Nutr. Mctabol. 18: 240 244 (1975)
Levels of Urea in the Blood and Urine of Rats Fed Raw and Heated Soybean Meal1 Zafrira Nitsan and Irvin E. Liener Department of Biochemistry, College of Biological Sciences. University of Minnesota, St. Paul, Minn.
Key Words. Blood urea ■Heated soybean meal • Raw soybean meal • Urea nitrogen Urinary urea Abstract. Proteins from many sources show negative correlation between their bio logical values and the levels of urea in the blood or urine of rats or pigs. On the basis of the difference in protein quality between raw and heated soybeans, it would be predicted that there should be a higher level of urea in the blood and urine of rats fed raw soybean meal. In the present study, however, little or no difference in the levels of urea in the blood and urine of animals fed raw or autoclaved soybean meal could be demonstrated. It is postulated that the increased catabolism of the poor quality protein of raw soybeans may be masked by a concomitant depression of liver arginase activity and/or a decrease in the total quantity of amino acids available for catabolism because of lower digestibility of the raw soybean protein.
Introduction Several investigators have reported that the level of urea in the blood de creases when a diet containing a deficient protein is supplemented with its first limiting amino acid (4, 8, 11, 14). The excretion of urea in the urine of rats (7) and pigs (1) on diets containing a poor quality protein is likewise reduced when such diets are supplemented with the missing amino acids. It has therefore been suggested that the measurement of urea in the blood (4) or urine (1) might provide a rapid and inexpensive technique for evaluating protein quality. Since it is a well-known phenomenon that heat treatment enhances the nutritive quality of the protein of soybean meal (9, 10), it was of interest to ascertain whether ' Supported by grants AM-13869 from the National Institutes of Health and GB-15385 from the National Science Foundation.
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Received: December 30, 1974; accepted: February 10. 1975.
241
N it sau I¡Jener
Table I. Composition of experimental diets Ingredient
Diet, %
Soybean meal, raw1 or heated2 Corn oil Mineral mix3 Vitamin mix3 Corn starch
18.6 6.0 4.0 1.0
70.4
1 Defatted soybean flour (Central Soya, Chicago). This product had a protein dispersity index of 90 and a protein content of 53.5 % (N X 6.25). 2 Autoclaved for 20 min at 15 psi (120°). 3 Purchased from General Biochemicals, Chagrin Falls, Ohio.
this difference in protein quality between raw and heated soybean meal would also be reflected in the levels of urea in the blood and urine of rats receiving such diets. Materials and Methods Three experiments were conducted with rats, using in all cases the basal ration shown in table 1 which provided a level of 10 % protein in the diet. In trial 1, 10 male albino rats (Holtzman Co., Madison. Wise.), weighing an average of 90 g, were divided equally into two groups and housed in individual metabolic cages in ordet to permit a separate collection of urine and feces. Each group was fed ad libitum diets containing cither raw or autoclaved soybean meal. Samples of blood were withdrawn from the tail vein on the 2nd, 5th, and 14th day of the experiment, and urine was collected daily. Deproteinized (2) blood and urine were kept at -20° until analyzed. Urea was determined by the method of Coulombe and Favreau (2). In trial II, 24 male weanling rats were divided into two groups, equalized as closely as possible with respect to weight (average 55 g), and housed in individual wire-bottom cages. Again, one group received the unheated meal, and the olher group the heated meal. Blood samples for urea determination were taken from 6 rats of each group on (he morning of the 18th day. Blood samples from the remaining 6 rats in each group were taken 24 h later during which time the animals had been fasted. In trial III, two groups of 6 male rats each, weighing an average of 120 g, were individ ually housed and fed raw and heated soybean meal diets, respectively. Prior to withdrawing blood samples, food was withheld for 18 h. The blood sample taken at this time was considered to be zero time, and additional samples were taken 2 and/or 5 h after refeeding. This procedure was repeated on the 1st, 3rd, and 9th day of (he experiment. All of the data were analyzed for statistical significance as described by Snedecor (15).
Rats fed on raw soybean diet grew 30 % less than those fed heated soybean diet although they consumed similar amounts of food as recorded elsewhere
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Results
Nitsan/Liener
242
Table 11. Urinary excretion of urea by rats fed raw and heated soybean meal (trial 1) Days on experiment Excretion, mg/day
raw 1 2 3 4 5 6 7 14 Mean
30.5 19.0 26.3 22.5 46.2 33.5 45.6 42.8 33.3
± 3.6' ± 5.4 ± 6.9 ± 4.4 ± 3.1 t 3.2 ± 6.8 ± 8.1 ± 2.41*3
Percent of N intake excreted as urea N
heated
raw
heated
42.4 28.8 49.7 21.3 61.7 42.1 48.7 51.5 43.3
12.5 ± 1.9 8.8 ± 3.1 8.7 ± 2.2 7.4 ± 2.5 17.6 ± 1.7 11.2 ± 1.5 19.6 ± 3.7 13.2 ± 2.4 12.4 ± 1.2
14.2 ± 4.8 14.3 ± 4.3 16.3 + 4.0 6.2 ± 2.1 17.2 ± 2.9 10.9 ± 0.8 17.1 ± 3.5 13.2 ± 2.4 13.7 ± 1.6
± 11.1 ± 6.2 ± 16.0 ± 10.3 ± 10.4 + 4.9 ± 2.8 ± 9.8 ± 3.73
1 Mean ± SEM. Each daily mean based on 5 animals. 3 Statistically different (p < 0.05).
Table III. Blood urea levels' of rats fed raw and heated soybean meal Days on experiment
Raw
Heated
Trial 1 2 5 14
44.1 t 8.8 29.0 ± 1.2 28.7 ± 5.4
38.7 ± 2.4 33.5 ± 2.6 27.2 ± 3.0
Trial II 18 19’
32.7 t 1.7 22.7 ± 3.4
32.7 ± 1.2 25.8 t 1.4
Trial 111 1 (0)3 1 (5)
31.9 ± 2.9 28.5 ± 3.7
32.8 ± 3.3 30.3 t 2.5
3(0) 3(2) 3(5)
47.5 ± 8.3 49.9 i 4.8 42.2 ± 8.8
39.2 ± 3.7 51.7 ± 10.1 33.7 ± 4.2
9(0) 9(5)
40.6 i 2.1 29.6 t 2.6
33.1 ♦ 2.4 30.5 t S.l
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1 Expressed as mg/100 ml blood. Values shown are means t SEM. 3 Includes an additional 24 It period of fasting. 3 Figures in parentheses denote the number of hours after refceding following a fasting period of 18 h.
Blood and Urine Urea Levels on Soybean Diets
243
(12). The protein digestibility (percent absorption of intake) was 86.4 and 76.4 for the heated and raw soybean proteins. The daily excretion of urea in the urine of rats fed raw or heated soybean in trial 1 is recorded in table II. In general the level of urea excreted by the animals receiving the unheated meal tended to be somewhat lower. The differences between the two groups, when compared on a daily basis, were not statistically significant. However, the mean value for the entire period was significantly lower (p < 0.05) for the group on raw soybean meal compared with that on heated soybean meal. Since the level of urea ex creted in the urine can be influenced by the quantity of protein ingested (4, 7), urinary urea was also calculated as the percentage of the nitrogen intake. As shown in table II, the differences in urea excretion between the two groups became even less pronounced, and the overall averages were no longer statisti cally significant. Determination of the level of urea in the blood of the animals in trial I failed to disclose any significant difference between the groups fed raw or auto claved soybean meal (table III). Repetition of these measurements in experi ments where samples of blood were taken before and after varying periods of fasting (trials 11 and III) likewise revealed no significant difference in the levels of urea in the blood of animals receiving raw or heated soybean meal.
Because of the well-established superiority of the protein quality of heated soybean meal over that of unheated meal (10, 13), one might have anticipated that the levels of urea would have been higher in the blood and urine of animals fed the raw soybean meal, as a high negative correlation between protein quality and urea levels were shown for many other proteins (4, 5, 11). The results of this study, however, do not support this prediction. No significant differences in the levels of urea in the blood or urine could be demonstrated, and, in fact, the excretion of urea in the urine was even somewhat lower in the group fed raw soybean meal when the level of significance was calculated on the basis of data obtained over the entire experimental period of 14 days. It would appear that the higher rate of urea production which usually accompanies the metabolism of a poor quality protein (1, 4, 5) is, in the case of raw soybean protein, com pensated for by factors which lead to a decrease in urea production. The lower levels of urea in the raw soybean fed group can perhaps be related to the fact that heat treatment enhanced the digestibility of the protein in 12 %. This means that in raw soybean meal a smaller fraction of the ingested protein is actually absorbed and hence a lesser quantity of the ingested amino acids is available for catabolic conversion to urea in the animal body. With other proteins, however, such as barley and rye (4, 6), protein digestibilities in rats were 82 and 77 %
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Discussion
Nitsan/Liener
244
while blood urea were 10 and 17 mg%, respectively. It seems, therefore, that soybeans have additional specific effect interfering with urea production. This might be the lower level of arginase activity in the liver of raw compared to heated soybean meal fed rats (3). Regardless of the interpretation, the observa tions recorded here serve to emphasize the fact that it cannot always be assumed that the level of urea in the blood or urine will be an accurate reflection of the quality of the protein in the diet.
References
Dr. Zafrira Nitsan, Department of Animal Nutrition, Agricultural Research Organization, The Volcani Center, Reliovot (Israel)
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1 Brown, J.A. and Cline. T.R.: Urea excretion in the pig: an indicator of protein quality and amino acid requirements. J. Nutr. 104: 542-545 (1974). 2 Coulombe, J.J. and Favreau, I..: A new simple semimicro method for calorimetric determination of urea. Clin. Chem. 9: 102 108 (1963). 3 Dror, Y. and Gertler, A.: Influence of raw and heated soybean meal and of soybean trypsin inhibitors on the activity of certain protein catabolism enzymes in kidneys and livers of chicks and rats. J. Nutr. 93: 401-410 (1967). 4 Eggum, B.O.: Blood urea measurement as a technique for assessing protein quality. Br. J. Nutr. 24: 983 988 (1970). 5 Eggum, B.O.: The levels of blood amino acids and blood urea as indicators of protein quality; in Porter and Rolls Proteins in human nutrition, pp. 317 327 (Academic Press, New York 1973). 6 Eggum, B.O.: A study of certain factors influencing protein utilization in rats and pigs, p. 406 (Beretening fra fors^gslaboratoriet, Ktfbenhavn 1973). 7 Kiriyama, S. and Iwao, H.: Effect of dietary amino acid balance on the excretion of urinary N compounds and their ratios. Agr. Biol. Chem. 28: 307 312 (1964). 8 Kumta, U.S. and Harper, A.E.: Amino acid balance and imbalance. VII. effects of dietary additions of amino acids on food intake and blood urea concentration of rats fed low-protein diets containing fibrin. J. Nutr. 74: 139- 147 (1961). 9 Liener, l.E. and Kakade, M.L.: Protease inhibitors: in Liener Toxic constituents of plant foodstuffs, pp. 8 -6 8 (Academic Press, New York 1969). 10 Liener, l.E.: Nutritional value of food protein products; in Smith and Circle Soybeans: chemistry and technology, pp. 203 - 277 (Avi Publishing. Westport 1972). 11 Munchow, H. und Bergner, H.: Empfchlung zur Proteinbewertung von Eiweissfuttermitteln anhand dcr Bestimmung der Harnstoff-konzentration in Blut von Ratte Oder Schwein. Nutr. Abstr. Rev. 39: 77 (1966). 12 Nitsan, Z. and Liener, l.E.: Studies on the digestibility and retention of nitrogen and amino acids in rats fed raw and heated soybean diets (in press). 13 Rackis, J.J.: Biological and physiological factors in soybeans. J. Am. Oil Chem. Soc. 51: 161-174 (1974). 14 Rose, W.C.: Johnson, J.E., and Haines, W.J.: The amino acid requirements of man. 1. The role of valine and methionine. J. biol. Chem. 182: 541 556 (1950). 15 Snedecor, G. IP..' Statistical methods; 5th ed. (Iowa State College Press, Ames 1962).
Levels of Urea in the Blood and Urine of Rats Fed Raw and Heated Soybean Meal1 Zafrira Nitsan and Irvin E. Liener Department of Biochemistry, College of Biological Sciences. University of Minnesota, St. Paul, Minn.
Key Words. Blood urea ■Heated soybean meal • Raw soybean meal • Urea nitrogen Urinary urea Abstract. Proteins from many sources show negative correlation between their bio logical values and the levels of urea in the blood or urine of rats or pigs. On the basis of the difference in protein quality between raw and heated soybeans, it would be predicted that there should be a higher level of urea in the blood and urine of rats fed raw soybean meal. In the present study, however, little or no difference in the levels of urea in the blood and urine of animals fed raw or autoclaved soybean meal could be demonstrated. It is postulated that the increased catabolism of the poor quality protein of raw soybeans may be masked by a concomitant depression of liver arginase activity and/or a decrease in the total quantity of amino acids available for catabolism because of lower digestibility of the raw soybean protein.
Introduction Several investigators have reported that the level of urea in the blood de creases when a diet containing a deficient protein is supplemented with its first limiting amino acid (4, 8, 11, 14). The excretion of urea in the urine of rats (7) and pigs (1) on diets containing a poor quality protein is likewise reduced when such diets are supplemented with the missing amino acids. It has therefore been suggested that the measurement of urea in the blood (4) or urine (1) might provide a rapid and inexpensive technique for evaluating protein quality. Since it is a well-known phenomenon that heat treatment enhances the nutritive quality of the protein of soybean meal (9, 10), it was of interest to ascertain whether ' Supported by grants AM-13869 from the National Institutes of Health and GB-15385 from the National Science Foundation.
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 7/18/2018 5:32:39 PM
Received: December 30, 1974; accepted: February 10. 1975.
241
N it sau I¡Jener
Table I. Composition of experimental diets Ingredient
Diet, %
Soybean meal, raw1 or heated2 Corn oil Mineral mix3 Vitamin mix3 Corn starch
18.6 6.0 4.0 1.0
70.4
1 Defatted soybean flour (Central Soya, Chicago). This product had a protein dispersity index of 90 and a protein content of 53.5 % (N X 6.25). 2 Autoclaved for 20 min at 15 psi (120°). 3 Purchased from General Biochemicals, Chagrin Falls, Ohio.
this difference in protein quality between raw and heated soybean meal would also be reflected in the levels of urea in the blood and urine of rats receiving such diets. Materials and Methods Three experiments were conducted with rats, using in all cases the basal ration shown in table 1 which provided a level of 10 % protein in the diet. In trial 1, 10 male albino rats (Holtzman Co., Madison. Wise.), weighing an average of 90 g, were divided equally into two groups and housed in individual metabolic cages in ordet to permit a separate collection of urine and feces. Each group was fed ad libitum diets containing cither raw or autoclaved soybean meal. Samples of blood were withdrawn from the tail vein on the 2nd, 5th, and 14th day of the experiment, and urine was collected daily. Deproteinized (2) blood and urine were kept at -20° until analyzed. Urea was determined by the method of Coulombe and Favreau (2). In trial II, 24 male weanling rats were divided into two groups, equalized as closely as possible with respect to weight (average 55 g), and housed in individual wire-bottom cages. Again, one group received the unheated meal, and the olher group the heated meal. Blood samples for urea determination were taken from 6 rats of each group on (he morning of the 18th day. Blood samples from the remaining 6 rats in each group were taken 24 h later during which time the animals had been fasted. In trial III, two groups of 6 male rats each, weighing an average of 120 g, were individ ually housed and fed raw and heated soybean meal diets, respectively. Prior to withdrawing blood samples, food was withheld for 18 h. The blood sample taken at this time was considered to be zero time, and additional samples were taken 2 and/or 5 h after refeeding. This procedure was repeated on the 1st, 3rd, and 9th day of (he experiment. All of the data were analyzed for statistical significance as described by Snedecor (15).
Rats fed on raw soybean diet grew 30 % less than those fed heated soybean diet although they consumed similar amounts of food as recorded elsewhere
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Results
Nitsan/Liener
242
Table 11. Urinary excretion of urea by rats fed raw and heated soybean meal (trial 1) Days on experiment Excretion, mg/day
raw 1 2 3 4 5 6 7 14 Mean
30.5 19.0 26.3 22.5 46.2 33.5 45.6 42.8 33.3
± 3.6' ± 5.4 ± 6.9 ± 4.4 ± 3.1 t 3.2 ± 6.8 ± 8.1 ± 2.41*3
Percent of N intake excreted as urea N
heated
raw
heated
42.4 28.8 49.7 21.3 61.7 42.1 48.7 51.5 43.3
12.5 ± 1.9 8.8 ± 3.1 8.7 ± 2.2 7.4 ± 2.5 17.6 ± 1.7 11.2 ± 1.5 19.6 ± 3.7 13.2 ± 2.4 12.4 ± 1.2
14.2 ± 4.8 14.3 ± 4.3 16.3 + 4.0 6.2 ± 2.1 17.2 ± 2.9 10.9 ± 0.8 17.1 ± 3.5 13.2 ± 2.4 13.7 ± 1.6
± 11.1 ± 6.2 ± 16.0 ± 10.3 ± 10.4 + 4.9 ± 2.8 ± 9.8 ± 3.73
1 Mean ± SEM. Each daily mean based on 5 animals. 3 Statistically different (p < 0.05).
Table III. Blood urea levels' of rats fed raw and heated soybean meal Days on experiment
Raw
Heated
Trial 1 2 5 14
44.1 t 8.8 29.0 ± 1.2 28.7 ± 5.4
38.7 ± 2.4 33.5 ± 2.6 27.2 ± 3.0
Trial II 18 19’
32.7 t 1.7 22.7 ± 3.4
32.7 ± 1.2 25.8 t 1.4
Trial 111 1 (0)3 1 (5)
31.9 ± 2.9 28.5 ± 3.7
32.8 ± 3.3 30.3 t 2.5
3(0) 3(2) 3(5)
47.5 ± 8.3 49.9 i 4.8 42.2 ± 8.8
39.2 ± 3.7 51.7 ± 10.1 33.7 ± 4.2
9(0) 9(5)
40.6 i 2.1 29.6 t 2.6
33.1 ♦ 2.4 30.5 t S.l
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1 Expressed as mg/100 ml blood. Values shown are means t SEM. 3 Includes an additional 24 It period of fasting. 3 Figures in parentheses denote the number of hours after refceding following a fasting period of 18 h.
Blood and Urine Urea Levels on Soybean Diets
243
(12). The protein digestibility (percent absorption of intake) was 86.4 and 76.4 for the heated and raw soybean proteins. The daily excretion of urea in the urine of rats fed raw or heated soybean in trial 1 is recorded in table II. In general the level of urea excreted by the animals receiving the unheated meal tended to be somewhat lower. The differences between the two groups, when compared on a daily basis, were not statistically significant. However, the mean value for the entire period was significantly lower (p < 0.05) for the group on raw soybean meal compared with that on heated soybean meal. Since the level of urea ex creted in the urine can be influenced by the quantity of protein ingested (4, 7), urinary urea was also calculated as the percentage of the nitrogen intake. As shown in table II, the differences in urea excretion between the two groups became even less pronounced, and the overall averages were no longer statisti cally significant. Determination of the level of urea in the blood of the animals in trial I failed to disclose any significant difference between the groups fed raw or auto claved soybean meal (table III). Repetition of these measurements in experi ments where samples of blood were taken before and after varying periods of fasting (trials 11 and III) likewise revealed no significant difference in the levels of urea in the blood of animals receiving raw or heated soybean meal.
Because of the well-established superiority of the protein quality of heated soybean meal over that of unheated meal (10, 13), one might have anticipated that the levels of urea would have been higher in the blood and urine of animals fed the raw soybean meal, as a high negative correlation between protein quality and urea levels were shown for many other proteins (4, 5, 11). The results of this study, however, do not support this prediction. No significant differences in the levels of urea in the blood or urine could be demonstrated, and, in fact, the excretion of urea in the urine was even somewhat lower in the group fed raw soybean meal when the level of significance was calculated on the basis of data obtained over the entire experimental period of 14 days. It would appear that the higher rate of urea production which usually accompanies the metabolism of a poor quality protein (1, 4, 5) is, in the case of raw soybean protein, com pensated for by factors which lead to a decrease in urea production. The lower levels of urea in the raw soybean fed group can perhaps be related to the fact that heat treatment enhanced the digestibility of the protein in 12 %. This means that in raw soybean meal a smaller fraction of the ingested protein is actually absorbed and hence a lesser quantity of the ingested amino acids is available for catabolic conversion to urea in the animal body. With other proteins, however, such as barley and rye (4, 6), protein digestibilities in rats were 82 and 77 %
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 7/18/2018 5:32:39 PM
Discussion
Nitsan/Liener
244
while blood urea were 10 and 17 mg%, respectively. It seems, therefore, that soybeans have additional specific effect interfering with urea production. This might be the lower level of arginase activity in the liver of raw compared to heated soybean meal fed rats (3). Regardless of the interpretation, the observa tions recorded here serve to emphasize the fact that it cannot always be assumed that the level of urea in the blood or urine will be an accurate reflection of the quality of the protein in the diet.
References
Dr. Zafrira Nitsan, Department of Animal Nutrition, Agricultural Research Organization, The Volcani Center, Reliovot (Israel)
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 7/18/2018 5:32:39 PM
1 Brown, J.A. and Cline. T.R.: Urea excretion in the pig: an indicator of protein quality and amino acid requirements. J. Nutr. 104: 542-545 (1974). 2 Coulombe, J.J. and Favreau, I..: A new simple semimicro method for calorimetric determination of urea. Clin. Chem. 9: 102 108 (1963). 3 Dror, Y. and Gertler, A.: Influence of raw and heated soybean meal and of soybean trypsin inhibitors on the activity of certain protein catabolism enzymes in kidneys and livers of chicks and rats. J. Nutr. 93: 401-410 (1967). 4 Eggum, B.O.: Blood urea measurement as a technique for assessing protein quality. Br. J. Nutr. 24: 983 988 (1970). 5 Eggum, B.O.: The levels of blood amino acids and blood urea as indicators of protein quality; in Porter and Rolls Proteins in human nutrition, pp. 317 327 (Academic Press, New York 1973). 6 Eggum, B.O.: A study of certain factors influencing protein utilization in rats and pigs, p. 406 (Beretening fra fors^gslaboratoriet, Ktfbenhavn 1973). 7 Kiriyama, S. and Iwao, H.: Effect of dietary amino acid balance on the excretion of urinary N compounds and their ratios. Agr. Biol. Chem. 28: 307 312 (1964). 8 Kumta, U.S. and Harper, A.E.: Amino acid balance and imbalance. VII. effects of dietary additions of amino acids on food intake and blood urea concentration of rats fed low-protein diets containing fibrin. J. Nutr. 74: 139- 147 (1961). 9 Liener, l.E. and Kakade, M.L.: Protease inhibitors: in Liener Toxic constituents of plant foodstuffs, pp. 8 -6 8 (Academic Press, New York 1969). 10 Liener, l.E.: Nutritional value of food protein products; in Smith and Circle Soybeans: chemistry and technology, pp. 203 - 277 (Avi Publishing. Westport 1972). 11 Munchow, H. und Bergner, H.: Empfchlung zur Proteinbewertung von Eiweissfuttermitteln anhand dcr Bestimmung der Harnstoff-konzentration in Blut von Ratte Oder Schwein. Nutr. Abstr. Rev. 39: 77 (1966). 12 Nitsan, Z. and Liener, l.E.: Studies on the digestibility and retention of nitrogen and amino acids in rats fed raw and heated soybean diets (in press). 13 Rackis, J.J.: Biological and physiological factors in soybeans. J. Am. Oil Chem. Soc. 51: 161-174 (1974). 14 Rose, W.C.: Johnson, J.E., and Haines, W.J.: The amino acid requirements of man. 1. The role of valine and methionine. J. biol. Chem. 182: 541 556 (1950). 15 Snedecor, G. IP..' Statistical methods; 5th ed. (Iowa State College Press, Ames 1962).
