagle, Boxer, Labrador Retriever, Pointer, Poodle, and two types of Dachshund) were included. From visual inspection of the scatterplot it could not be concluded that either breed or sex differences affected the relationship of daily ME required for maintenance of body weight. The data were fitted to a linear model and an allometric model. The simple linear model (ME = b0 + b1W, where b0 is the intercept, b1 is the slope, and W is weight in kilograms) described the within-species relationship between basal heat production and weight. The allometric model used was ME = b0Wb1 (where b0 is the mass coefficient and b1 is the exponent). The allometric approach to energy data was first used by Kleiber (1932) on data in which the lightest animal (rat) weighed close to zero compared to the heaviest (cattle). This model implies that the relationship will intersect the origin. There is no reason to use the allometric model unless a curved line that intersects the origin fits the data better than a simple straight line. Since large weight differences comparable to those in the data used by Kleiber (1932) do not exist within most species (including dogs), it is unlikely that the best model is one that requires the relationship to intersect the origin. The results of fitting the two models to the data are shown in Figure 1. The linear equation, ME = 144.4 + 62.2 W, and the allometric equation, ME = 99.56 W0.879, both explained 85.6 percent of the variation with a standard deviation of 260 kcal. The daily ME requirement predicted by these equations is shown in Table 5 (see p. 45) for dogs varying in weight from 1 to 60 kg. Either equation may be used. The NRC (1974) equation (ME = 132 W0.75) is also shown in Figure 1, but it explains less of the variation than the allometric and linear equations actually derived from the data. It underpredicts the ME requirements of the larger dogs and, thus, supports the work of Blaza (1981). More data are needed, however, to predict accurately the energy needs of dogs greater than 35 kg mature body weight. Therefore, until more data are available, it may be advisable to determine feeding levels based on the 1974 equation predictions. These values or any others cannot be taken as absolute ME requirements for any individual or breed of dog, since needs vary with age, activity, body condition, insulative characteristics of the hair coat, temperature, acclimatization, external environmental circumstances, and psychological temperament. Finally, it would not appear to serve much practical purpose to further refine energy requirements of even ''average" dogs, since the ME concentration of foods available to such "average" dogs is frequently either unknown or can only be calculated by methods resulting in no greater precision. Generally, adult dogs adjust their food intake to energy requirements. Cowgill (1928) found that dogs previously adjusted to an appropriate intake of a particular diet consumed fewer grams, but a similar number of calories, when a higher-energy-density diet was offered. Durrer and Hannon (1962) reported that caloric intake varied inversely with long-term changes in environmental temperature. In July, when the mean temperature was 17°C, Beagles consumed approximately 163 kcal of ME per W per day, while Huskies consumed 127. In November, when mean temperatures were - 17°C, the respective daily ME intakes for Beagles and Huskies were 278 and 205 kcal per W. Huskies exhibited a marked increase in hair growth during November and December, while little seasonal change in hair growth was seen in Beagles. Dogs of both breeds minimized heat loss during extremely cold weather (less than - 40°C) by curling into a ball and tucking their noses and tails underneath their bodies. While Huskies showed no evidence of shivering and refused to sleep in plywood shelters, Beagles shivered and sought shelter. These data illustrate the marked effect on energy requirements imposed by the environment and the additional influence of differences in breed and behavior. While weight changes were small, both Beagles and Huskies were heavier in the summer than in the winter.

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Page 4 Figure 1 Relationship between metabolizable energy and adult body weight. These data support what is increasingly recognized in practice, namely, the inability of many dogs to accurately adjust food intake to meet requirements for biological energy. When dogs are kept in a controlled environment with limited opportunity to exercise and are fed highly palatable, nutrient-dense foods, the ability to regulate food intake for optimum body weight may be compromised. Obesity, common to many pets, is the ultimate result of caloric intake in excess of metabolic requirements. Regardless of the accuracy of any prediction of energy requirements, the judgment of how much to feed must ultimately be with the individual feeding the dog. This judgment is based on weight, conformation, and general appearance of the dog in question. Requirements for Growth Growing puppies require about 2 times as much energy per unit of body weight as adult dogs of the same breed (Arnold and Elvehjem, 1939). The newly weaned dog can readily adapt to this level of feeding, particularly when the food is offered in multiple judiciously spaced meals. However, an arbitrary decrease to 1.6 times maintenance is recommended when 40 percent of adult body weight is achieved, and 1.2 times maintenance when 80 percent of adult weight is reached. This reduction compensates for the decline in energy required from weaning to adult age. Excessive nutrient intake from weaning to adolescence, resulting in maximal growth rates, is incompatible with proper skeletal development (Hedhammar et al., 1974). Requirements for Reproduction and Lactation Limited studies and practical experience suggest that energy requirements of the normal pregnant bitch are only slightly more than those recommended for maintenance for the first two-thirds of gestation. During the last trimester, energy requirements may increase to as much as 150 to 160 percent of preconception values (Romsos et al., 1981). Energy requirements during lactation increase greatly and are influenced by size of the litter. Bitches with large litters may require 3 or more times the maintenance energy requirement. The ability to meet these requirements may be limited by the capacity of the bitch to consume sufficient food. Foods of high

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Page 5 nutrient density are recommended for feeding at this time. Requirements for Work and Adverse Environmental Conditions The range of work performed by dogs may vary from the limited exercise characteristic of the apartment pet to the intense effort of the working sled dog. Systematic schedules for meeting such diverse requirements are impractical. Thus, it is recommended to feed to thrifty body condition. It should be pointed out, however, that while a conditioned racing Greyhound may require energy only 10 to 20 percent above that of maintenance, a sled dog working under polar conditions may require 2 to 4 times the maintenance requirement in order to avert significant weight loss (Kronfeld et al., 1977). Conversely, working dogs in hot, humid environments may require 50 to 100 percent more energy than similar dogs in less stressful circumstances (McNamara, 1971). Signs of Deficiency Signs of energy deficiency are frequently nonspecific, and diagnosis may be complicated by a simultaneous shortage of several nutrients. The most conspicuous and reliable sign of uncomplicated energy deficiency is a generalized loss of body weight. Under conditions of partial or complete starvation, most internal organs exhibit some atrophy. A loss of subcutaneous, mesenteric, perirenal, uterine, testicular, and retroperitoneal fat is an early sign. Low fat content of the marrow in the long bones is a good indicator of prolonged inanition. Brain size is least affected, but the size of gonads may be greatly decreased. Hypoplasia of lymph nodes, spleen, and thymus leads to a marked reduction in their size. The adrenal glands are usually enlarged. The young skeleton is extremely sensitive to energy deficiency, and growth may be slowed or stopped completely. In the adult the skeleton may become osteoporotic. Lactation and the ability to perform work are also impaired. As muscle proteins are catabolized for energy, endogenous nitrogen losses increase. Parasitism and bacterial infections frequently occur under such circumstances and may superimpose other clinical signs. Fat Dietary fat is a concentrated source of energy and provides essential fatty acids. These essential fatty acids serve structural functions in cell membranes and in metabolic regulation, e.g., as precursors of prostaglandins and related metabolites. Fat also serves as a carrier of fat-soluble vitamins and lends palatability and a desirable texture to dog food. Dietary Fat Concentration Proper formulation of diets containing fat requires an adjustment for the high energy value of fats. Because the ME concentration of digestible fat is approximately 2.25 times the ME concentration of digestible carbohydrate or protein, substitution of fat on an equal weight basis for these nutrients will increase the energy density of the diet. As a result, dogs fed high-fat diets formulated without consideration for the higher energy value of fat may exhibit nutrient deficiencies. This occurs because dogs generally respond by eating less of a high-fat diet to maintain near-normal energy intake (Cowgill, 1928). Consequently, daily intake of protein, minerals, and/or vitamins may not be adequate. The adverse consequences of improper formulation of high-fat diets on the growth rate of puppies has been demonstrated (Elvehjem and Krehl, 1947; Campbell and Phillips, 1953; Ontko et al., 1957; Crampton, 1964). When the formulation of high-fat diets is adjusted to ensure adequate intake of protein, minerals, and vitamins, diets with wide ranges in fat concentration appear compatible with good health of dogs. (See Newberne et al., 1978, for examples of procedures for formulating high-fat diets.) Apparent digestibility of fat by dogs varies from approximately 80 to 95 percent when mixtures of glycerides from plant and animal sources are fed (James and McCay, 1950; Orr, 1965). The growth rate of Beagle puppies between 2 and 6 months of age was unaffected by feeding purified, canned diets ranging in composition from 13 to 76 percent of energy from fat and containing 20 to 25 percent of energy from protein (Romsos et al., 1976). Between 6 and 10 months of age, puppies fed the lower-fat diet (13 percent fat) gained less body weight because they gained less body fat than did puppies fed diets containing 38, 55, or 76 percent of energy from fat. Results of this study and several others (Siedler and Schweigert, 1952; Campbell and Phillips, 1953) demonstrated that puppies exhibit satisfactory growth when fed diets with rather widely varying concentrations of fat if amino acid requirements are met. Reproductive performance of bitches fed diets varying in fat concentration has received only limited attention. Siedler and Schweigert (1954) indicated that reproductive performance was somewhat better when Cocker Spaniel bitches were fed a diet containing 7.7 percent fat rather than a diet containing either 3.7 or 11.7 percent fat. Because their diets were formulated by adding 4 and 8 percent fat to the diet containing 3.7 percent fat, the ratio of essential nutrients to ME was

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Page 6 progressively decreased in the higher-fat diets. This may have adversely affected performance of bitches fed the diet containing 11.7 percent fat. Ontko and Phillips (1958) fed diets containing 8 or 16 percent fat and observed satisfactory reproductive performance. Provided appropriate adjustments are made to maintain an adequate nutrient-to-energy ratio, it would appear that diets with rather widely varying concentrations of fat will also permit satisfactory reproductive performance. Sedentary adult dogs have a greater tendency to become obese when fed high-fat diets ad libitum rather than high-carbohydrate diets. When adult female Beagles were fed ad libitum a diet containing 51 percent of energy from fat for 25 weeks, they gained twice as much body fat as dogs fed a diet containing 23 percent of energy from fat (Romsos et al., 1978). Other animals also tend to gain more body fat when fed a high-fat diet than when fed a low-fat diet (Sclafani, 1980). A slight restriction in food intake, however, will prevent development of obesity even if high-fat diets are fed. The concentration of fat in the diet may affect work performance of dogs. Fatty acids are the primary source of energy for skeletal muscle during exhaustive exercise (Therriault et al., 1973). When adult Beagles that had been maintained in a high state of physical conditioning and had been fed a cereal-based diet with 7 percent fat were fasted for 5 days, their endurance capacity increased by 74 percent relative to their endurance in the fed state (Young, 1959). It was concluded that this improvement in work performance was mediated by an enhanced ability to mobilize body fat. Consumption of a high-fat diet rather than a high-carbohydrate diet has been shown to lengthen the time to exhaustion of Beagles on a treadmill by approximately 30 percent (Downey et al., 1980) and to cause a greater elevation in plasma-free fatty acid concentration during exercise of sled dogs (Hammel et al., 1977). Essential Fatty Acids Dogs, like other animals, have a dietary requirement for certain polyunsaturated fatty acids. These fatty acids have been shown to stimulate growth and cure the dermatitis characteristic of dogs fed a diet very low in fat or a diet in which the fat is completely saturated (Hansen et al., 1948, 1954; Hansen and Wiese, 1951; Wiese et al., 1965, 1966). Members of the linoleic acid n-6 (denotes the position of the first double bond from the terminal end of the chain) family including linoleic acid, C18:2 (n-6); -linolenic acid, C18:3 (n-6); and arachidonic acid, C20:4 (n-6) all exhibit essential fatty acid activity. The shorthand terminology used denotes the number of carbon atoms in the fatty acid (followed by a colon) and the number of double bonds. Because C18:2 (n-6) can be desaturated and elongated to form C18:3 (n-6) and C20:4 (n-6) (Mead, 1980), and because the latter two fatty acids are only minor components of most natural fats, the requirement for fatty acids of the n-6 family is usually expressed as linoleic acid. Linoleic acid concentrations of a variety of ingredients used in dog foods are shown in Table 6 (see p. 46). The minimum amount of linoleic acid (or other members of the n-6 family of fatty acids) required by the dog has not been precisely determined. The pathological and biochemical changes in the skin produced by an essential fatty acid deficiency can be reversed when 2 to 6 percent of the ME requirement is provided by linoleic acid or arachidonic acid (Hansen and Wiese, 1951; Wiese et al., 1966). One percent of the ME requirement as linoleic acid does not appear to be adequate for growing puppies (Wiese et al., 1966). Several factors including rate of growth and concentrations of saturated fatty acids, trans fatty acids, and monounsaturated fatty acids in the diet influence the requirement for essential fatty acids (Mead, 1980; Holman, 1981). Beagle puppies fed a low-fat diet at 200 kcal ME per kilogram of body weight per day exhibited skin lesions within 2 to 3 months (Wiese et al., 1962). When the level of intake was reduced to 150 kcal ME per kilogram body weight per day, lesions appeared in 3 to 4 months. Puppies fed 100 kcal ME per kilogram of body weight per day did not grow, nor did they exhibit gross or histological evidence of fat deficiency during the 5-month study. Based on data obtained with rodents (Mead, 1980; Holman, 1981), one would predict that high intakes of saturated fatty acids, trans fatty acids, or oleic acid [C18:1 (n-9)] by dogs would compete with the metabolism of essential fatty acids and thereby increase the requirement for essential fatty acids. However, data are unavailable for estimating the extent to which these fatty acids might increase the requirement for essential fatty acids in dogs. There is speculation that fatty acids of the -linolenate [C18:3 (n-3)] family also exhibit essential fatty acid activity in animals (Mead, 1980; Budowski, 1981). In rats C18:3 (n-3) promotes normal growth but does not prevent the skin lesions associated with lack of n-6 fatty acids (Mead, 1980). Fatty acids of the n-3 family have been suggested to serve some special function in the nervous system (Mead, 1980; Holman et al., 1982). They also are precursors of prostaglandins that may play an important role in control of blood clotting (Budowski, 1981). Data are unavailable to indicate if dogs have a requirement for fatty acids of the n-3 family. Recommendation It is recommended that a dog food contain at least 5 percent fat on a dry basis, including 1 percent of the diet

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Page 7 as linoleic acid. Because not all fats are rich in linoleic acid (Table 6), supplemental fats must be chosen judiciously when total fat is limited to 5 percent. Although these concentrations appear sufficient for normal physiological functions, higher concentrations of fat may be desirable in practical dog foods to enhance acceptability and to improve hair coat sheen. If such increases are made, the concentrations of other nutrients should be appropriately increased to maintain a satisfactory nutrient-to-energy ratio, i.e., when substantial fat supplementation is implemented, total dietary reformulation is in order to prevent nutrient imbalances from occurring. Signs of Deficiency Puppies fed a low-fat diet (probably less than 0.01 percent linoleic acid) but with a high energy intake per day began to show coarse, dry hair and desquamation on the ventrum after 2 to 3 months. After 4 to 5 months (6 to 7 months of age), these lesions became severe. With normal energy intake, appearance of the lesions was delayed about 1 month (Wiese et al., 1962). The earliest gross lesions appeared on the abdomen, then on the thigh, and last in the interscapular area. Histologically, the epidermis was edematous and thickened, with up to 12 layers of cells in the most severely affected areas. Keratinization was deranged and, as the deficiency advanced, parakeratosis became evident. Maturation of the epidermal cells seemed impaired. Affected areas were invaded first by mononuclear cells, followed later by polymorphonuclear neutrophils. The epidermis appeared ulcerated and was more susceptible to infection. Linoleic acid and arachidonic acid concentrations in the skin decreased markedly. Carbohydrates All animals have a metabolic requirement for glucose to supply energy for organs, including the central nervous system, and to supply substrate for synthesis of compounds such as pentoses and glycoproteins. Provided the diet contains sufficient glucose precursors (amino acids and glycerol), the glucogenic capacity of the liver and kidneys is usually sufficient to meet the metabolic need of growing animals for glucose without inclusion of carbohydrate in the diet (Brambila and Hill, 1966; Chen et al., 1980). Beagle puppies have been fed purified, canned diets ranging in composition from 0 to 62 percent of metabolizable energy from carbohydrate (cornstarch) and from 13 to 76 percent of metabolizable energy from fat (corn oil, tallow, and lard) to determine if dietary carbohydrate is required for growth and maintenance of normal blood glucose levels (Romsos et al., 1976). Protein (20 to 25 percent of energy) in these diets was derived from approximately equal proportions of lean beef and isolated soybean protein. Weight gain of the 2-month-old Beagles fed the carbohydrate-free diet containing 24 and 76 percent of energy from protein and fat, respectively, for 8 months was comparable to the gain of pupies fed diets containing 20 to 62 percent of energy from carbohydrate. Pups fed the carbohydrate-free diet also maintained normal plasma glucose concentrations and normal rates of glucose utilization (estimated by disappearance of [2-3H] glucose) (Belo et al., 1976). These results agree with earlier reports demonstrating that growing rats (Chen et al., 1980) and chickens (Brambila and Hill, 1966) do not appear to have a dietary requirement for carbohydrate provided adequate dietary glucose precursors are available in the form of glucogenic amino acids and glycerol. A dietary requirement for carbohydrate has been demonstrated in growing rats (Chen et al., 1980) by reducing the concentration of protein in the diet to 13 percent and by providing unesterified fatty acids, rather than glycerol-containing triglycerides, as the only nonprotein energy source. Rats fed this diet gained less weight and had depressed blood glucose concentrations. Either adding 6 percent glucose or doubling the concentration of protein in the diet to 26 percent increased their growth rate to equal the growth rate of control rats. Similar experiments have not been reported with dogs, but it seems probable that consumption of a diet devoid of carbohydrate and low in glucose precursors would also cause hypoglycemia and limit growth rate of dogs. During gestation and lactation the metabolic requirement for glucose is increased to supply needs for fetal development and lactose synthesis, respectively. When, starting 3½ to 4 weeks after conception, Beagle bitches were fed a diet containing 26, 74, and 0 percent of energy from protein, fat, and carbohydrate, respectively, their food intake, body weight increase, and plasma glucose concentrations during the first two trimesters of gestation were comparable to values in bitches fed a diet containing 26, 30, and 44 percent of energy from protein, fat, and carbohydrate, respectively (Romsos et al., 1981). However, during the week before whelping, bitches fed the carbohydrate-free diet developed hypoglycemia and had depressed plasma concentrations of two key glucose precursors (lactate and alanine). Total number of pups whelped by the bitches was unaffected by diet, but fewer pups from bitches fed the carbohydrate-free diet were alive at birth (63 percent) than from bitches fed the carbohydrate-containing diet (96 percent). Only 35 percent of the pups whelped by bitches fed the carbohydrate-free diet were alive at 3 days of age. The cause of death of the pups was not established, but they probably had less ability to maintain their plasma glucose concentrations immediately after delivery

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Page 8 than did control pups (Kliegman et al., 1980). Additionally, the lethargic condition of the hypoglycemic bitches reduced their mothering ability immediately after delivery. The severe hypoglycemia (plasma glucose concentrations as low as 15 to 20 mg/dl) and ketosis (blood -hydroxybutyrate concentrations as high as 2.5 mM) that develop at whelping in some bitches fed a carbohydrate-free diet (Romsos et al., 1981) are not unique to dogs. Hypoglycemia and ketosis are also often observed during the last trimester of gestation in ewes carrying twins or triplets (Bergman, 1973). Since most of the carbohydrate consumed by ewes is fermented in the rumen, they absorb only small amounts of glucose and therefore depend on endogenous synthesis to supply their need for glucose. Under conditions of accelerated need for glucose such as occur in the last trimester of gestation, both dogs and ewes sometimes fail to meet these needs from endogenous synthesis. It is possible, based on observations with pregnant rats (Taylor et al., 1983), that bitches also require some carbohydrate in their diet during the period immediately postconception. Rats fed a carbohydrate-free diet from the day of conception exhibit an increase in early embryonic abnormalities and resorption (Taylor et al., 1983). Because the bitches were not switched to their experimental diets until 3 ½ to 4 weeks after conception (Romsos et al., 1981), effects of a carbohydrate-free diet on the early stages of their gestation remain unknown. It is recommended that diets of pregnant bitches contain some available carbohydrate for optimal reproductive performance. Lactating Beagle bitches have also been fed a carbohydrate-free diet containing 26 percent of energy from protein and 74 percent from fat to determine if they require dietary carbohydrate for lactation (Romsos et al., 1981). To maximize the need for milk synthesis, each bitch nursed six pups, and the pups were not allowed to consume the bitches' diet during the first 4 weeks of lactation. Pups suckling bitches fed the carbohydrate-free diet grew as well as those suckling bitches fed the carbohydrate-containing diet. Likewise, plasma glucose concentrations of the lactating bitches were unaffected by the absence of carbohydrate in the diet. Because the milk of Beagles contains less than 20 percent of energy from lactose (Luick et al., 1960; Romsos et al., 1981), the need for glucose in lactating Beagles is less than the need during gestation when glucose is a major energy source for fetal development. This may explain why consumption of a carbohydrate-free diet adversely affected performance of the Beagles during gestation but not during lactation. In breeds of dogs that have larger litters and a greater milk production than Beagles it is possible that the metabolic demand for glucose could exceed the ability of the bitch to synthesize it. At least in high-producing dairy cows, hypoglycemia and ketosis have been shown to develop during peak lactation (Bergman, 1973). Carbohydrates provide an economical source of energy in the diet of dogs. Cooked starch is well digested by adult dogs (Roseboom and Patton, 1929; Ivy et al., 1936; James and McCay, 1950; Heiman, 1959) and by growing Beagles (Romsos et al., 1976). Growing Beagles fed a purified, canned diet containing 62 percent of energy from cornstarch exhibited an apparent energy digestibility of 84 percent, whereas energy digestibility was slightly higher (87 to 89 percent) for dogs fed diets containing less starch (0 to 42 percent of metabolizable energy) and more fat (38 to 76 percent of metabolizable energy). Weight gain of Beagles fed the diet containing 62 percent cornstarch from 2 to 10 months of age was somewhat less than for dogs fed diets with less starch and more fat. This difference in weight gain occurred because dogs fed the high-starch diet gained less body fat; gain in fat-free mass was unaffected by concentration of starch in the diet. Raw starch is less well utilized by dogs than is starch that has been subjected to some dextrinization by processing (for example, cooking, baking, or toasting) (Heiman, 1959). Utilization of simple carbohydrates such as lactose, dextrin-maltose, and sucrose has also been evaluated in dogs. Suckling puppies obviously utilize lactose, although as mentioned above, the milk of Beagles contains a relatively low percentage of energy from lactose. Sudden introduction of large amounts of lactose into the diet of adult Beagles causes diarrhea (Bennent and Coon, 1966). This response is not unique to dogs—adults of a number of other species would also exhibit diarrhea if abruptly challenged with large amounts of lactose. Diets containing as much as 49 percent by weight of sucrose support satisfactory body weight gain in immature Beagles (Milner, 1979). Young adult Beagles show a preference for sucrose-containing diets when allowed to self-select between a sucrose-supplemented diet and a control, starch-containing diet (Houpt et al., 1979). Although sucrose is well utilized by dogs in the postweaning period, there is the possibility, based on research in other species (Becker et al., 1954), that intestinal sucrase activity is low immediately after birth, in which case sucrose should not be included as a significant component in artificial milk for puppies. Fiber is not generally considered essential for simple-stomached mammals, including dogs, although inclusion of some fiber in the diet may be beneficial. Energy density of the diet is reduced by fiber, and therefore inclusion of some fiber in the diet may contribute to the maintenance of ideal body weight in adult sedentary dogs fed ad libitum. Fiber reduces gastrointestinal transit time in simple-stomached mammals. But because

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Page 9 dogs have a relatively short gastrointestinal tract, ingesta transit time is relatively rapid even when a low-fiber diet is fed (Banta et al., 1979). As a result of their short gastrointestinal tract, dogs have a low colonic-rectal surface area per unit body weight. For example, Beagle dogs have only 17 cm2 colonic-rectal mucosal area per kilogram of body weight, whereas the comparable value for pigs is 200 cm2 per kilogram of body weight (Herschel et al., 1981). Consequently, dogs are unlikely to absorb significant amounts of energy from the fermentation of fiber that might occur during its relatively rapid transit through the lower gastrointestinal tract. Finally, although data are unavailable for the dog, it should be recognized that inclusion of large amounts of fiber in the diet may adversely affect nutrient availability. Protein And Amino Acids Dietary protein is required to supply specific essential amino acids that cannot be synthesized in sufficient quantities by tissues to allow for optimum performance. Additionally, dietary nitrogen is required to allow for optimal biosynthesis of the dispensable amino acids and other nitrogenous compounds. Recent studies by Milner (1979 a,b) using purified diets containing 4.1 kcal ME/g have established that the following amino acids are required for optimum growth and nitrogen balance in the immature Beagle: arginine histidine isoleucine leucine lysine methionine phenylalanine threonine tryptophan valine Studies of Rose and Rice (1939) established that all of the above amino acids except for arginine were also required to maintain nitrogen equilibrium in adult female dogs. However, recent studies of Burns et al. (1981) have shown that dietary arginine is required by the mature dog to maintain body weight and to prevent emesis and other signs associated with hyperammonemia. Numerous factors may modify the percentage of protein required in the diet. In establishing this requirement, factors such as digestibility, amino acid composition, availability of the protein source, caloric density of the diet, and physiological state of the dog must be considered. The quantity, including excesses and deficiencies, of essential (indispensable) and dispensable amino acids, plus other nonspecific nitrogen sources are factors that may influence the minimal percentage of dietary protein required for optimum growth and health. Estimates of the protein requirement of the dog can also vary depending on the methods and criteria used in their derivation. Signs of Deficiencies Protein deficiency in the dog results in depressed food intake, severe growth retardation or weight loss, hypoproteinemia, depletion of protein reserves, muscular wasting, emaciation, and, ultimately, death (Chow et al., 1945; Allison et al., 1946; Allison and Wannemacher, 1965; Burns et al., 1982). Edema sometimes accompanies the hypoproteinemia. Generally, during limited access to protein the hair coat becomes rough and dull in appearance, antibody formation is impaired, and milk production is depressed. Although the signs of protein deficiency are nonspecific and can be created by other dietary deficiencies, including caloric restriction, these signs do indicate the severity of dietary limitations on the dog's health and performance. Removal of a single essential amino acid results in a prompt reduction in food consumption leading to a negative nitrogen balance. Generally there is a return to normal within a few days after replacing the limiting amino acid. Prolonged deficiency of any of the essential amino acids leads to a syndrome similar to that occurring during protein deficiency. Limitation in a dietary essential amino acid tends to be reflected by lowered concentrations of the specific amino acid in the blood plasma (Longnecker and Hause, 1959). Specific signs characteristic of a deficiency of individual amino acids in the dog have not been adequately documented. Amino acid imbalances or antagonisms are known to increase the requirements for individual amino acids (Harper and Rogers, 1965; Harper et al., 1970). Some adaptation to minor imbalances and antagonisms appears to occur. Data obtained in other species indicate that the effects of imbalances or antagonisms are greater when suboptimal dietary nitrogen is offered but of lesser importance if all amino acids are in excess in the diet. Amino Acid Requirements Indispensable The dietary requirement of a particular protein or a mixture of proteins is determined by the ability of the protein(s) to meet the dog's metabolic requirements for amino acids and nitrogen. The closer the supply of the complement of amino acids to the requirement, the lower the percentage of protein required in the dog's diet (Allison et al., 1947; Kade et al., 1948; Arnold and Schad, 1954). Amino acid requirements, as a percentage of the diet, decline from birth to maturity. However, Wannemacher and McCoy (1966) have suggested that

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Page 10 amino acid needs of adult dogs may increase from maturity to old age. Unfortunately, the influence of age has not been adequately studied. Research with other species reveals that in estimating requirements for amino acids, consideration must be given to the energy and total protein concentration of the diet (Wretlind and Rose, 1950; Bressani and Mertz, 1958; Milner, 1981). Similarities in metabolism among mammalian species suggest that it is prudent to consider these as important dietary factors for the dog until research proves otherwise. Arginine Arginine has been shown to elicit the release of a number of metabolic hormones including insulin, glucagon, growth hormone, and prolactin. Recent evidence also indicates that arginine can stimulate the immune response (Barbul et al., 1981). LeFebvre et al. (1976) showed that arginine stimulated the release of glucagon and gastrin from the stomach of the dog. The importance of physiological concentrations of arginine in maintaining body homeostasis needs further investigation. Using purified L-amino acid diets, arginine has been shown to be an essential amino acid for the immature and mature dog (Ha et al., 1978; Burns et al., 1981). Consumption of a diet devoid of arginine resulted in signs of ammonia toxicity within 1 hour following voluntary consumption of a single meal. Signs of ammonia intoxication included emesis, frothing at the mouth, muscle spasms, and altered cellular metabolism. Consumption of an arginine-deficient diet was accompanied by an elevation in the concentration of the pyrimidine metabolite, orotic acid, in both blood and urine. These studies revealed that 0.56 percent dietary arginine supported optimum growth, nitrogen balance, and prevention of orotic aciduria in the immature Beagle fed an L-amino acid diet equivalent to 14 percent protein. Czarnecki and Baker (1984) reported that the dietary arginine requirement for English Pointer puppies was 0.40 percent for maximum weight gain. Consumption of equimolar quantities of ornithine failed to prevent signs of arginine deficiency, which included growth failure, emesis, hyperammonemia, and orotic aciduria. Consumption of equimolar quantities of citrulline, however, resulted in near-normal growth, but blood and urine metabolites did not parallel those in puppies fed arginine. Although the dietary arginine requirement in the immature dog appears to increase with increasing nitrogen concentration of the diet (Ha et al., 1978), the recommendations have been set at 274 mg per kilogram of body weight per day to allow for optimum growth and prevention of abnormal metabolism. This corresponds to 1.37 g/1,000 kcal ME. Signs of ammonia intoxication in mature Pointers were prevented by feeding a purified diet containing 0.28 percent arginine (Burns et al., 1981). In the absence of other data, 21 mg per kilogram of body weight per day has been proposed as the requirement for mature dogs. These results are consistent with the concept that the mature dog has a lower dietary amino acid requirement than that of the immature dog. Histidine Diets containing 0.11 percent or less histidine and the equivalent of 14 percent protein resulted in depressions in food intake. Analysis of growth, feed efficiency, and nitrogen retention data of Beagles fed purified L-amino acid diets revealed that 0.21 percent histidine (193 mg/d/kg0.75) was required to meet these parameters (Burns and Milner, 1982). Therefore, the requirement for immature dogs has been given as 98 mg per kilogram of body weight per day or 0.49 g/1,000 kcal ME of dietary energy intake. Although Rose and Rice (1939) reported that histidine was an essential amino acid for the adult dog, no data were presented. Recently Cianciaruso et al. (1981) confirmed that histidine was required in the diet of adult female dogs. Mature dogs tube-fed a purified L-amino acid diet devoid of histidine for approximately 60 days had reduced plasma and muscle histidine concentrations, decreased muscle carnosine, reduced hematocrit, depressed serum albumin, and weight loss. The minimum requirement for mature dogs has not been established but has been estimated to be 22.0 mg/kg/d by Ward (1975). Cianciaruso et al. (1981) reported that signs of histidine deficiency may not appear for many days, but then progress rapidly. Factors that modify the onset of signs of histidine deficiency may include the release of histidine from carnosine (b-alanyl-L-histidine) and hemoglobin, a reduction in the rate of histidine degradation, and possibly enhanced histidine reabsorption in the kidney (Cianciaruso et al., 1981). Leucine, Isoleucine, and Valine Leucine, isoleucine, and valine are classified as branched-chain amino acids. Plasma concentrations of branched-chain amino acids have been observed to increase during prolonged fasting in the dog (Brady et al., 1977). Since muscle is a site of metabolism of the branched-chain amino acids, this increase may represent decreased muscular uptake or increased muscular release of these amino acids. Branched-chain amino acids are not only substrates for protein biosynthesis, but have also been implicated in the regulation of protein turnover and energy metabolism (Adibi, 1980). Purified L-amino acid diets containing 0.41 percent isoleucine support optimum growth in immature Beagles (Milner, 1979b). Furthermore, similar studies have shown that diets containing more than

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Page 11 0.55 percent leucine and 0.42 percent valine are required to optimize growth and nitrogen balance in immature dogs (Milner, 1979a). Since probably all branched-chain amino acids in the dog are metabolized by a common enzyme, it is possible that an excess of one of these amino acids will increase the dietary requirements for the others. Burns et al. (1984) have recently reported that 10- to 12-week-old Beagles require 0.65 percent leucine, 0.40 percent isoleucine, and 0.43 percent valine to support optimal growth, feed efficiency, and nitrogen retention. These values and proposed recommendations correspond to 159 mg leucine, 98 mg isoleucine, and 105 mg valine per 1,000 kcal dietary metabolizable energy. Ward (1975) has estimated that the leucine, isoleucine, and valine requirements for the mature dog are 84, 48, and 60 mg/kg/d, respectively. Lysine Milner (1979a, 1981) has shown that lysine is an essential amino acid and that the dietary requirement for maximum growth and nitrogen balance occurred in immature male Beagles fed 0.58 percent or more lysine. In the absence of other data, 280 mg per kilogram of body weight per day has been set as the lysine recommendation for growth. This requirement would be met by a diet supplying 1.40 g lysine per 1,000 kcal ME. Dogs given diets containing excess lysine (1.73 percent) had significantly lower growth rates than dogs given diets containing optimal (0.58 percent) lysine. Ward (1975) estimated the minimum lysine required to maintain nitrogen equilibrium in the adult dog is approximately 50 mg/kg/d. Methionine Methionine was established as an essential amino acid for optimum growth and nitrogen balance in immature Beagle dogs using diets containing purified L-amino acids (Milner, 1979b; Burns and Milner, 1981; Blaza et al., 1982). These studies revealed that diets containing 4 kcal ME/g and 0.20 percent methionine and 0.15 percent cystine meet the total sulfur amino acid requirement of the immature Beagle based on growth and nitrogen balance (Burns and Milner, 1981). Expressing methionine and cystine on an isosulfurous basis, the total dietary sulfur amino acid requirement for immature Beagles fed a 16 percent L-amino acid diet was found to be 0.39 percent. The total sulfur amino acid requirement was estimated to be 373 mg/d/kg0.75 in diets containing an estimated 4.4 kcal gross energy per gram dry weight (Burns and Milner, 1981). Support for this requirement for methionine comes from studies showing intake of diets containing more than 12 percent protein as casein did not improve the growth of the immature dog (Burns et al., 1982). Cystine could supply approximately 50 percent of this requirement. These studies also demonstrated that the dog can effectively utilize D-methionine, DL-methionine, OH-L-methionine, N-acetyl-L-methionine, but not N-acetyl-D-methionine, to replace dietary L-methionine (Burns and Milner, 1981). Hirakawa and Baker (1984) found that the dietary sulfur amino acid requirement of English Pointer puppies for maximal growth rate and efficiency of weight gain was approximately 0.45 percent. In dogs fed diets containing limited methionine, excess cystine caused anorexia, growth depression, and severe skin lesions on the neck, tail, and foot pads. Blaza et al. (1982) studied the sulfur amino acid requirements of growing Labrador and Beagle dogs in three experiments. In two of these experiments the diets were based on isolated soy protein supplemented with methionine, while in the third experiment a crystalline amino acid diet was used. For the isolated soy protein diet supplemented with 0.15 percent cystine, the addition of 0.39 percent methionine gave significantly lower body weight gains, nitrogen retention, and food intakes than 0.57 or 0.74 percent methionine. Intakes of 116 mg total sulfur amino acids (TSAA)/100 kcal ME were inadequate, but 154 mg TSAA/100 kcal ME appeared adequate for Labrador dogs. These studies indicated that the dog's breed may influence methionine requirements, since Labradors but not Beagles responded to increasing the methionine content from 0.36 to 0.71 percent by increased weight gains and food intakes. These data also indicate that methionine available from isolated soy preparations must be considered in evaluating the adequacy of diets. Estimates of minimal total sulfur amino acid requirements for normal growth have ranged from 0.39 to 0.71 percent in diets of comparable caloric value. The minimal recommendation has been set at 1.06 g/1,000 kcal ME. Methionine is often considered a limiting amino acid in many normal protein sources. Methionine supplementation is known to reduce the quantity of these protein sources required for adult dogs (Allison et al., 1947). Kade et al. (1948) reported that a daily intake of 140 mg nitrogen from casein per kilogram of body weight was adequate to support nitrogen balance in adult dogs. The quantity of nitrogen could be reduced to 90 mg per kilogram of body weight when the casein was supplemented with methionine. Arnold and Schad (1954) reported that the addition of 1 g DL-methionine to 100 g casein protein reduced the quantity of nitrogen required for nitrogen equilibrium from 139 to 102 mg per kilogram of body weight. Addition of 3 g DL-methionine reduced the quantity of nitrogen required for equilibrium to 72 mg per kilogram of body weight. These authors concluded that the sulfur amino acid requirement of the mature dog was approximately 30 mg

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Page 12 per kilogram of body weight per day when the sulfur amino acid component contained about 89 percent methionine and 11 percent cystine. This quantity is lower than the minimum of 43 mg/kg/d reported by Ward (1975) to maintain nitrogen equilibrium in 15-kg adult dogs and has been considered the requirement. Similar to rats, pigs, and rabbits, the mature dog (Cho et al., 1980) apparently utilizes D-methionine as efficiently as L-methionine. Phenylalanine and Tyrosine Phenylalanine is a dietary essential for the immature dog (Milner, 1979a). Dietary phenylalanine requirements have been reported to be less than 0.58 percent when 0.35 percent tyrosine was present in the diet (Milner, 1979a). Deletion of tyrosine from purified L-amino acid diets did not significantly influence growth when excess phenylalanine (1.16 percent) was present in the diet. The percentage of the phenylalanine requirement that can be met by tyrosine remains to be determined, but it is probably similar to the 50 percent observed in other mammals. Based on data with other species, the recommendations are for 1.95 g total aromatic amino acids per 1,000 kcal ME, of which 50 percent can be supplied as tyrosine. Ward (1975) suggested that the phenylalanine requirement for maintainance of nitrogen equilibrium in the mature dog is 51 mg/kg/d or 85.8 mg/kg/d of phenylalanine and tyrosine, and this has been taken as the requirement. Threonine The threonine requirement of the immature dog has been investigated by Milner (1979b) and Burns and Milner (1982). Studies utilizing purified L-amino acid diets containing the equivalent of 14 percent protein have revealed that 0.52 percent threonine meets the requirement for optimal growth, feed efficiency, and nitrogen retention. This intake is equivalent to 254 mg per kilogram of body weight per day, which is proposed as the requirement (1.27 g/1,000 kcal ME). Signs of neurological dysfunction and/or lameness have been reported in kittens fed a threonine-deficient or imbalanced diet (Titchenal et al., 1980). However, these signs have not been reported in dogs. The estimate of Ward (1975), that the threonine requirement to maintain nitrogen balance of the mature dog is 44 mg/kg/d, was chosen as the requirement. Tryptophan The dietary tryptophan requirement of the immature dog has been met by supplying 145 mg/d/kg0.75 or by supplying 0.17 percent dietary tryptophan (Burns and Milner, 1982). Czarnecki and Baker (1982) reported that the tryptophan requirement for optimal growth of weanling English Pointers 6 to 10 weeks old was at least 0.16 percent, between 0.12 and 0.16 percent for 10- to 12-week-old puppies, and 0.12 percent for 12- to 14-week old puppies. Their studies also revealed that D-tryptophan utilization was 36 ± 6 percent (X ± SD) that of L-tryptophan. Based on the above studies, the requirement for tryptophan has been placed at 82 mg per kilogram of body weight per day or 0.41 g/1,000 kcal ME of dietary energy. The metabolism of D- and L-tryptophan by mature dogs was compared by Triebwasser et al. (1976). Kynurenic acid was shown to be a major urinary metabolite of L-tryptophan (Brown and Price, 1956; Triebwasser et al., 1976). However, unchanged D-tryptophan, D-kynurenine, and kynurenic acid were the major metabolites of D-tryptophan. The inversion of D-tryptophan to L-tryptophan via indolepyruvic acid appeared to be one of the major fates of ingested D-tryptophan in the mature dog. The minimum quantity of tryptophan that is required to maintain nitrogen equilibrium in adult dogs was estimated by Ward (1975) to be 13 mg per kilogram of body weight per day, and has been set as the daily requirement. Protein Digestibility The digestibility of some sources of protein has been evaluated in the dog. Hegsted et al. (1947) found that the apparent digestibility of proteins in an all-vegetable diet containing white bread, corn, rice, potatoes, lettuce, carrots, onions, tomatoes, and applesauce was 80.0 ± 7.7 percent (X ± SD). James and McCay (1950) reported that the apparent protein digestibility of commercial, dry-type food, containing both vegetable and animal proteins, ranged from 67 to 82 percent for adult dogs. Kendall and Holme (1982) reported the apparent crude protein (N × 6.25) digestibility coefficients for textured soy protein, extracted soy meal, full-fat soy flour, and micronized whole soybeans ranged from 71 to 87 percent. Moore et al. (1980) reported apparent digestibility values of soybean meal, corn, rice, and oats by mature Pointers to be in the range of 77 to 88 percent. Their data revealed that normal cooking procedures did not significantly influence the digestibility of rice, oat, or corn protein. Their data also indicated that increasing the fat content of the diet from 10 to 20 percent did not alter the digestibility of nitrogen in a corn-soybean-based diet. Studies of Burns et al. (1982) have shown that the apparent digestibilities of lactalbumin, casein, soy protein, and wheat gluten are 87, 85, 78, and 77 percent, respectively. The digestibilities of these proteins were 5 to 10 percent greater in the immature rat than observed in the immature dog.

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Page 28 3: 22 µg; the fourth group was given 115 µg twice a week. All dogs except for those in groups 1 and 4 were maintained on this protocol for 29 weeks. For the latter two groups, the supplemental thiamin was reduced for the last 8 weeks to 11 µg per kilogram of body weight daily and 77 µg per kilogram of body weight twice weekly, respectively. No clinical abnormalities were detected in the dogs except for one bitch in group 3, which began to lose weight after 9 weeks of receiving 22 µg thiamin per kilogram of body weight per day, and died at 22 weeks. This dog exhibited transitory diarrhea at week 19, and the terminal signs were a sudden loss of appetite and body weight, accompanied by weakness. The transketolase activity of the erythrocytes of this dog were stimulated markedly by thiamin pyrophosphate, indicating gross thiamin deficiency (Brin and Vincent, 1965). No other dogs showed changes in transketolase activity. These authors concluded that 22 µg thiamin per kilogram of body weight per day was too low to support Beagles in a satisfactory state of health during the first year of life. Cowgill (1934) reported that a daily intake of 6 µg thiamin per kilogram of body weight was sufficient for maintenance of mature dogs. Slightly higher values were suggested by Street et al. (1941), who reported that adult dogs could be maintained in apparent good health for 129 to 386 days on a 25 percent fat diet when supplied daily with 6.7 to 9.4 µg thiamin per kilogram of body weight. They suggested an allowance of approximately 8 µg thiamin per kilogram of body weight for maintenance of dogs of 6 to 10 kg. Maass et al. (1944) found that less than 10 µg per kilogram of body weight per day was inadequate for weight maintenance of adult dogs and that the thiamin requirement of adult and growing dogs did not appear to be increased by phlebotomy. These workers were the first to use a semipurified diet fortified with crystalline riboflavin, pyridoxine, calcium pantothenate, niacin, and choline chloride to investigate thiamin requirements. Previous workers such as Arnold and Elvehjem (1939) and Street et al. (1941) had relied on autoclaved yeast to supply B vitamins other than thiamin. Thiamin requirements are influenced by both physiological and dietary factors. Experimental hyperthyroidism has been shown (Drill 1941; Drill and Hays, 1942; Drill and Shaffer, 1942) to increase thiamin requirements of the dog on a body weight basis. There is limited evidence from the dog and other mammals that thiamin requirements per kilogram of diet are greater for pregnancy and lactation than for growth (e.g., Voegtlin and Lake, 1919). The level of fat and protein in the diet and the inclusion of penicillin, lactose, sorbitol, and ascorbic acid are reported to be inversely related to thiamin requirement in other species (Evans and Lepkovsky, 1929 a, b, 1935; Scott and Griffith, 1957; Haenel et al., 1959). However, a subsequent study with rats did not support the effect of fat (Murdock et al., 1974). Thiamin requirements of the cat were reported by Deady et al. (1981) to be increased by feeding a diet high in glutamic acid (an amino acid present in high concentrations in some vegetable proteins). Factors favoring intestinal microbial synthesis of thiamin, e.g., by diets containing starch rather than sucrose, and practice of coprophagy can markedly influence dietary requirements for this vitamin. Natural feeds may contain compounds with antithiamin activity that increase dietary needs. Thiaminases are present in animal tissues, notably in some freshwater and saltwater fish, shellfish, and crustaceans; some plants, e.g., ferns; and some bacteria and fungi. In general, these thiaminases are heat-labile, so are inactivated by cooking. Some plants contain small thermostable molecules (e.g., o-dihydroxyphenols such as caffeic acid and catechol) which react with thiamin and prevent its giving the thiochrome reaction (Davis and Somogyi, 1969). Evans (1975) suggests that the main product formed from the reaction of these thermostable ''antithiamin" factors is thiamin disulfide, which is biologically inactive. Further, he suggests that thermostable antithiamin factors appear to have little nutritional significance to animals. Thiamin is readily destroyed by heat, especially under basic conditions. Losses of 74 percent of thiamin have been reported for some canned dog foods due to retorting and storage for 14 days (Roche, 1981). Naturally occurring clinical cases of thiamin deficiency in dogs attributed to thermal destruction of thiamin in meat have been reported (Read et al., 1977). Therefore, intake of thiamin should be calculated from analyses of diets taken at the time of consumption. The thiamin requirements of the normal adult dog for maintenance can be met by 20 µg per kilogram of body weight daily, and the growing dog by 40 µg per kilogram of body weight. There do not appear to be any published data to permit definition of a requirement for pregnancy or lactation. All evidence suggests that 270 µg thiamin/1,000 kcal ME is adequate for maintenance and growth. Signs Of Deficiency And Pathology Because of the body's limited capacity to store thiamin, clinical signs may be observed after a relatively short period of ingestion of a thiamin-deficient diet. Anorexia has been consistently observed as an early clinical sign of thiamin deficiency. Read and Harrington (1981)

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Page 29 fed 2- to 5-month-old Beagle dogs a thiamin-deficient diet (20 to 30 µg thiamin per kilogram of diet) and described three clinical stages of the disease: an initial short (18 ± 8 days) stage of induction, during which the dogs grew suboptimally, but were otherwise healthy; an intermediate stage characterized by a variable period (59 ± 37 days) of progressive inappetence, failure to grow, loss of body weight, and coprophagy; and a terminal period that in most dogs was abrupt and short (8 ± 6 days) and consisted of either a neurological syndrome or sudden unexpected death. The neurological syndrome was characterized by anorexia, emesis, central nervous system depression, paraparesis, sensory ataxia, torticollis, circling, tonic-clonic convulsions, profound muscular weakness, and recumbency. Erythrocyte transketolase activity was depressed in deficient dogs. In vitro addition of thiamin pyrophosphate to red cells from deficient dogs gave a stimulation of transketolase activity above the normal of 11 ± 4 percent (Brin and Vincent, 1965; Noel et al., 1971; Read, 1979). Pathological changes due to thiamin deficiency predominately involve the nervous system and heart. The pattern of changes depends on the period of induction; acute deficiencies tend to involve the brain and produce severe neurological signs, whereas chronic deficiencies produce pathological changes of the myocardium and peripheral nerves (Read, 1979). Brain lesions include symmetrical necrosis of the gray matter of the inferior colliculi, medial vestibular nuclei, cerebellar nodulus, claustra and cerebral cortex (Read et al., 1977; Read, 1979). Histologically, the peripheral neuropathy reported by Cowgill (1921) and subsequent workers is characterized by diffuse bilateral myelin degeneration and axonal disintegration (Voegtlin and Lake, 1919; Street et al., 1941; Read, 1979). In contrast to thiamin deficiency in humans, cardiac hypertrophy is not a constant lesion in dogs. Andrews (1912) described hypertrophy of the right heart in one of seven puppies suckled by mothers whose babies had died from beriberi. Voegtlin and Lake (1919) and Street et al. (1941) also reported several cases of enlargement of the heart. Read (1979) described the cardiac lesion as nonspecific multifocal myocardial necrosis, and suggested primary vascular damage may be involved. The in vitro measurement of erythrocyte transketolase stimulation by thiamin pyrophosphate (Brin and Vincent, 1956; Noel et al., 1971; Read, 1979) has been used to diagnose thiamin deficiency in the dog. However, a decrease in the concentration of thiamin pyrophosphate in the blood of rats has been shown to precede changes in transketolase activity (Warnock et al., 1978) and may be a superior test for the dog. Hypervitaminosis Thiamin Rapid intravenous injection of 5 to 50 mg thiamin per kilogram of body weight causes a transient fall in blood pressure, with more severe effects from higher dosages. The lethal dose is approximately 350 mg per kilogram of body weight (Neal and Sauberlich, 1980), and death is due to depression of the respiratory center. Under ether anesthesia, blood thiamin concentrations of 7 to 10 mg/ml were fatal. The ratios of lethal intravenous doses to those administered subcutaneously or orally were estimated to be 1:6:40 (Unna, 1954). Riboflavin Microbial biosynthesis of riboflavin and other alloxazines has been shown to occur in the gastrointestinal tract of a number of animal species. However, utilization of this endogenously synthesized riboflavin varies from species to species. Within a single species, utilization depends on the composition of the diet (Christensen, 1973) and incidence of coprophagy. Young rats fed a riboflavin-free, purified diet with sucrose as the only carbohydrate will cease to grow. However, when sucrose is replaced by starch, sorbitol, or lactose, growth is comparable to that of rats supplied with riboflavin (Fridericia et al., 1927; Haenel et al., 1959). Excretion of riboflavin in urine and feces is also dependent on the carbohydrate in the diet and is suppressed by inclusion of sulfa drugs in the diet (De and Roy, 1951). The contribution of symbiotically synthesized riboflavin to the dog's requirement is not known. Street and Cowgill (1939) fed adult dogs a basal diet containing 30 percent casein, 36 percent sucrose, and 27 percent fat and an extract of rice polishings to supply B vitamins other than riboflavin. Another group of dogs was pair-fed the same diet, plus rice polishing extract and 25 µg riboflavin per kilogram of body weight. Those dogs pair-fed the supplemented diet lost body weight, because of food restriction, but remained healthy for 130 to 196 days. Dogs fed the unsupplemented diet collapsed after 120 ± 18 days of consuming the basal diet and generally responded to treatment with 0.75 mg riboflavin per kilogram of body weight. Street et al. (1941) confirmed, using the same casein-sucrose-fat diet as that used in their 1939 study, that 4 to 8 µg riboflavin per kilogram of body weight daily was inadequate for adult dogs, but 25 µg per kilogram of body weight maintained dogs without clinical signs of deficiency. Estimates of the minimal riboflavin requirement of the growing dog were made by Axelrod et al. (1940). When riboflavin was supplemented once weekly, 2 mg per kilogram of diet was inadequate, but 4 mg per kilogram

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Page 30 of diet was sufficient. In a later study (Axelrod et al., 1941) they reported a minimal requirement of 2 mg per kilogram of diet, but tissue storage was low, which suggested that 4 mg per kilogram of diet was a satisfactory level. Potter et al. (1942) reported that 60 to 100 µg per kilogram of body weight appeared to give comparable growth rates but marked differences in tissue storage. These authors also calculated that the dietary riboflavin requirement of the growing dog was more than 2 mg per kilogram of diet. They further showed that isocaloric substitution of lard for sucrose in the diet did not increase riboflavin requirements of the growing dog. Spector et al. (1943) subjected both young and adult dogs given variable riboflavin intakes to repeated phlebotomy. They suggested that 30 µg riboflavin per kilogram of body weight per day is necessary for growing dogs for good hemoglobin production and rapid recovery from anemia and that 15 µg riboflavin per kilogram of body weight per day is required by adult dogs. Heywood and Partington (1971), however, reported corneal lesions in dogs 4.5 to 5 months of age given diets containing various levels of riboflavin for 17 weeks. Without riboflavin supplementation corneal edema was seen in the fifth week, which developed to superficial vascularization at the eleventh week. Bilateral corneal opacities were also observed in one of four dogs receiving 30 µg riboflavin/kg/d. Two out of three male dogs receiving 55 µg/kg/d developed bilateral corneal lesions. Noel et al. (1972) reported corneal opacities without vascularization in two growing male Beagles receiving a diet providing 39 to 60 µg riboflavin per kilogram of body weight. It is concluded that a daily intake of 50 µg riboflavin per kilogram of body weight for adult dogs and 100 µg riboflavin per kilogram of body weight for growing puppies will provide adequate levels of the vitamin for reasonable tissue storage. No data derived from dogs are available to give a dietary requirement for gestation and lactation. This translates to 0.68 mg riboflavin per 1,000 kcal of dietary ME. Signs Of Deficiency Acute riboflavin deficiency may result in anorexia, hypothermia, a decreased respiratory rate, apathy, progressive weakness, ataxia, sudden collapse to a semicomatose condition, and death (Street and Cowgill, 1939). Chronic hyporiboflavinosis has been associated with anorexia; loss of body weight; muscular weakness, particularly of the hind quarters; dry, flaky dermatitis, accompanied by erythema of the hind legs, chest, and abdomen; and ocular lesions. The ocular lesions are generally bilateral, and progress from a watery or purulent discharge accompanied by conjunctivitis to opacity and vascularization of the cornea (Street et al., 1941; Potter et al., 1942; Heywood and Partington, 1971; Noel et al., 1972). Hyporiboflavinosis is accompanied by a reduction in erythrocyte riboflavin concentration, a reduced urinary excretion of riboflavin, and a low urinary recovery of a riboflavin test load (Axelrod et al., 1941; Potter et al., 1942; Noel et al., 1972). The anemias and fatty livers that early workers associated with hyporiboflavinosis were probably induced by the diet's lacking other essential factors, e.g., choline, folic acid, or vitamin B12, required for normal hematopoiesis and lipid transport (Noel et al., 1972). Erythrocyte glutathione reductase assay is currently the preferred test for diagnosis of riboflavin deficiency in humans. Factors affecting the assay are described by Thurnham and Rathkette (1982). Hypervitaminosis Riboflavin Riboflavin has a low toxicity, which may be a result of its low solubility. Dogs given a single oral dose of 2 g riboflavin per kilogram of body weight showed no ill effects (Unna and Greslin, 1942). Similarly, four 10-week-old puppies were fed 25 mg riboflavin per kilogram of body weight for 5 months, and neither toxic signs were observed, nor pathological changes in the organs at necropsy. Pantothenic Acid McKibben et al. (1939, 1940), Morgan and Simms (1940), and Fouts et al. (1940) demonstrated the necessity of pantothenic acid in the diet of the dog. Schaefer et al. (1942) fed weanling puppies and adult dogs a diet of 66 percent sucrose, 19 percent casein, 11 percent fats and oils, minerals and equal amounts of thiamin, riboflavin, niacin, pyridoxine, and choline, and varying levels of calcium pantothenate. These authors concluded that 100 µg calcium pantothenate per kilogram of body weight per day was adequate to prevent deficiency signs in growing puppies, but 60 µg per kilogram of body weight was insufficient. Adult dogs required less calcium pantothenate per unit of body weight than growing dogs. Sheffy (1964) depleted Beagle puppies 4 to 5 weeks of age of pantothenate by feeding a purified diet not supplemented with pantothenate for variable periods of time. Supplements of calcium pantothenate approximating 0, 50, 100, 200, 500, and 1,000 µg per kilogram of body weight per day were given. Coincidental with the initiation of supplementation, a single inoculation of

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Page 31 a virus was given, and change in body weight and antibody response were measured. Puppies receiving the 0-or 50-µg levels of calcium pantothenate died. No significant difference in growth rate occurred between those receiving 200, 500, and 1,000 µg/kg body weight. Dogs receiving either 500 or 1,000 µg/kg had higher antibody responses at 7 days, but not at 21 days following vaccination, than those receiving 200 µg/kg. As the antigen was given on the first day of supplementation, it is not clear whether a longer period of repletion would have given different results. Sheffy concluded that the daily requirement for growth was between 100 and 200 µg per kilogram of body weight. Free pantothenate appears to be efficiently absorbed by the dog, as Taylor et al. (1974) found that between 81 and 94 percent of an oral dose of sodium (14C) pantothenate was absorbed. Urinary secretion represents the major route of loss from the body, principally as the b-glucuronide (Taylor et al. 1972). The kidney of the dog is distinct from that of other animals in that it excretes little of the free vitamin, but the excretion of the b-glucuronide approaches the glomerular filtration rate. From the limited data available on dogs and the requirements of other species, it would appear prudent to provide 200 µg pantothenic acid per kilogram of body weight for adult maintenance and 400 µg per kilogram of body weight for growth of dogs as suggested by NRC (1974). No data are available to give estimates for reproduction and lactation. A dietary concentration of 2.6 mg/1,000 kcal ME is considered adequate. Signs Of Deficiency Pantothenic acid-deficient dogs exhibit erratic appetites, depressed rates of growth, reduced urinary excretion of the vitamin (Silber, 1944), lowered antibody response (Sheffy, 1964), and reduced blood concentrations of cholesterol, cholesterol esters, and total lipids (Scudi and Hamlin, 1942). Deficient dogs have reduced concentrations of pantothenate in blood, liver, muscle, and brain (Silber, 1944). In the terminal stages of pantothenic acid deficiency, dogs exhibit spasticity of the hind quarters, sudden prostration or coma, usually accompanied by rapid respiratory and cardial rates and possibly convulsions. Hypervitaminosis Pantothenic Acid Large amounts (10 to 20 g) of calcium pantothenate have been administered to humans without evidence of toxicity other than occasional diarrhea (Gershberg et al., 1949). Niacin (Nicotinic Acid, Nicotinamide) Historically the dog played an important role as a model for the study of pellagra in humans and in testing antipellagra vitamin preparations (Harvey et al., 1938). In a text on nutrition of humans, Chittenden (1907) described clinical signs of disturbances of the gastrointestinal tract with bloody discharge and inflammation of the mucous membrances of the mouth in a dog given a diet of bread and lard. Goldberger and Wheeler (1928) recognized the similarity between these signs of the disease known as "black tongue" in dogs and those of pellagra in humans (Goldberger and Wheeler, 1920). Elvehjem et al. (1937, 1938) demonstrated that nicotinic acid and nicotinamide were equally effective in curing black tongue and in preventing it in dogs given a black tongue-producing diet. Street and Cowgill (1937) also confirmed that nicotinic acid cured black tongue in dogs. Sebrell et al. (1938) gave variable amounts of nicotinic acid by intramuscular injection semiweekly to dogs of 6 to 8 kg body weight fed a corn-based diet (modified Goldberger diet). On a body weight basis, 340 µg nicotinic acid per kilogram per day prevented appearance of black tongue, while 126 µg per kilogram per day produced incipient signs of the disease. Margolis et al. (1938), also using a modified Goldberger diet, reported that black tongue induced in adult dogs was cured by a daily dose of 500 µg or greater of nicotinic acid per kilogram of body weight. When doses were reduced to 200 µg per kilogram of body weight daily the response was delayed, and 100 µg per kilogram of body weight was ineffective. Birch (1939) also used a corn-based diet and found that 130 µg nicotinic acid per kilogram of body weight gave protection against signs of black tongue and some increase in body weight of depleted adult dogs, whereas 250 µg per kilogram allowed for a rapid weight gain. Both 84 and 27 µg per kilogram of body weight gave no protection. Prior to the report of Schaefer et al. (1942), all attempts to define the nicotinic acid requirement of the dog had used a Goldberger-type diet, based largely on corn. These authors were the first to use a semipurified diet, which in this study contained sucrose, 66 percent; acid-washed casein, 19 percent; cottonseed oil, 8 percent; cod liver oil, 3 percent; and salt mixture, 4 percent. Consequently, this diet contained a lower content of niacin than diets based on natural food ingredients. The diet was fortified with thiamin, riboflavin, pyridoxine, pantothenic acid, and choline. The requirement of nicotinic acid (calculated from single-dose feedings) for adult dogs was 200 to 225 µg per kilogram of body weight per day and for growing dogs 250 to 365 µg per kilogram of body weight per day. In most animals nicotinic acid is a minor end product

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Page 32 of tryptophan degradation. Hence, dietary niacin requirements are dependent on the level of tryptophan in the diet. Singal et al. (1948) fed growing dogs a semipurified niacin-deficient diet based on sucrose, 66 percent, and casein, 19 percent. When they replaced 21 percent of the sucrose in the basal diet with an equal weight of either zein or gelatin (proteins low in tryptophan) the time that elapsed before the dogs were depleted was not prolonged. In contrast, when 21 percent of the sucrose was replaced by casein (i.e., 40 percent casein in the diet) about twice as much weight gain occurred before the dogs' weight plateaued. Also, the response to an injection of 10 mg nicotinic acid per kilogram of body weight was greater for dogs fed the 40 percent casein diet than the basal diet alone or with 21 percent gelatin or zein. While none of the above diets prevented eventual onset of clinical signs of nicotinic acid deficiency, complete protection was obtained by substituting 42 percent of the sucrose with casein. Complete protection was also obtained by adding 0.5 percent L-tryptophan (calculated tryptophan in 42 percent casein) to the basal diet. Further work suggested that the D-isomer of tryptophan was poorly utilized by the dog for niacin synthesis, but increments of 0.3, 0.2, or 0.1 percent DL-tryptophan added to the basal diet gave protection. The efficiency of utilization of D-tryptophan for growth by the dog is about one third that of L-tryptophan (Czarnecki and Baker, 1982). If one assumes comparable relative efficiencies between the isomers for niacin synthesis, then the lowest total dietary level of tryptophan giving complete protection was equivalent to 0.37 percent of L-isomer. This conclusion is difficult to reconcile with the finding that a diet of 40 percent casein containing 0.48 percent L-tryptophan resulted in the disease. From a bioassay procedure, these workers concluded that for the dog 132 mg L-tryptophan is equivalent to approximately 1 mg nicotinic acid. This ratio is considerably wider than that proposed for the rat or human. Hankes et al. (1948) calculated that 33 to 40 mg tryptophan yield 1 mg niacin for the rat, and Horwitt et al. (1956) proposed that 60 mg tryptophan are equivalent to 1 mg nicotinic acid for humans. However, this ratio is probably not constant but varies with the tryptophan and niacin concentration of the diet (Anonymous, 1974). In naturally occurring foods, particularly cereal grains, a considerable proportion of the niacin may be in the bound form, which is unavailable or only partly available unless hydrolyzed. Ghosh et al. (1963), using a microbiological assay, reported that 85 to 90 percent of the total nicotinic acid in cereals was in a bound form. Mason et al. (1973) showed that extraction of wheat bran under neutral conditions yielded 62 percent of the bound niacin in solution; of this, 90 percent was nondiffusible. Bound nicotinic acid was found to be linked to macromolecules of which 60 percent were polysac-charides and 40 percent peptides or glycopeptides. Oil seeds contain about 40 percent of their total niacin in bound form, while only a small proportion of the niacin in pulses, yeast, crustacea, fish, animal tissue, or milk is bound. By use of a rat assay procedure, Carter and Carpenter (1982) showed that for eight samples of mature cooked cereals (corn, wheat, rice, and milo), only about 35 percent of the total niacin was available. In the calculation of the niacin content of formulated diets, probably all niacin from cereal grain sources should be ignored or at least given a value no greater than one-third of the total niacin. The association of pellagra in humans and of black tongue in dogs with consumption of diets based on corn appears at least in part due to the combination of two factors—the low availability of niacin in corn and the deficiency in tryptophan of the main protein in corn (zein). Researchers from India (Belavady and Gopalan, 1965, 1966; Belavady et al., 1967; Gopalan et al., 1969) have suggested that high levels of leucine in jowar (Sorghum vulgare) may induce black tongue in dogs and pellagra in humans. However, researchers from other laboratories (Truswell et al., 1963; Nakagawa and Sasaki, 1977; Manson and Carpenter, 1978) have been unable to reproduce the reported induction of niacin deficiency by high levels of dietary leucine. Whether the condition existing in India associated with consumption of jowar involves a concurrent pyridoxine deficiency or feedback control of tryptophan pyrrolase activity as suggested by Hankes et al. (1971) has not been resolved. There are a number of reports in the literature (e.g., Handler, 1943; Krehl et al., 1945) to suggest that niacin requirements of dogs fed corn-based diets may be higher than those given purified diets. For usual diets with minimal quantities of tryptophan, the daily requirement of the adult dog will be met by 225 µg niacin per kilogram of body weight and for the growing dog by 450 µg per kilogram of body weight. These amounts will be supplied by diets containing 3 mg of niacin equivalents per 1,000 kcal ME. No experimental data are available to give a requirement for niacin during pregnancy and lactation. However, for humans the rate of conversion of tryptophan to niacin appears to be enhanced in pregnancy due to higher levels of circulating estrogen (Rose and Braidman, 1971; Horwitt et al., 1975). Battistacci et al. (1979) have shown that urinary excretion of metabolites of the tryptophan-niacin pathway are markedly increased in dogs following surgery. Anesthesia alone had no significant effect on metabolite

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Page 33 levels. The response to surgery is probably mediated by elevated corticosteroid levels and induction of tryptophan pyrrolase activity. Signs Of Deficiency Niacin-deficient dogs exhibit anorexia; loss in weight; erythema; severe inflammation and ulceration of the oral and pharyngeal mucosa; profuse salivation with ropy, bloodstained saliva drooling from the mouth; and foul breath. There is bloody diarrhea, inflammation and hemorrhagic necrosis of duodenum and jejunum with shortening and clubbing of villi, and inflammation and degeneration of the mucosa of the large intestine. Intestinal absorption of water, glucose, sodium, and potassium is reduced. Hepatic periportal fatty metamorphosis, neuronal degeneration of the spinal cord, and distortion of conditioned reflexes are evident. Urinary excretion of N-methylnicotinamide is decreased, and there are decreased liver and skeletal muscle concentrations of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. Uncorrected deficiencies lead to dehydration, emaciation, and death (Dann and Handler, 1941; Sarett, 1942; Schaefer et al., 1942; Handler, 1943; Smith et al., 1943; Layne and Carey, 1944; Efremov et al., 1954; Nelson et al., 1962; Belavady and Gopalan, 1965; Greengard et al., 1966; Madhavan et al., 1968; Manson and Carpenter, 1978). Hypervitaminosis Niacin High doses of nicotinic acid (but not nicotinamide) have been shown to cause vasodilatation and increased intracranial blood flow in humans. A cutaneous flush in dogs appeared within 10 minutes of intravenous injection of 1 to 100 mg nicotinic acid per kilogram of body weight (Pereira, 1967). Intravenous nicotinic acid also increases the flow of gastric secretions in dogs (Bailey et al., 1972). In rats, neutralized nicotinic acid injections produced a transient decline in plasma-free fatty acid concentrations possibly due to an inhibitory effect on norepinephrine-induced lipolysis (Pereira and Mears, 1971). Intravenous nicotinic acid administered to dogs prior to thermal trauma reduces plasma volume loss (Hilton and Wells, 1976). Although there are variable reports on the effect of nicotinic acid upon hypercholesterolemia in the dog (Grande and Amatuzio, 1960; Zanetti and Tennent, 1963), Grande (1966) established that nicotinic acid has a plasma cholesterol-depressing effect in normal dogs that depends upon the dose used and the initial cholesterol level. Vitamin B6 Vitamin B6, usually in the form of pyridoxal phosphate and occasionally as the amine, acts as a cofactor for a large number of enzymes involved in various aspects of amino acid metabolism including aminotransferases (transaminases), decarboxylases, racemases, dehydratases, synthetases, and hydroxylases. Pyridoxal phosphate is required for the synthesis of g-aminolevulinic acid, a precursor of heme. Hematological parameters have been the main criteria used to determine the requirement of the dog for vitamin B6 rather than the more recent and sensitive indices of adequate status, based on analysis of plasma pyridoxal 5'-phosphate, urinary 4-pyridoxic acid, and urinary tryptophan metabolite excretion following a tryptophan load (Leklem and Reynolds, 1980). Requirements Fouts et al. (1938) showed that weaned puppies given a semipurified diet lacking vitamin B6 developed a severe microcytic hypochromic anemia. This anemia could be overcome in adult dogs by oral administration of 60 µg per kilogram of body weight of crystalline pyridoxine isolated from natural sources (Fouts et al., 1939); or in puppies (McKibben et al., 1939–1940) and in adults (Borson and Mettier, 1940) by administration of the synthetic vitamin. Michaud and Elvehjem (1944) quoted unpublished experiments in which growing dogs given 5 µg pyridoxine per kilogram of body weight died before evidence of anemia appeared, whereas 10 µg per kilogram gave fairly good growth, but not equal to that with 60 µg per kilogram. They suggested that the level of 10 µg per kilogram of body weight per day may be sufficient for maintenance. Vitamin B6 deficiency was induced in adult dogs given a semipurified diet by Street et al. (1941). Control dogs pair-fed to those given the deficient diet and supplemented with a pyridoxine concentrate lost less body weight and had normal hematology. The potency of the pyridoxine concentrate given was not determined, but a similarly prepared concentrate to that used would have supplied the equivalent of 5 µg pyridoxine per kilogram per day. This intake of vitamin B6 was inadequate for a single ad libitum control dog, which required 10 µg pyridoxine per kilogram of body weight per day. Besides blood dyscrasia, anorexia, and body weight loss, Street et al. (1941) described a range of pathological changes including ataxia, cardiac dilatation and hypertrophy, congestion of various tissues, and demyelination of peripheral nerves.

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Page 34 Axelrod et al. (1945) showed that following a tryptophan load, young vitamin B6-deficient dogs excreted xanthurenic acid and kynurenine in their urine. Dogs supplemented with pyridoxine excreted kynurenine and kynurenic acid but no xanthurenic acid. They raised young dogs on vitamin B6-deficient diets containing either high or moderate levels of protein (casein). Dogs given the high level exhibited a greater decline in hemoglobin concentration and more rapid appearance of clinical signs following tryptophan loading than dogs raised on moderate levels of protein. The foregoing experimental data do not permit the derivation of a definitive requirement for either maintenance or growth of the dog. The requirement for maintenance appears to be at least 10 µg per kilogram of body weight per day, and for growth, greater than 10 µg per kilogram of body weight per day but probably less than 60 µg per kilogram of body weight per day. Values of 60 µg per kilogram per day for the growing dog and 22 µg per kilogram of body weight per day for the adult are suggested as the requirement. On a dietary basis these requirements are satisfied by 300 µg pyridoxine per 1,000 kcal ME. By comparison, recommended allowances for growth of the rat are 6.0 mg per kilogram of diet (NRC, 1978) and the pig 1.5 mg per kilogram of diet (NRC, 1979). Pyridoxal in serum from dogs appears to be more heat-stable than in the serum from horse, rabbit, and man (Davis and Smith, 1975). A naturally occurring heat-stable vitamin B6 antagonist (linatine) has been isolated from flaxseed (Klosterman et al., 1967). The presence of B6 antagonists in ingredients used in dog foods does not appear to have been examined. Signs Of Deficiency Acute deficiency of vitamin B6 in growing puppies may lead to sudden death without untoward clinical signs other than anorexia, slow growth, or body weight loss. Vitamin B6 deficiency has been associated with microcytic hypochromic anemia, generalized convulsions, and elevated plasma iron concentration (Fouts et al., 1938, 1939; McKibbin et al., 1939–1940; Borson and Mettier, 1940; Street et al., 1941; McKibbin et al., 1942). Because of the involvement of pyridoxal phosphate in amino acid metabolism, altered metabolites from tryptophan have been observed in urine (Axelrod et al., 1945). Dietary deficiencies of vitamin B6 or metabolic deficiencies induced by deoxypyridoxine have been shown to result in prolonged tolerances of skin grafts and renal transplants (Humphries et al., 1961; Fisher et al., 1963). The toxicity of the tuberculostatic drug isoniazid to dogs has been alleviated by injections of pyridoxine (Chin et al., 1978). Hypervitaminosis Vitamin B6 The vitamins B6 are not considered highly toxic and have been used in a relatively large dose (20 mg pyridoxine per kilogram of body weight intravenously) as an antidote to a rodenticide "Castrix" (Ullrich, 1967), and to protect against the toxic pressor effects of strophanthin K (Eremeev, 1968). Folacin Although dogs have been extensively used as a model for humans in the study of absorption of folic acid from the gut (Baugh et al., 1971, 1975; Bernstein et al., 1972, 1975), critical studies from which a requirement for folic acid can be derived are not available. Most of the early experiments on folic acid supplementation were undertaken before the isolation of vitamin B12 and before the interrelationships between folic acid and vitamin B12 were recognized. Krehl and Elvehjem (1945) proposed that folic acid may be synthesized in significant amounts by the intestinal bacteria and may contribute to the dog's requirement. Krehl and Elvehjem (1945) and Krehl et al. (1946) also demonstrated that dogs given niacin-deficient diets showed an enhanced response to nicotinic acid when the diet contained folic acid. Folic acid seemed to play a role in maintaining a more adequate blood picture. While intestinal synthesis in dogs has been elegantly demonstrated by Bernstein et al. (1972, 1975), the extent to which it contributes to meeting body needs has not been quantified. Furthermore, data from other species indicate the contribution is likely to be dependent on the type of diet (Teply et al., 1947; Miller and Luckey, 1963; Klipstein and Samloff, 1966). The jejunum is the preferential site of folic acid absorption (Hepner et al., 1968; Bernstein et al., 1972). Naturally occurring dietary folates are mostly in the form of polygultamates, which are not absorbed intact into the circulation. Cleavage of the polyglutamate side chain to monoglutamate or diglutamate seems to occur in the interior of intestinal epithelial cells (Baugh et al., 1975). There is no evidence of significant intraluminal conjugase activity in mammals, nor is the enzyme found in isolated brush border fractions (Hoffbrand and Peters, 1969 a, b; Halsted et al., 1975). Polyglutamates are readily absorbed by the intestinal mucosal cells and are apparently cleaved by conjugases that occur in the lysosomal particles. Afonsky (1954) reported weight loss and a progressive

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Page 35 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

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Page 36 biotin deficiencies in poultry (Johnson, 1967; Marusick et al., 1970) and swine (Adams et al., 1967) fed practical diets. While a definitive requirement for biotin for the dog cannot be stated, the inclusion in a diet of 30 µg biotin per 1,000 kcal ME may be prudent as a safeguard against a possible deficiency. This level is similar to that suggested for the growing pig (National Research Council, 1979). Signs Of Deficiency No adequate descriptions of biotin deficiency in the dog are available. Greve (1963) reported scurfy skin (due to hyperkeratosis of the superficial and follicular epithelia) and a marked decline in urinary biotin concentration. No alopecia or achromotrichia was noted. Vitamin B12 Requirements In mammals vitamin B12 is required as a factor for the transmethylation of 5-methyl tetrahydrofolate to homocysteine and the formation of tetrahydrofolate and methionine. Vitamin B12 also participates as a coenzyme for the conversion of methylmalonyl-CoA to succinyl-CoA (Weissbach and Taylor, 1970). It has been hypothesized that a metabolic deficiency of vitamin B12 results in folic acid being "trapped" as methyl tetrahydrofolate and that it depletes 5- to 10-methylene tetrahydrofolate required for thymidylate synthesis and therefore DNA biosynthesis (Herbert and Zalusky, 1962; Mertz et al., 1968; Butterworth and Krumdieck, 1975). This hypothesis explains much, but not all, of the interaction of vitamin B12 in hematopoiesis (Chanarin et al., 1981). The dog has been used intensively as a model in the study of the mechanism of vitamin B12 absorption (Reizenstein et al., 1960; Fleming et al., 1962; Hermann et al., 1964; Bryant and Stafford, 1965; Gazet and McColl, 1967; Weisberg et al., 1968; Lavrova, 1969; Taylor et al., 1969; Yamaguchi et al., 1969a,b; Weisberg and Rhodin, 1970), plasma transport of vitamin B12 (Markelova, 1960; Rappazzo and Hall, 1972; Sonneborn et al., 1972; Hall and Rappazzo, 1974), and tissue vitamin B12 distribution (Cooperman et al., 1960; Woods et al., 1960; Skeggs et al., 1963; Rosenblum et al., 1963); however, no definitive data on dietary vitamin B12 requirements for the dog are available. Arnrich et al. (1952) fed a semipurified diet containing 20 percent vitamin-free casein without supplemental vitamin B12 to weanling Cocker Spaniel puppies for 20 weeks. No anemia developed and gains were satisfactory, although a supplement of 50 µg vitamin B12 per kilogram of diet appeared to increase gains (primarily fat). Likewise, urinary vitamin B12 excretion has been studied in the dog (Nelp et al., 1964; Coppi et al., 1970; and Silverman, 1979), but no data were presented that would provide a guide to vitamin B12 status in relation to vitamin B12 intake. In rats the toxicity of methionine can be reduced by the inclusion of vitamin B12 in the diet at about 3 times the requirement (Areshkina et al., 1973). The requirement for dietary vitamin B12 varies with the dietary content of choline, methionine, and folic acid. In the absence of information on the requirement of dogs for vitamin B12, it is suggested that the recommended requirement for the baby pig (NRC, 1979) of 0.5 µg vitamin B12 per kilogram of body weight be adopted for maintenance of the adult dog and twice this level (1.0 µg per kilogram of body weight) be used for growth of puppies. These amounts would be supplied by 26 µg vitamin B12 per kilogram of dry diet containing 3.67 kcal ME per gram, or 7 µg per 1,000 kcal dietary ME. No data are available to make a recommendation of the requirement during pregnancy and lactation. The requirement for bitches fed diets based on soy protein may be greater during pregnancy than the above recommendation, as Woodward and Newberne (1966) reported hydrocephaly in rat pups from female rats fed a soy-based diet. This condition was prevented by supplementation of the diet by 50 µg per kilogram of vitamin B12 recommended by NRC (1978) for the rat. Signs Of Deficiency Uncomplicated vitamin B12 deficiency has not been described in the dog. Lavrova (1969) reported an anemia in dogs with an internal biliary fistula, which may have been associated with a failure in vitamin B12 absorption. The anemia was generally macrocytic hypochromic, macrocytic normochromic, normocytic hypochromic, or normocytic normochromic in type. The bone marrow erythropoietic centers appeared hypoplastic. Serum and liver vitamin B12 concentrations were decreased. In vitamin B12 deficiency there is an enhanced urinary excretion of methylmalonic acid. Vitamin B12 is required as a coenzyme for the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A. A clinical test for vitamin B12 deficiency is to load the animal with a precursor of methylmalonic acid (e.g., valine) and measure urinary excretion of methylmalonic acid (Williams et al., 1969; Chanarin et al., 1973). Hypervitaminosis B12 Although frank vitamin B12 toxicity has not been described in the dog, Pshonik and Gribanov (1961) noted

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Page 37 disturbances of reflex activity in the form of reduction in size of vascular conditioned reflexes, exaggeration of unconditioned reflexes, and intensification of successive inhibition when vitamin B12 was injected subcutaneously in doses of 2 to 33 µg per kilogram of body weight. Choline Requirements The importance of choline in the nutrition of the dog was suggested by its lipotrophic action on the liver of the depancreatized dog, according to Best et al. (1933). The dietary requirement for choline is markedly affected by the concentration of other methyl donors in the diet, of which the most important is methionine. High levels of methionine in the diet will obviate the need for dietary choline. Schaefer et al. (1941) pointed out that a number of workers have found that dietary casein concentrations of 40 percent or more tend to obviate the need for dietary choline. In the studies of Schaefer et al. (1941) themselves, puppies receiving a 19 percent casein diet became choline-deficient. Controls receiving 50 mg choline per kilogram of body weight per day grew satisfactorily over the 37-day experimental period. On a 15 percent casein diet, Fouts (1943) found that 10 or 20 mg choline per kilogram of body weight would not prevent or cure the deficiency state in puppies, while a 100-mg level would. When 41 percent casein was provided, no choline deficiency nor any response to supplemental choline could be shown. McKibbin et al. (1944) fed a diet containing natural proteins low in sulfur amino acids (10 percent protein from peanut flour plus 10 percent casein) to puppies and concluded that choline requirements were probably not greater than 1,000 mg per kilogram of diet or 50 mg per kilogram of body weight per day. Complex interactions occur involving single carbon transfer by choline, methionine, folate, and vitamin B12. Choline requirements can only be determined when the diet contains a minimal, but adequate level of methionine and adequate levels of folate and vitamin B12. When the previous experiments were undertaken the minimal requirement of the dog for methionine was not known, and isolated sources of vitamin B12 and folic acid were not available. Furthermore, for rats the dietary requirement of choline is influenced by the lipid content of the diet, the chain length and degree of saturation of the fatty acids, and the total caloric content of the diet (Best et al., 1954; Salmon and Newberne, 1962; Zachi et al., 1965; Patek et al., 1966). It is concluded that the choline requirements for adult maintenance may be met by 25 mg per kilogram of body weight per day and those for growth of puppies by 50 mg per kilogram of body weight per day. Diets containing 340 mg choline per 1,000 kcal ME will supply these quantities of choline when fed to adult or growing dogs. Signs Of Deficiency Dutra and McKibbin (1945) described the pathology of "uncomplicated" choline deficiency in young puppies. They reported fatty metamorphosis of the liver and atrophic changes of the thymus. The morphologic changes in the liver, correlated with impairment in liver function as measured by delayed bromsulfalein elimination, were reported by McKibbin et al. (1944, 1945) and Anonymous (1945a,b). Plasma phosphatase activity and blood prothrombin times were also elevated in the choline-deficient puppies. Choline-deficient dogs with fatty livers show an increased rate of hepatic phospholipid synthesis following choline supplementation (Di Luzio and Zilversmit, 1959). Excess Dietary Choline Acara and Rennick (1973) reported renal clearance studies on dogs that indicated that only one-thirtieth of the choline filtered at the glomerulus was excreted in the urine, suggesting active tubular reabsorption. When exogenous choline was infused intravenously, choline renal clearance exceeded glomerular filtration rate, indicating active tubular excretion. Solomon (1966) has reported that infusion of choline results in urinary alkalinization, primarily from an increased urinary bicarbonate output. At the same time, there is a decrease in ammonia output. Vitamin C Innes (1931) demonstrated that the dog, unlike the guinea pig, was independent of an exogenous supply of vitamin C. Puppies fed a diet devoid of vitamin C for 147 to 154 days showed neither growth impairment nor lesions of bones or teeth, although the same diet killed guinea pigs within 25 days with severe signs of scurvy. Furthermore, the livers of dogs on the deficient diet contained the vitamin in sufficient amounts to prevent the onset of scurvy in guinea pigs, indicating that the dog can synthesize its own vitamin C. Naismith (1958) showed that this synthetic ability is present in puppies during the first weeks of postnatal life. Litters were divided: some puppies were left with the bitch; others were fed a synthetic diet minus vitamin C, or plus vitamin C. No significant differences in blood ascorbic acid concentration were evident, regardless of treatment. Naismith and Pellet (1960) reported that the concentration

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Page 38 of ascorbic acid in the milk from bitches is approximately 4 times that of the blood. The comparative rates of hepatic synthesis of ascorbic acids in dogs and cats appear to be lower than that in ruminants, rodents, and lagomorphs (Chatterjee et al., 1975). Despite the above evidence, a number of clinical case history reports (Garlick, 1946; Meier et al., 1957; Ditch-field and Phillipson, 1960; Holmes, 1962; Hunt, 1962; Sadek, 1962; Bendefy, 1965; Belfield, 1967, 1976; Vaananen and Wikman, 1979) have been published purporting to describe scurvy in the dog with or without concomitant hip dysplasia or osteodystrophy. None of these reports included observations on control untreated animals. Also, the effect on the dog of pharmacological doses of ascorbic acid (e.g., 3,000 mg intravenously per day) may be quite distinct from its nutritional contribution. Teare et al. (1980) reported that 600 mg of ascorbic acid twice daily only aggravated the skeletal disease induced by overfeeding protein, energy, and calcium to Labrador Retriever puppies. In addition, vitamin C has been proposed as a prophylactic agent against canine distemper (Belfield, 1967; Leveque, 1969), and some veterinary practitioners apparently advocate vitamin C for the treatment of kennel cough. Sheffy (1972) conducted some carefully controlled studies concerned with these issues and established that exogenous vitamin C was of no benefit in alleviating clinical signs of illness, mortality, or gross or microscopic pathology associated with experimentally produced canine herpes virus infection, kennel cough, or infectious canine hepatitis. In addition, as determined by measuring blood ascorbic acid levels, the latter disease did not affect vitamin C synthesis. Other data on blood and urine ascorbic acid values in the dog have been published by Majumdar et al. (1964), Kleit et al. (1965), Crilly et al. (1976), and Robinson et al. (1979). Csaba and Toth (1966), in controlled studies, established that ascorbic acid given before antigen challenge in dogs has no protective action against anaphylactic shock and does not influence histamine release. Weintraub and Griner (1974) found that high doses of ascorbic acid had no effect on biological half-life or kinetics of Warfarin-induced hypoprothrombinemia. It is concluded that there is no adequate evidence to justify recommendation of routine vitamin C additions to the diet of the normal dog. However, dogs with hepatic dysfunction may have lowered plasma concentrations of ascorbic acid (Strombeck et al., 1983). Whether lower plasma concentrations are of clinical significance remains to be demonstrated.