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Biochemical Changes in the Pancreases of Chicks Fed Raw Soybeans and Soybean Meal1
Biochemical Changes in the Pancreases of Chicks Fed Raw Soybeans and Soybean Meal1 M.
L. KAKADE,2 T.
L.
BARTON,3 P. J.
SCHAIBLE4 AND R.
Departments of Poultry Science and Biochemistry, Michigan State East Lansing, Michigan 48823
J.
EVANS 5
University,
(Received for publication May 4, 1967)
T
' J o u r n a l Article No. 4047 Michigan Agricultural Experiment Station. 'Present address: Department of Biochemistry, University of Minnesota, St. Paul 55101. 'Present address: Department of Animal Sciences, University of Arkansas, Fayetteville 72701. * Professor, Department Michigan State University.
of
Poultry
Science,
"Department of Biochemistry, Michigan State University.
TABLE 1.—Composition oj basal diets used in the experiments Diets
Ground yellow corn Defatted soybean flakes* Dehulled soybean meal Alfalfi meal, 20% protein Fish meal with solubles, 57% prot. Dried corn distillers solubles Dried whey Salt Ground limestone Dicalcium phosphate Vitamin trace mineralst Methionine hydroxy analogue, Ca Pro-Strept Total Calculated protein (%)
1
2
3
% 61.21 — 27.44
%
64.10 24.55
%
63.825 24.55
2.00
— . 2.00
— 2.00
3.00 2.00 2.00 0.50 0.90 0.70 0.25
3.00 2.00 2.00 0.50 0.90 0.70 0.25
3.00 2.00 2.00 0.50 0.90 0.70 0.25
— —
•— —
0.15 0.125
100.00
100.00
100.00
21.0
21.0
21.0
* Soybean flakes contributed by Central Soya Company, • Decatur, Ind. Soybeans are tempered by steam to permit flaking and removal of hulls. They are fat extracted at 69° C. The flakes retard growth slightly less than raw soybeans. t Supplied per kilogram of feed: Vitamin A, 3,850 I.U.; Vitamin Dj, 1,100 I.C.U.; Vitamin E, 1.1 I.U.; Riboflavin, 3.3 mg.; Niacin, 22.0 mg.; d-Pantothenic acid, 5.5 mg.; Choline chloride, 220 mg.; Vitamin Bn, 5.5 meg.; Menadione sodium bisulfite complex, .55 mg.; Zinc bacitracin, 4.4 mg.; BHT, 125 mg.; Manganese, 60 mg.; Zinc, 27.5 mg.; Iodine, 1.2 mg.; Iron, 20 mg.; Copper, 2 mg.; Cobalt, 0.2 mg. J This level supplied 13.75 mg. procaine penicillin and 41.25 mg. streptomycin per kg. of feed.
dislocation. Pancreases were removed, weighed, and frozen as quickly as possible. For analysis, the frozen tissues from each treatment were pooled and homogenized in ten volumes of deionized water. Aliquots of the homogenates were used to determine total nitrogen (Kjeldahl), deoxyribose nucleic acid (Schneider, 1957), amylase (Bernfeld, 1955), and trypsin (Kunitz, 1947). Trypsin was determined after treating the trypsinogen with esterokinase as described by Pubols et al. (1964). The growth rate and pancreatic data for the various treatments at different time in-
1578
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
HE morphological studies on the tissues of animals fed raw soybean meal indicate that all tissues were normal except pancreas which was hypertrophic (Booth et al., 1960). A recent report from Goldberg and Guggenheim (1964) suggests the direct action of antibiotics in reducing the pancreatic hypertrophy and changes in the enzymes of pancreases of rats fed raw soybeans. The change in the pancreatic enzyme levels due to feeding raw soybeans to chicks have also been observed by Pubols et al. (1964). Haines and Lyman (1961) observed no changes in protease or lipase activity in the pancreases of rats fed raw or heated soybeans. The present investigation was undertaken, therefore, to examine the biochemical changes associated with the pancreases of chicks fed raw soybeans unsupplemented and supplemented with antibiotics and methionine. Three lots of chicks, each consisting of 120 Cobb strain White Rock cockerels, were fed diets containing soybean meal, raw soybean flakes and raw soybean flakes with methionine and antibiotics (Table 1). At the end of 1, 3, 5 and 8 days, twenty chicks from each treatment were killed by cervical
1579
PANCREAS AND SOYBEANS TABLE 2.—Average body weight and size of pancreases of chicks (20) at specified intervals*
Diet
1 2 3
Weight (gm.) 50.7a 48.8a 51.9a
5 days
3 days
1 day Pa
|«
s
Weight
Pa
"«efas
.45a .58b .64b
s
Weight S
71 ,3a 64 .5a 69.3a
8 days
Pa cr
wt.
83 .5a 81 .9a 81 .3a
.50A .84B .73B
12 days
,,, • , . Pancreas (gm.) 119.3a 110.0a 116.1a
%t ,47A .75B .86C
„ , • , . Pancreas W«ght % o f (gm.) ^ t 195.9a 156.5b
—
.44A .78B
—
* Values not followed by the same letter within the same column are significantly different. Capital letter P < 0 . 0 1 ; small letter P<0.05. Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
meal. No chicks on diet 3 were available at this age. Supplementation of raw soybean flakes with methionine and antibiotics permitted almost normal weight at eight days of age but did not prevent enlargement of the pancreases of the chicks. Nitrogen and DNA contents of the pancreases (Table 3) were fairly uniform throughout the experimental period of eight days. The former appeared to increase with age but there were no profound differences between dietary treatments. If we assume that the DNA content of the cell is constant, then it appears that the enlargement of pancreases of chicks fed raw soybeans is associated with the increase in cell number (hyperplasia), a conclusion which has also TABLE 3.—Nitrogen, deoxyribose nucleic acid, been reached by Singh et al. (1964) No amylase, and trypsin contents of pancreases of differences were apparent in the amylase acchicks fed the diets shown in Table 1 tivity except for chicks fed raw soybean Diet 1 day 3 days 5 days 8 days flakes; these were lower at eight days of Total nitrogen1 1 2.41 2.57 3.09 3.21 age. 2 2.98 2.70 3.00 3.24 3 2.49 2.87 3.09 The trypsin activity (in terms of proteoDeoxyribose nucleic acid1 lytic activity) showed significant changes .168 1 .155 .170 .160 .205 2 .155 .178 due to feeding raw soybean diet. .182 3 .160 .160 The raw soybean produced a considerable Amylase activity* 1 1.02 1.05 1.31 1.72 drop of trypsin activity in 1 to 3 days and 2 1.22 1.35 1.22 1.49 3 1.00 .85 1.42 then an increase during 5 to 8 days. The Trypsin activity* methionine and antibiotics supplementation .42 1 .40 .45 .28 .22 2 .28 .50 .54 caused similar changes in the pancreatic .29 3 .10 .64 enzymes as those obtained on unsupple1 2 Milligrams per 100 mg. of wet tissue. Amylase activity is expressed in terms of mg. of maltose mented raw soybean diet. These results are liberated in 3 min. at 25°C. by 1 ml. enzyme solution. Calculated contradictory to those of Goldberg and per8100 mg. of wet tissue. Trypsin activity was expressed as an increase in O.D. at 280 ntyt. against a blank prepared by the addition of 5% TCA Guggenheim (1964) who reported the dito the substrate prior to the addition of enzyme solution. Calrect action of antibiotics on pancreas in culated per mg. wet tissue.
tervals are shown in Table 2. Raw soybean flakes caused enlarged pancreases compared to soybean meal. The enlargement of pancreas was noticeable as early as three days of the experimental period. There was no statistically significant difference in weight of chicks among treatments at eight days. One soybean meal replicate was lower than expected. Previous experiments by one of the authors have indicated that chicks fed raw soybean flakes were significantly smaller at ten days of age than those fed soybean meal (Al Soudi and Schaible, 1965). Body weights of chicks fed raw soybean flakes were significantly lower at twelve days of age than those fed soybean
1580
M. L. KAKADE, T. L. BARTON, P. J. SCHAIBLE AND R. J. EVANS
REFERENCES Al Soudi, K. A., and P. J. Schaible, 1965. Effects of certain soybean treatments upon growth and egg production of chickens. Michigan Agr. Expt. Sta. Quart. Bui. 48(2): 184-194. Bernfeld, P. 19SS. Analyses a and fj. In: Meth-
ods of Enzymology, Vol. I, Eds. S. B. Colowick and N. O. Kaplan. Academic Press, Inc., New York. p. 149-150. Booth, A. M., D. J. Robbins, W. E. Ribelin and F. DeEds, i960. Effect of raw soybean meal and amino acids on pancreatic hypertrophy in rats. Proc. Soc. Exp. Biol. Med. 140: 681-683. Braham, J. E., H. R. Bird and C. A. Maumann, 1959. Effect of antibiotics on the weight of chicks and rats fed raw or heated soybean meal. J. Nutr. 67: 149-158. Goldberg, A., and K. Guggenheim, 1964. Effect of antibiotics on pancreatic enzymes of rats fed soybean flour. Arch. Biochem. Biophys. 108: 250-254. Haines, P. C , and R. L. Lyman, 1961. Relationship of pancreatic enzyme secretion to growth inhibition in rats fed soybean trypsin inhibitor. J. Nutr. 74: 445-452. Kunitz, M., 1947. Crystalline soybean trypsin inhibitor. II. .General properties. J. Gen. Physiol. 30: 291-310. Pubols, M. H., H. C. Saxena and J. McGinnis, 1964. Pancreatic enzyme levels in chicks fed unheated soybean meal. Proc. Soc. Exp. Biol. Med. 117:713-717. Schneider, W. B., 1957. Determination of nucleic acids in tissues by pentose analysis. I n : Methods in Enzymology, Vol. I l l , S. B. Colowick and N. O. Kaplan. Academic Press, Inc., New York, p. 680-684. Singh, H., P. J. Schaible, H. C. Zindel and R. K. Ringer, 1964. The effects of cooked and raw soybeans supplemented with niacin or a multi-enzyme preparation upon the nutrition of chicks. Michigan Agr. Expt. Sta. Quart. Bui. 47(1): 17-23.
NEWS AND NOTES (Continued from page 1531) of America, held in St. Louis, Missouri, in May, the following officers were elected: President— Fred Moultrie, Arbor Acres Farms, Glastonbury, Connecticut; First Vice-President—John E. Williams, Williams Turkey Breeding Farm, Oakdale, California; Second Vice-President—Bruce Babcock, Babcock Poultry Farms, Ithaca, New York; and Secretary-Treasurer—Don M. Turnbull, Kansas City, Missouri. A.H.I. NOTES At the annual meeting of the Animal Health In-
stitute held in Biloxi, Mississippi, the following officers were elected: President—L. S. Roehm, Merck Chemical Division, Merck & Co. Inc., Railway, N. J.; First Vice-President—L. R. Patton, Sterwin Chemicals, Inc.; Second Vice-President—• D. A. Phillipson, Agricultural Products Division, The Upjohn Co.; and Treasurer, D. A. Peterson, Fort Dodge Laboratories. HALL OF FAME Nominations for the Poultry Hall of Fame may be made by anyone prepared to sponsor the in-
(Continued on page 1608)
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
lowering the fall of trypsin and amylase. Methionine and antibiotics added to the diet with raw soybeans did not alter the changes in pancreatic enzyme brought about by raw soybeans alone. The basal diet contained zinc bacitracin at the level of 4.4 mg. per kilogram of the feed. Braham et al. (1959) reported that the addition of zinc bacitracin (10 mg. per kilogram) did not stimulate the growth of chicks and that the high level of antibiotic (1000 mg. per kilogram) was required to observe the significant growth stimulation of chicks fed raw soybean meal. Our results are in agreement with those of Pubols et al. (1964) who found lower amylase and higher trypsin in the pancreases of chicks receiving raw soybeans than that obtained on heated soybeans. The answer as to whether the increase in the pancreatic trypsin activity is due to the specific increased synthesis of zymogen, the accumulation of zymogen, or due to both, must await further experimentations.
L. KAKADE,2 T.
L.
BARTON,3 P. J.
SCHAIBLE4 AND R.
Departments of Poultry Science and Biochemistry, Michigan State East Lansing, Michigan 48823
J.
EVANS 5
University,
(Received for publication May 4, 1967)
T
' J o u r n a l Article No. 4047 Michigan Agricultural Experiment Station. 'Present address: Department of Biochemistry, University of Minnesota, St. Paul 55101. 'Present address: Department of Animal Sciences, University of Arkansas, Fayetteville 72701. * Professor, Department Michigan State University.
of
Poultry
Science,
"Department of Biochemistry, Michigan State University.
TABLE 1.—Composition oj basal diets used in the experiments Diets
Ground yellow corn Defatted soybean flakes* Dehulled soybean meal Alfalfi meal, 20% protein Fish meal with solubles, 57% prot. Dried corn distillers solubles Dried whey Salt Ground limestone Dicalcium phosphate Vitamin trace mineralst Methionine hydroxy analogue, Ca Pro-Strept Total Calculated protein (%)
1
2
3
% 61.21 — 27.44
%
64.10 24.55
%
63.825 24.55
2.00
— . 2.00
— 2.00
3.00 2.00 2.00 0.50 0.90 0.70 0.25
3.00 2.00 2.00 0.50 0.90 0.70 0.25
3.00 2.00 2.00 0.50 0.90 0.70 0.25
— —
•— —
0.15 0.125
100.00
100.00
100.00
21.0
21.0
21.0
* Soybean flakes contributed by Central Soya Company, • Decatur, Ind. Soybeans are tempered by steam to permit flaking and removal of hulls. They are fat extracted at 69° C. The flakes retard growth slightly less than raw soybeans. t Supplied per kilogram of feed: Vitamin A, 3,850 I.U.; Vitamin Dj, 1,100 I.C.U.; Vitamin E, 1.1 I.U.; Riboflavin, 3.3 mg.; Niacin, 22.0 mg.; d-Pantothenic acid, 5.5 mg.; Choline chloride, 220 mg.; Vitamin Bn, 5.5 meg.; Menadione sodium bisulfite complex, .55 mg.; Zinc bacitracin, 4.4 mg.; BHT, 125 mg.; Manganese, 60 mg.; Zinc, 27.5 mg.; Iodine, 1.2 mg.; Iron, 20 mg.; Copper, 2 mg.; Cobalt, 0.2 mg. J This level supplied 13.75 mg. procaine penicillin and 41.25 mg. streptomycin per kg. of feed.
dislocation. Pancreases were removed, weighed, and frozen as quickly as possible. For analysis, the frozen tissues from each treatment were pooled and homogenized in ten volumes of deionized water. Aliquots of the homogenates were used to determine total nitrogen (Kjeldahl), deoxyribose nucleic acid (Schneider, 1957), amylase (Bernfeld, 1955), and trypsin (Kunitz, 1947). Trypsin was determined after treating the trypsinogen with esterokinase as described by Pubols et al. (1964). The growth rate and pancreatic data for the various treatments at different time in-
1578
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
HE morphological studies on the tissues of animals fed raw soybean meal indicate that all tissues were normal except pancreas which was hypertrophic (Booth et al., 1960). A recent report from Goldberg and Guggenheim (1964) suggests the direct action of antibiotics in reducing the pancreatic hypertrophy and changes in the enzymes of pancreases of rats fed raw soybeans. The change in the pancreatic enzyme levels due to feeding raw soybeans to chicks have also been observed by Pubols et al. (1964). Haines and Lyman (1961) observed no changes in protease or lipase activity in the pancreases of rats fed raw or heated soybeans. The present investigation was undertaken, therefore, to examine the biochemical changes associated with the pancreases of chicks fed raw soybeans unsupplemented and supplemented with antibiotics and methionine. Three lots of chicks, each consisting of 120 Cobb strain White Rock cockerels, were fed diets containing soybean meal, raw soybean flakes and raw soybean flakes with methionine and antibiotics (Table 1). At the end of 1, 3, 5 and 8 days, twenty chicks from each treatment were killed by cervical
1579
PANCREAS AND SOYBEANS TABLE 2.—Average body weight and size of pancreases of chicks (20) at specified intervals*
Diet
1 2 3
Weight (gm.) 50.7a 48.8a 51.9a
5 days
3 days
1 day Pa
|«
s
Weight
Pa
"«efas
.45a .58b .64b
s
Weight S
71 ,3a 64 .5a 69.3a
8 days
Pa cr
wt.
83 .5a 81 .9a 81 .3a
.50A .84B .73B
12 days
,,, • , . Pancreas (gm.) 119.3a 110.0a 116.1a
%t ,47A .75B .86C
„ , • , . Pancreas W«ght % o f (gm.) ^ t 195.9a 156.5b
—
.44A .78B
—
* Values not followed by the same letter within the same column are significantly different. Capital letter P < 0 . 0 1 ; small letter P<0.05. Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
meal. No chicks on diet 3 were available at this age. Supplementation of raw soybean flakes with methionine and antibiotics permitted almost normal weight at eight days of age but did not prevent enlargement of the pancreases of the chicks. Nitrogen and DNA contents of the pancreases (Table 3) were fairly uniform throughout the experimental period of eight days. The former appeared to increase with age but there were no profound differences between dietary treatments. If we assume that the DNA content of the cell is constant, then it appears that the enlargement of pancreases of chicks fed raw soybeans is associated with the increase in cell number (hyperplasia), a conclusion which has also TABLE 3.—Nitrogen, deoxyribose nucleic acid, been reached by Singh et al. (1964) No amylase, and trypsin contents of pancreases of differences were apparent in the amylase acchicks fed the diets shown in Table 1 tivity except for chicks fed raw soybean Diet 1 day 3 days 5 days 8 days flakes; these were lower at eight days of Total nitrogen1 1 2.41 2.57 3.09 3.21 age. 2 2.98 2.70 3.00 3.24 3 2.49 2.87 3.09 The trypsin activity (in terms of proteoDeoxyribose nucleic acid1 lytic activity) showed significant changes .168 1 .155 .170 .160 .205 2 .155 .178 due to feeding raw soybean diet. .182 3 .160 .160 The raw soybean produced a considerable Amylase activity* 1 1.02 1.05 1.31 1.72 drop of trypsin activity in 1 to 3 days and 2 1.22 1.35 1.22 1.49 3 1.00 .85 1.42 then an increase during 5 to 8 days. The Trypsin activity* methionine and antibiotics supplementation .42 1 .40 .45 .28 .22 2 .28 .50 .54 caused similar changes in the pancreatic .29 3 .10 .64 enzymes as those obtained on unsupple1 2 Milligrams per 100 mg. of wet tissue. Amylase activity is expressed in terms of mg. of maltose mented raw soybean diet. These results are liberated in 3 min. at 25°C. by 1 ml. enzyme solution. Calculated contradictory to those of Goldberg and per8100 mg. of wet tissue. Trypsin activity was expressed as an increase in O.D. at 280 ntyt. against a blank prepared by the addition of 5% TCA Guggenheim (1964) who reported the dito the substrate prior to the addition of enzyme solution. Calrect action of antibiotics on pancreas in culated per mg. wet tissue.
tervals are shown in Table 2. Raw soybean flakes caused enlarged pancreases compared to soybean meal. The enlargement of pancreas was noticeable as early as three days of the experimental period. There was no statistically significant difference in weight of chicks among treatments at eight days. One soybean meal replicate was lower than expected. Previous experiments by one of the authors have indicated that chicks fed raw soybean flakes were significantly smaller at ten days of age than those fed soybean meal (Al Soudi and Schaible, 1965). Body weights of chicks fed raw soybean flakes were significantly lower at twelve days of age than those fed soybean
1580
M. L. KAKADE, T. L. BARTON, P. J. SCHAIBLE AND R. J. EVANS
REFERENCES Al Soudi, K. A., and P. J. Schaible, 1965. Effects of certain soybean treatments upon growth and egg production of chickens. Michigan Agr. Expt. Sta. Quart. Bui. 48(2): 184-194. Bernfeld, P. 19SS. Analyses a and fj. In: Meth-
ods of Enzymology, Vol. I, Eds. S. B. Colowick and N. O. Kaplan. Academic Press, Inc., New York. p. 149-150. Booth, A. M., D. J. Robbins, W. E. Ribelin and F. DeEds, i960. Effect of raw soybean meal and amino acids on pancreatic hypertrophy in rats. Proc. Soc. Exp. Biol. Med. 140: 681-683. Braham, J. E., H. R. Bird and C. A. Maumann, 1959. Effect of antibiotics on the weight of chicks and rats fed raw or heated soybean meal. J. Nutr. 67: 149-158. Goldberg, A., and K. Guggenheim, 1964. Effect of antibiotics on pancreatic enzymes of rats fed soybean flour. Arch. Biochem. Biophys. 108: 250-254. Haines, P. C , and R. L. Lyman, 1961. Relationship of pancreatic enzyme secretion to growth inhibition in rats fed soybean trypsin inhibitor. J. Nutr. 74: 445-452. Kunitz, M., 1947. Crystalline soybean trypsin inhibitor. II. .General properties. J. Gen. Physiol. 30: 291-310. Pubols, M. H., H. C. Saxena and J. McGinnis, 1964. Pancreatic enzyme levels in chicks fed unheated soybean meal. Proc. Soc. Exp. Biol. Med. 117:713-717. Schneider, W. B., 1957. Determination of nucleic acids in tissues by pentose analysis. I n : Methods in Enzymology, Vol. I l l , S. B. Colowick and N. O. Kaplan. Academic Press, Inc., New York, p. 680-684. Singh, H., P. J. Schaible, H. C. Zindel and R. K. Ringer, 1964. The effects of cooked and raw soybeans supplemented with niacin or a multi-enzyme preparation upon the nutrition of chicks. Michigan Agr. Expt. Sta. Quart. Bui. 47(1): 17-23.
NEWS AND NOTES (Continued from page 1531) of America, held in St. Louis, Missouri, in May, the following officers were elected: President— Fred Moultrie, Arbor Acres Farms, Glastonbury, Connecticut; First Vice-President—John E. Williams, Williams Turkey Breeding Farm, Oakdale, California; Second Vice-President—Bruce Babcock, Babcock Poultry Farms, Ithaca, New York; and Secretary-Treasurer—Don M. Turnbull, Kansas City, Missouri. A.H.I. NOTES At the annual meeting of the Animal Health In-
stitute held in Biloxi, Mississippi, the following officers were elected: President—L. S. Roehm, Merck Chemical Division, Merck & Co. Inc., Railway, N. J.; First Vice-President—L. R. Patton, Sterwin Chemicals, Inc.; Second Vice-President—• D. A. Phillipson, Agricultural Products Division, The Upjohn Co.; and Treasurer, D. A. Peterson, Fort Dodge Laboratories. HALL OF FAME Nominations for the Poultry Hall of Fame may be made by anyone prepared to sponsor the in-
(Continued on page 1608)
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 7, 2015
lowering the fall of trypsin and amylase. Methionine and antibiotics added to the diet with raw soybeans did not alter the changes in pancreatic enzyme brought about by raw soybeans alone. The basal diet contained zinc bacitracin at the level of 4.4 mg. per kilogram of the feed. Braham et al. (1959) reported that the addition of zinc bacitracin (10 mg. per kilogram) did not stimulate the growth of chicks and that the high level of antibiotic (1000 mg. per kilogram) was required to observe the significant growth stimulation of chicks fed raw soybean meal. Our results are in agreement with those of Pubols et al. (1964) who found lower amylase and higher trypsin in the pancreases of chicks receiving raw soybeans than that obtained on heated soybeans. The answer as to whether the increase in the pancreatic trypsin activity is due to the specific increased synthesis of zymogen, the accumulation of zymogen, or due to both, must await further experimentations.