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Gastric Hydrogen Ion Concentration and Acidity in the Domestic Fowl
Gastric Hydrogen Ion Concentration and Acidity in the Domestic Fowl DONALD
FARNER1
S.
Department of Zoology, University of Wisconsin, Madison, Wisconsin (Received for publication July 13, 1942)
NFORMATION on the hydrogen ion concentration in the gizzard of the domestic fowl has been recorded by McLaughlin (1931),.Mussehl et al. (1933), Kerr and Common (1935), Heller and Penquite (1936), and Farner (1942). In each case, however, the determinations were made on the fluid removed from the gizzards of freshly killed birds. Collip (1922) removed gizzard fluid from live birds by lumbar puncture and determined the titratable acidity to Toepfer's reagent before and after injections of certain extracts. Cheney (1938) removed gizzard fluid by means of a tube inserted through the esophagus, crop, and proventriculus of anesthetized birds. Free and total acidity were determined by titration to end points with Toepfer's reagent and phenolphthalein, respectively. It was noted that the gizzard contents removed from live birds contained "free acid" whereas those from autopsied birds did not. Preliminary investigations by the author showed that marked differences in hydrogen ion concentration existed between the gizzard fluids as removed from living and freshly killed birds. Consequently it seemed desirable to conduct a series of experiments to find the hydrogen ion concentration in the gizzard of normal live birds. MATERIALS AND METHODS
Adult Rhode Island Red and Barred ' F r o m the Zoological and Veterinary Science Laboratories.
Plymouth Rock hens from the pedigreed stocks of the department of Poultry Husbandry were used. These birds were fed on a ration of equal weights of corn, oats, and wheat. This diet was selected because the domestic fowl is naturally a granivorous bird. Gizzard fluid was removed in the following manner: The bird was placed on its right side on the operating table with its wings raised and held together by means of a strap. The left leg was drawn anteriorly and tied; the right leg was drawn posteriorly and tied. It was then possible to palpate the gizzard posterior to the last rib and dorsal to the sternum. A spinal puncture needle with trochar was inserted into the gizzard. The trochar was then removed and the fluid drawn from the gizzard with a 20 c.c. syringe. It was frequently possible to obtain 10 c.c. of fluid although in routine determinations 3-5 c.c. were taken. This method was found to be superior to that of Collip (1922) since it required a single operator and caused little if any struggling on the part of the bird. It was superior to that of Cheney (1938) in that it did not require anesthesia. Many of the birds became so accustomed to the operation that they would peck at the ring or wrist watch of the operator during the process of the operation. Several birds were subjected to the operation more than 30 times with no ill effects. It has been performed several thousand times with less than 10 casualties due to the operation.
[79]
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
I
80
DONALD S. FARNER EXPERIMENTS AND RESULTS
Number of birds 22 Number of pH determinations 130 Mean pH 2.05 Range of S.D. of mean pH 1.82-2.62 Range of S.E. of mean pH 2.02-2.07 Number of "free HCl" titrations 127 Mean "total acidity" as m.e./l 59.2 ± 1.1 S.D. of mean "free HCl" 11.4 Number of "total acidity" titrations 127 Mean "free HCl" as m.e./l 25.0 ± 1.0 S.D. of mean "total acid" 13.6
In the titration of acidity in gastric juice with standard hydroxide, the acid which is neutralized to Toepfer's reagent is usually regarded as the uncombined HCl. That which is measured in titration to phenolphthalein is the acidity due to HCl, acid protein, organic acids, acid salts, and other acidic substances many of which are only partially or slightly ionized hence exerting little effect on the actual hydrogen ion concentration. Theoretically gastric fluid with a "free HCl" titration of 2S.0 milli-
equivalents per liter (0.025 N HCl) should have a hydrogen ion concentration of pH 1.60. The discrepancy between this and the actual hydrogen ion concentration, pH 2.OS, as determined with the quinhydrone electrode may possibly be due to the release during titration of HCl from combinations with protein and protein hydrolysis products produced by peptic activity. This would result in a "free HCl" figure which is too high. Since the end-point with Toepfer's reagent is reached at pH 2.9-4.0 it is also possible that a certain amount of buffering may be encountered toward the end of the titration. In order to learn whether or not there was any correlation between the general activity of the birds and the gastric acidity and hydrogen ion concentration the data were examined according to the time of day at which the samples were taken (Table 1 and Fig. 1). It can be seen that there is TABLE 1.—Mean gastric acidity and hydrogen ion concentration for 22 hens. Samples taken at 12-24 hour intervals Time 12:00- 1:00 3:00- 4:00 6:00- 7:00 10:30-11:30 1:00- 2:00 3:00- 4:00 6:00- 7:00 9:00-10:00
a.m. a.m. a.m. a.m. p.m. p.m. p.m. p.m.
PH
Free HCl*
Total acidity*
2.13 2.37 2.15 1.92 1.96 1.98 1.97 2.12
24.9 25.2 23.8 30.9 33.2 28.7 29.0 27.1
63.4 59.5 45.3 . 62.1 61.1 58.1 64.2 54.9
* Expressed in milli equivalents of HCl per liter gastric fluid.
at least a diurnal trend in the hydrogen ion concentration. 2. A second series of observations were made in order to verify this apparent diurnal trend in pH. Samples from the gizzards of eight birds were removed at intervals of two or three hours for 24 hours. The results agree with those in experiment 1 (Table 2 and Fig. 1).
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
1. Samples of gastric (gizzard) fluid were drawn from 22 birds at various times during the day and night for a period of one week. The times of sampling were so arranged that an interval of at least 12 hours elapsed between each sample. The hydrogen ion concentration was determined with a quinhydrone electrode. "Free HCl" and "total acidity" were determined by titration of 1 ml. samples with N/100 NaOH to Toepfer's reagent and phenolphthalein, respectively. The data from these titrations are expressed in milli-equivalents of HCl per liter of gastric fluid. In statistical treatments of the data the pH values were converted to their corresponding hydrogen ion concentrations expressed as numbers. Following statistical analyses the data were reconverted to pH. In the expression of standard deviation (S.D.) and standard error (S.E.) as pH it is necessary to use the "range of standard error" and "range of standard deviation" since pH is a. logarithmic expression. The results:
GASTRIC HYDROGEN ION CONCENTRATION AND ACIDITY IN THE FOWL
81
variations in pH may be produced by variations in other substances in the fluid while the titratable acidity remains more or less constant. In comparing the results of this investigation with the data obtained from the gastric fluid of freshly killed birds (Table 3) there can be little doubt that a
Time i£
4 AR
S M
IS Time
4 Pk
8 PM
1:45 4:45 7:30 11:30 12:30 2:30 5:00 8:00 10:45
12
FIG. 1. Diurnal variations in gastric pH. DISCUSSION
Northrup (1922) showed that the .optimum pH for peptic activity is from 1.82.2 depending on the nature of the substrate. It is evident that the gizzard of fowls on grain diet is at an optimum hydrogen ion concentration for peptic digestion. It is interesting to note that the diurnal trend in gastric hydrogen ion concentration corresponds roughly with the general activity of the fowl and that the most optimum conditions for peptic digestions exist during the day (Fig. 1). The fact that the diurnal variation in pH is not reflected in the titratable acidity perhaps may be explained by the large standard deviations in the acidity data. On the other hand the
a.m. a.m. a.m. a.m. p.m. p.m. p.m. p.m. p.m.
pH
Free HCl*
Total acidity*
2.50 2.32 2.19 2.09 1.94 2.06 2.10 2.14 2.19
9.6 20.1 18.0 26.1 23.8 25.6 19.2 21.7 16.2
62.2 63.9 56.6 63.9 59.8 63.8 55.0 61.0 61.2
* Expressed in milli-equivalents per liter gastric fluid.
decrease in hydrogen ion concentration in the gizzard occurs with death. This agrees with the observation of Cheney (1938). Possible sources of the variations in the data in Table 3 have been discussed by the author in a previous paper (1942). Collip (1922) stated that the gastric fluid from live birds was frequently alkaline to Toepfer's reagent. The data obtained in this investigation do not confirm this. Heller and Penquite (1936) using the gizzard fluid from freshly killed birds
TABLE 3.—Hydrogen ion concentration of the gizzard contents from freshly killed birds. Results of various investigators Investigator
No. of birds
McLaughlin Mussehl et al. Kerr and Common Heller and Penquite Farner
8 20 11 20
Description
Diet
Mean pH
hens Leghorn cockerels pullets, cockerels 10 month cocks, hens
grain complete ration* "normal diet" complete ration f grain
3.39 2.9 3.0 2.99 2.60
* Yellow cornmeal, shorts, bran, barley, alfalfa meal, meat meal, dried buttermilk, NaCl, limestone, t Same plus cottonseed meal, bone meal, cod liver oil.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
TABLE 2.—Mean gastric acidity and hydrogen ion concentration for eight hens. Samples^taken at 2-3 hour intervals
82
DONALD S. FARNEE
ACKNOWLEDGMENTS The author wishes to thank Prof. C. A. Herrick for his cooperation and interest and for making certain laboratory facilities available; and also the department of
poultry husbandry for providing the experimental animals used in this investigation. REFERENCES
Cheney, G., 1938. Gastric acidity in chickens with experimental gastric ulcers. Amer. Jour. Digest. Dis. 5:104-107. Collip, J. B., 1922. The activation of the glandular stomach of the fowl. Amer. Jour. Physiol. 59: 435-438. Farner, D. S., 1942. The hydrogen ion concentration in avian digestive tracts. Poultry Sci. 2 1 : 445-450. Heller, V. G., and R. Penquite, 1936. Effect of minerals and fiber on avian intestinal pH. Poultry Sci. IS :397-399. Kerr, W. R., and R. H. Common, 1935. The effect of certain acid treatments for coccidiosis on the hydrogen ion concentration of the fowl intestine. Vet. Jour. 91:309-311. McLaughlin, A. R., 1931. pH of the alimentary tracts of fowl, cat, and rabbit. Science 73:191192. Mussehl, F. E., M. J. Blish, and C. W. Ackerson, 1933. Effect of dietary and environmental factors on the pH of the intestinal tract of fowls. Poultry Sci. 12 :120-123. Northrup, J. H., 1922. The mechanism of the influence of acids and alkalis on the digestion of proteins by trypsin or pepsin. Jour. Gen. Physiol. 5:263-274.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
showed that gastric hydrogen ion concentration could be altered by feeding certain rations. Preliminary experiments by the author with live birds have confirmed this. This is being subjected to further investigation. CONCLUSIONS 1. The mean hydrogen ion concentration in the gizzard of 22 live hens was found to be pH 2.05. The mean "free HC1" was 25.0 milli-equivalents per liter and the mean "total acidity" was 59.2 milli-equivalents per liter. 2. The hydrogen ion concentration in the gastric fluid removed from gizzards of live birds is higher than that in the fluid removed from the gizzards of freshly killed kinds. 3. The hydrogen ion concentration showed a tendency to be higher in the daytime and lower at night.
FARNER1
S.
Department of Zoology, University of Wisconsin, Madison, Wisconsin (Received for publication July 13, 1942)
NFORMATION on the hydrogen ion concentration in the gizzard of the domestic fowl has been recorded by McLaughlin (1931),.Mussehl et al. (1933), Kerr and Common (1935), Heller and Penquite (1936), and Farner (1942). In each case, however, the determinations were made on the fluid removed from the gizzards of freshly killed birds. Collip (1922) removed gizzard fluid from live birds by lumbar puncture and determined the titratable acidity to Toepfer's reagent before and after injections of certain extracts. Cheney (1938) removed gizzard fluid by means of a tube inserted through the esophagus, crop, and proventriculus of anesthetized birds. Free and total acidity were determined by titration to end points with Toepfer's reagent and phenolphthalein, respectively. It was noted that the gizzard contents removed from live birds contained "free acid" whereas those from autopsied birds did not. Preliminary investigations by the author showed that marked differences in hydrogen ion concentration existed between the gizzard fluids as removed from living and freshly killed birds. Consequently it seemed desirable to conduct a series of experiments to find the hydrogen ion concentration in the gizzard of normal live birds. MATERIALS AND METHODS
Adult Rhode Island Red and Barred ' F r o m the Zoological and Veterinary Science Laboratories.
Plymouth Rock hens from the pedigreed stocks of the department of Poultry Husbandry were used. These birds were fed on a ration of equal weights of corn, oats, and wheat. This diet was selected because the domestic fowl is naturally a granivorous bird. Gizzard fluid was removed in the following manner: The bird was placed on its right side on the operating table with its wings raised and held together by means of a strap. The left leg was drawn anteriorly and tied; the right leg was drawn posteriorly and tied. It was then possible to palpate the gizzard posterior to the last rib and dorsal to the sternum. A spinal puncture needle with trochar was inserted into the gizzard. The trochar was then removed and the fluid drawn from the gizzard with a 20 c.c. syringe. It was frequently possible to obtain 10 c.c. of fluid although in routine determinations 3-5 c.c. were taken. This method was found to be superior to that of Collip (1922) since it required a single operator and caused little if any struggling on the part of the bird. It was superior to that of Cheney (1938) in that it did not require anesthesia. Many of the birds became so accustomed to the operation that they would peck at the ring or wrist watch of the operator during the process of the operation. Several birds were subjected to the operation more than 30 times with no ill effects. It has been performed several thousand times with less than 10 casualties due to the operation.
[79]
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
I
80
DONALD S. FARNER EXPERIMENTS AND RESULTS
Number of birds 22 Number of pH determinations 130 Mean pH 2.05 Range of S.D. of mean pH 1.82-2.62 Range of S.E. of mean pH 2.02-2.07 Number of "free HCl" titrations 127 Mean "total acidity" as m.e./l 59.2 ± 1.1 S.D. of mean "free HCl" 11.4 Number of "total acidity" titrations 127 Mean "free HCl" as m.e./l 25.0 ± 1.0 S.D. of mean "total acid" 13.6
In the titration of acidity in gastric juice with standard hydroxide, the acid which is neutralized to Toepfer's reagent is usually regarded as the uncombined HCl. That which is measured in titration to phenolphthalein is the acidity due to HCl, acid protein, organic acids, acid salts, and other acidic substances many of which are only partially or slightly ionized hence exerting little effect on the actual hydrogen ion concentration. Theoretically gastric fluid with a "free HCl" titration of 2S.0 milli-
equivalents per liter (0.025 N HCl) should have a hydrogen ion concentration of pH 1.60. The discrepancy between this and the actual hydrogen ion concentration, pH 2.OS, as determined with the quinhydrone electrode may possibly be due to the release during titration of HCl from combinations with protein and protein hydrolysis products produced by peptic activity. This would result in a "free HCl" figure which is too high. Since the end-point with Toepfer's reagent is reached at pH 2.9-4.0 it is also possible that a certain amount of buffering may be encountered toward the end of the titration. In order to learn whether or not there was any correlation between the general activity of the birds and the gastric acidity and hydrogen ion concentration the data were examined according to the time of day at which the samples were taken (Table 1 and Fig. 1). It can be seen that there is TABLE 1.—Mean gastric acidity and hydrogen ion concentration for 22 hens. Samples taken at 12-24 hour intervals Time 12:00- 1:00 3:00- 4:00 6:00- 7:00 10:30-11:30 1:00- 2:00 3:00- 4:00 6:00- 7:00 9:00-10:00
a.m. a.m. a.m. a.m. p.m. p.m. p.m. p.m.
PH
Free HCl*
Total acidity*
2.13 2.37 2.15 1.92 1.96 1.98 1.97 2.12
24.9 25.2 23.8 30.9 33.2 28.7 29.0 27.1
63.4 59.5 45.3 . 62.1 61.1 58.1 64.2 54.9
* Expressed in milli equivalents of HCl per liter gastric fluid.
at least a diurnal trend in the hydrogen ion concentration. 2. A second series of observations were made in order to verify this apparent diurnal trend in pH. Samples from the gizzards of eight birds were removed at intervals of two or three hours for 24 hours. The results agree with those in experiment 1 (Table 2 and Fig. 1).
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
1. Samples of gastric (gizzard) fluid were drawn from 22 birds at various times during the day and night for a period of one week. The times of sampling were so arranged that an interval of at least 12 hours elapsed between each sample. The hydrogen ion concentration was determined with a quinhydrone electrode. "Free HCl" and "total acidity" were determined by titration of 1 ml. samples with N/100 NaOH to Toepfer's reagent and phenolphthalein, respectively. The data from these titrations are expressed in milli-equivalents of HCl per liter of gastric fluid. In statistical treatments of the data the pH values were converted to their corresponding hydrogen ion concentrations expressed as numbers. Following statistical analyses the data were reconverted to pH. In the expression of standard deviation (S.D.) and standard error (S.E.) as pH it is necessary to use the "range of standard error" and "range of standard deviation" since pH is a. logarithmic expression. The results:
GASTRIC HYDROGEN ION CONCENTRATION AND ACIDITY IN THE FOWL
81
variations in pH may be produced by variations in other substances in the fluid while the titratable acidity remains more or less constant. In comparing the results of this investigation with the data obtained from the gastric fluid of freshly killed birds (Table 3) there can be little doubt that a
Time i£
4 AR
S M
IS Time
4 Pk
8 PM
1:45 4:45 7:30 11:30 12:30 2:30 5:00 8:00 10:45
12
FIG. 1. Diurnal variations in gastric pH. DISCUSSION
Northrup (1922) showed that the .optimum pH for peptic activity is from 1.82.2 depending on the nature of the substrate. It is evident that the gizzard of fowls on grain diet is at an optimum hydrogen ion concentration for peptic digestion. It is interesting to note that the diurnal trend in gastric hydrogen ion concentration corresponds roughly with the general activity of the fowl and that the most optimum conditions for peptic digestions exist during the day (Fig. 1). The fact that the diurnal variation in pH is not reflected in the titratable acidity perhaps may be explained by the large standard deviations in the acidity data. On the other hand the
a.m. a.m. a.m. a.m. p.m. p.m. p.m. p.m. p.m.
pH
Free HCl*
Total acidity*
2.50 2.32 2.19 2.09 1.94 2.06 2.10 2.14 2.19
9.6 20.1 18.0 26.1 23.8 25.6 19.2 21.7 16.2
62.2 63.9 56.6 63.9 59.8 63.8 55.0 61.0 61.2
* Expressed in milli-equivalents per liter gastric fluid.
decrease in hydrogen ion concentration in the gizzard occurs with death. This agrees with the observation of Cheney (1938). Possible sources of the variations in the data in Table 3 have been discussed by the author in a previous paper (1942). Collip (1922) stated that the gastric fluid from live birds was frequently alkaline to Toepfer's reagent. The data obtained in this investigation do not confirm this. Heller and Penquite (1936) using the gizzard fluid from freshly killed birds
TABLE 3.—Hydrogen ion concentration of the gizzard contents from freshly killed birds. Results of various investigators Investigator
No. of birds
McLaughlin Mussehl et al. Kerr and Common Heller and Penquite Farner
8 20 11 20
Description
Diet
Mean pH
hens Leghorn cockerels pullets, cockerels 10 month cocks, hens
grain complete ration* "normal diet" complete ration f grain
3.39 2.9 3.0 2.99 2.60
* Yellow cornmeal, shorts, bran, barley, alfalfa meal, meat meal, dried buttermilk, NaCl, limestone, t Same plus cottonseed meal, bone meal, cod liver oil.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
TABLE 2.—Mean gastric acidity and hydrogen ion concentration for eight hens. Samples^taken at 2-3 hour intervals
82
DONALD S. FARNEE
ACKNOWLEDGMENTS The author wishes to thank Prof. C. A. Herrick for his cooperation and interest and for making certain laboratory facilities available; and also the department of
poultry husbandry for providing the experimental animals used in this investigation. REFERENCES
Cheney, G., 1938. Gastric acidity in chickens with experimental gastric ulcers. Amer. Jour. Digest. Dis. 5:104-107. Collip, J. B., 1922. The activation of the glandular stomach of the fowl. Amer. Jour. Physiol. 59: 435-438. Farner, D. S., 1942. The hydrogen ion concentration in avian digestive tracts. Poultry Sci. 2 1 : 445-450. Heller, V. G., and R. Penquite, 1936. Effect of minerals and fiber on avian intestinal pH. Poultry Sci. IS :397-399. Kerr, W. R., and R. H. Common, 1935. The effect of certain acid treatments for coccidiosis on the hydrogen ion concentration of the fowl intestine. Vet. Jour. 91:309-311. McLaughlin, A. R., 1931. pH of the alimentary tracts of fowl, cat, and rabbit. Science 73:191192. Mussehl, F. E., M. J. Blish, and C. W. Ackerson, 1933. Effect of dietary and environmental factors on the pH of the intestinal tract of fowls. Poultry Sci. 12 :120-123. Northrup, J. H., 1922. The mechanism of the influence of acids and alkalis on the digestion of proteins by trypsin or pepsin. Jour. Gen. Physiol. 5:263-274.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 23, 2015
showed that gastric hydrogen ion concentration could be altered by feeding certain rations. Preliminary experiments by the author with live birds have confirmed this. This is being subjected to further investigation. CONCLUSIONS 1. The mean hydrogen ion concentration in the gizzard of 22 live hens was found to be pH 2.05. The mean "free HC1" was 25.0 milli-equivalents per liter and the mean "total acidity" was 59.2 milli-equivalents per liter. 2. The hydrogen ion concentration in the gastric fluid removed from gizzards of live birds is higher than that in the fluid removed from the gizzards of freshly killed kinds. 3. The hydrogen ion concentration showed a tendency to be higher in the daytime and lower at night.