Acute respiratory acidosis in the domestic fowl

Comp. Biochem. Physiol., 1967, Vol. 21, pp. 223 to 230. Pergamon Press Ltd. Printed in Grmt Britabl

SHORT COMMUNICATION ACUTE RESPIRATORY ACIDOSIS IN THE DOMESTIC FOWL J. R. H U N T * and K. S I M K I S S Department of Physiology and Biochemistry, Reading University, Berkshire, England (Received 8 November 1966)

Abstract--1. Laying fowl and cockerels have been placed in 7% or 20% carbon dioxide for about 12 hr. 2. Bicarbonate, base excess, carbon dioxide tension and pH levels of the blood have been assessed in relation to acute hypercapnia and eggshell formation. 3. There is a fall in base excess at the start of respiratory acidosis. 4. Eggshells laid by fowl restrained and breathing carbon dioxide were from 13 to 24 per cent thinner than normal. INTRODUCTION THE acid-base status of the laying fowl has recently attracted considerable attention. The eggshell contains about 5 g calcium carbonate and is formed in about 20 hr. Neither calcium nor carbonate ions are stored in the oviduct prior to eggshell formation so both must be derived from some other source presumably via the bloodstream. Calcium ions are removed from the blood as it passes through the oviduct (Hunsaker & Sturkie, 1961) but they are rapidly replenished from the diet and from a special store of medullary bone (Simkiss, 1961). The bicarbonate content of the blood also falls during eggshell formation, thereby inducing a metabolic acidosis (Mongin & Lacassagne, 1964) which is partially compensated by hyperventilation (Mongin & Lacassagne, 1965). The mineralization of the shell is therefore related to a fall in pH, bicarbonate and pCO~ (Mongin & Laeassagne, 1966a, b). Many factors affect the acid-base balance of an animal and these might, therefore, be expected to influence eggshell formation. Thus metabolic acidosis, induced by feeding ammonium chloride to laying hens, produces a fall in pH and plasma bicarbonate plus a reduction in shell thickness (Hall & Helbacka, 1959; Hunt & Aitken, 1962). Respiratory acidosis, induced by breathing various carbon dioxide : air mixtures, produces a similar decreased pH but an elevated plasma bicarbonate. Birds treated with from 2 to 5% carbon dioxide for periods of from 12 to 54 hr were found to lay thin-shelled eggs (Helbacka et al., 1963) although * Visiting Scientist from Animal Research Institute, Ottawa, Canada. Contribution No. 247. 223

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J.R. HUNTANDK. SIMKISS

lower concentrations of up to 2"5% given over a period of 21 days were found to result in thicker eggshells (Frank & Burger, 1965). There is therefore some controversy as to whether it was the change in the hydrogen ion or bicarbonate ion concentration which modified shell formation. The discussion of these problems is to some extent hampered by an absence of fundamental data on the changes which occur in birds during acidosis. Respiratory acidosis may be compensated by a renal effect which excretes hydrogen ions into the urine and passes bicarbonate into the blood. This excretion of hydrogen ions is limited by the buffering ability of the urine but is potentially greater during eggshell formation, because the resorption of medullary bone liberates phosphate ions which could be used to excrete protons. The following preliminary experiments were therefore undertaken to study the changes in the blood of laying fowl exposed to acute respiratory acidosis. MATERIALS AND METHODS The domestic fowl used throughout these experiments were Shavers 288 aged from 6 to 8 months. Respiratory acidosis was induced by exposing the birds to 7% or 20% carbon dioxide in oxygen. A plastic face mask was fitted over the head and gas was supplied at a rate of 2-3 l/rain. The results indicate that some rebreathing occurred. Anaerobic blood samples were taken in heparinized syringes from the brachial veins at roughly 3-hr intervals and during the acidotic part of the experiment the birds were restrained. The pH of the blood was read immediately using a Radiometer microelectrode unit thermostated at 41.5°C. Samples of blood were also equilibrated with known COJO2 mixtures using the Astrup microtonometer after which their pH was measured. The pCO2 of the blood was derived from pH/log pCOz plots. The overall reaction of carbon dioxide with the blood buffers is given by the reaction H20 + CO2 + Buf- = H Buf+ HCO3-, so that there is no change in the buffering ability of the blood. If, however, extra bicarbonate is removed or added to the blood then the position of the pH/pCO~ curve shifts to the left or right of its original position (Fig. 1). The extent of the shift is quantitatively related to the amount of base removed or added to the blood and can be determined as mequiv./1 of base excess. Both base excess and bicarbonate were derived from nomograms prepared for human blood and the bicarbonate was also calculated from the Henderson-Hasselbalch equation pH= pKl+log

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using a value of 6.090 for pK1 in avian blood (Helbacka et al., 1964) and 0.033 for c~. The principles of these methods of analysis are discussed in detail by SiggaardAndersen (1965).

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Twenty birds (nine male and eleven female) were studied through at least 12 hr of induced acidosis. Six eggs were laid after being calcified while the birds were breathing 7% carbon dioxide and three were formed during 20% carbon dioxide inhalation. The intact eggs and the isolated shells were weighed and the shell thickness calculated as mg/cm 2. IO0 - •

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RESULTS The venous blood of the male birds had an average pH of 7.402+_0.042 and a pCO2 of 39.6 + 3.16 mm Hg before the start of the treatment. Females had a pH of 7-390 + 0"053 and a pCO2 of 37-6 + 5.36. The lower values and greater variability of the hens' blood may be related to the fact that shell calcification was in progress when the experiment started. The analyses of the blood revealed two phenomena which were common to all the birds. These are illustrated (Fig. 2) by reference to Cock 2204. When the bird was exposed to 7% COs in 93% O3 there was a fall in base excess which occurred within the first hour and later disappeared. When the mask was removed and the bird was allowed to breath air a similar phenomenon occurred. These results indicate that at the times when the pCO2 of the blood is changed there is either a fall in the blood bicarbonate or a rise in the fixed acids of the blood beyond what would be expected to occur with a simple change in the pCO= of the blood in vitro.

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Fio. 2. The base excess, pCOa, bicarbonate and pH content of the blood of Cock 2204 in relation to the inhalation of 7% carbon dioxide in 93% oxygen. The results obtained with a laying bird are again best discussed in relation to individual animals. Hen 2217 was actually exposed to 7% COs in 93% OI and 9 days later to 20% COs in 80% Os. Both treatments will therefore be discussed in relation to this one bird although the phenomena observed are typical of those from the other birds studied. The results of the blood analyses for 7% COs are shown in Fig. 3 and those for 20% COs in Fig. 4. The bicarbonate and base excess values tended to fall during the 7 per cent treatment as the eggshell was being formed. The gas treatments were only started when an egg could be palpated in the shell gland but they were then continued throughout shell formation. In the case of Hen 2217 in 7% COs oviposition occurred 15 rain after the gas treatment was stopped. On the 20% COs treatment oviposition occurred prematurely while under acidosis. After oviposition the base excess and bicarbonate increased again until the gas treatment was terminated. The weights per unit area of the eggshells formed while the birds were breathing 7% COs are shown in Table 1 with the average of the three previous eggs. The results for aU the eggs formed under 7% COs showed an average decrease in weight per unit area of 18.3%. Many of the eggs laid while the birds were breathing 20% COs were premature. The shells were thin but this could obviously not be directly related to changes in blood composition and detailed results are therefore not given.

227

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T A B L E 1 - - C H A N G E IN W E I G H T / U N I T AREA (mg/cm ~') OF SHELL OF EGGS LAID UNDER NORMAL CONDITIONS AND DURING EXPOSURE TO 7 % C O S IN 9 3 % O 3

Bird No. 2216 Average value for 3 eggs preceding acidosis 78.40 Shell formed and laid during or immediately after 7% COs 59.06 % change -24.7

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63.25 -16"6

DISCUSSION

A deficiency in "base excess" shortly after the initiation of respiratory acidosis has been noted by a number of other workers using mammals (Brown, 1960; Cunningham et al., 1962) and wrongly interpreted as being due to the production of metabolic acidosis. The true situation has been revealed by comparing in vitro and in vivo titrations of dog's blood (Cohen et al., 1964). The blood is able to buffer large increases in carbon dioxide much more completely than can the tissues so that on inducing respiratory acidosis in vivo there is a greater rise in the concentration of plasma bicarbonate than is found in the tissues. The result is a diffusion of bicarbonate from the blood. This would account for the fall in base excess at the initiation of respiratory acidosis in the fowl. This phenomenon was not detected in all our experiments for it appears to be transitory and its detection depends upon the time of sampling the blood in relation to the start of the experiment. The deficiency in "base excess" which occurs when the birds are recovering from the respiratory acidosis is interpreted as being a genuine metabolic acidosis. The birds hyperventilate when returned to air because of the high pCOs and low pH of the blood, and the increased muscular effort or the diminished pCO 2 of the blood (Eichenholz et al., 1962) may have led to an increased production of lactic acid in these animals. The blood of two birds were in fact analysed for lactic acid before, during and after they had been exposed to 7% CO, in 93% O~. Average values of 13.5, 17.3 and 27.1 mg per cent were obtained and these are in keeping with the above interpretation. The birds used in our experiments were put into respiratory acidosis during the night, i.e. at a time when shell formation was occurring and when dietary calcium would be minimal. Under these conditions there is usually a resorption of medullary bone and an excretion of urinary phosphate (Taylor, 1962). The avian kidney is capable of excreting protons by means of urinary phosphate (Wolbach, 1955) but it seems unlikely that this occurred in our experiments since there was no increase in the base excess of the blood. It is more likely that, as in the mammal, the ability to compensate for reapiratory acidosis by means of renal responses requires several days for the adaptation to occur (Sullivan & Dorman, 1955; Polak et al., 1961; Schwartz, 1966). Thus although phosphate

ACUTE RESPIRATORY ACIDOSIS I N THE DOMESTIC FOWL

229

ions are released from the bones the bird is probably unable to use them to overcome the metabolic acidosis which occurs at the time of shell formation. The relationship of blood bicarbonate to shell formation has attracted some attention recently. It has been suggested that a deficiency of bicarbonate (Hunt & Aitken, 1962; Frank & Burger, 1965) or a fall in pH (Helbacka et al., 1963) may be the limiting factor. In our experiments the pH fell and the bicarbonate was increased by the acute respiratory acidosis. The weight/unit area of the eggshell fell by almost 20 per cent although the significance of this figure is to some extent concealed since shell formation had always started prior to the experimental treatment. It is, however, likely that previous analyses have tended to oversimplify the relationship of shell carbonate to blood pH or bicarbonate. In the final analysis the oviduct has to form, by some unknown mechanism, the carbonate ions for the shell. The formation of carbonate ions will depend upon both pH and bicarbonate concentration. If in fact one takes the apparent second dissociation constant of carbonic acid as 10 47s (Sendroy & Hastings, 1926) it is possible to calculate the carbonate concentration of the blood under various conditions. Using the data of Mongin & Lacassagne (1964) for a normal egg cycle one finds the carbonate concentration of the blood faUs from 0.059 m M to 0.032 m M shortly before oviposition. In our experiment on Hen 2217 in 7% CO~ the carbonate fell from 0.038 m M before acidosis to 0.020 m M towards the end of shell formation. If one accepts that this decrease in blood carbonate reflects the difficulty of forming shell carbonate during acidosis then this could explain the occurrence of thin-shelled eggs even when the blood bicarbonate is elevated. A similar explanation would account for the decreased shell thickness found by Helbacka et al. (1963) and Hunt & Aitken (1962). An increased shell thickness was found by Frank & Burger (1965) when they progressively increased the pCOs of birds during a 3-week period. They reported that this produced a fall in the pH of arterial blood of from 7.48 to 7.40 and a rise in bicarbonate of 20.8-21-8 mequiv./l. Unfortunately, they do not state when these blood samples were taken in relation to eggshell formation and one is not certain whether any renal compensation occurred during the period of the experiment. If, however, one calculates the carbonate concentration for these two blood samples one obtains values of 0-038 and 0.032 raM, i.e. within the range of values for a normal egg-cycle. It is known that the oviduct can regulate its pH independent of that of the blood (Ogasawara et al., 1964; Winget et al., 1965) in which case the above arguments would provide only a crude estimate of the ability of the bird to obtain carbonate ions for shell formation, which would be superimposed upon the metabolic activities of the oviduct. Acknow/edgements---One of us (K. S.) would like to thank the S.R.C. for a grant (B/SR/2227) covering the cost of the equipment used in this research. REFERENCES Bnow~r E. B. (1960) Plasma electrolyte composition in dogs breathing high COt mixtures: source of bicarbonate deficit in severe respiratory acidosis, j~. Lab. din. Med. 55, 767-775.

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COHEN J. J., BRACKETN. C. & SCHWAnTZW. B. (1964) The nature of the carbon dioxide titration curve in the normal dog. ,y. din. Invest. 43, 777-786. CUNNINGHAMD. J. C., LLOYDB. B. & MiCHeL C. C. (1962) Acid-base changes in the blood during hypercapnia and hypocapnia in normal man..7. Physiol. 161, 26P-27P. EICHSNHOLZA., MULHAUSENR. O., ANnEmON W. E. & MACVONALnF. M. (1962) Primary hypocapnia: a cause of metabolic acidosis. ?. appl. Physiol. 17, 283-288. FnANK F. R. & BunGEn R. E. (1965) The effect of carbon dioxide inhalation and sodium bicarbonate ingestion on eggshell deposition. Poult. Sci. 44, 1604-1606. HALL K. N. & I-IELnACKAN. V. (1959) Improving albumen quality. Poult. ,Sci. 38, 111-114. HELBAeKAN. V., CAST~LXNEJ. L. & SMITH C. J. (1963) The effect of high carbon dioxide atmosphere on the laying hen. Poult. Sci. 42, 1082-1084. HSLnACKAN. V., CASTm~LXNEJ. L., SMXTRC. J. & SHAFFNERC. S. (1964) Investigation of plasma carbonic acid pK of the chicken. Poult Sci. 43, 138-144. HUNSAKEaW. G. & STURKmP. D. (1961) Removal of calcium from uterine blood during shell formation in the chicken. Poult. Sci. 40, 1358-1352. HUNT J. R. & AITKENJ. R. (1962) The effect of ammonium and chloride ions in the diet of hens and egg shell quality. Poult. Sci. 41, 434-438. MONGiN P. & LACASSAONEL. (1964) Physiologie de la formation de la coquille de l'~uf de Poule et 6quilibre acido-basique du sang. C. r. Acad. Sci., Paris, 258, 3093-3094. MONGIN P. & LACASSAGh'EL. (1965) Physiologie de la formation de la coquille de l'ceuf de Poule et ventilation pulmonaire. C. r. Acad. Sci., Paris 261, 4-228-4229. MONOXN P. & LACASSAGNEL. (1966a) Equilibre acido-basique du sang et formation de la coquille de l'ceuf. Ann. Biol. anita., Biochira., Biophys. 6, 93-100. MONGIN P. & LACASSAGNEL. (1966b) Rhythme respiratoire et physiologic de la formation de la coquiUe de l'0~uf. Ann. Biol. anita., Biochim., Biophys. 6, 101-111. OGASAWARAF. X., VAN K ~ Y H. P. & LGa~NZ F. W. (1964) Hydrogen ion concentration of the oviduct of the laying domestic fowl Poult. ,Sci. 43, 3-6. POLAKA., HAYNEG. D., HAYS R. M. & SCHWARTZW. B. (1961) Effects of chronic hypercapnia on electrolytes and acid-base equilibrium--1. Adaptation. J. clin. Invest. 40, 1223-1237. SCHWARTZW. B. (1966) Defense of extra-cellular pH during acute and chronic hypercapnia. Ann. N . Y . Acad. Sci. 133, 125-131. SENnnGY J. & HASTINGSA. B. (1926) Studies of the solubility of calcium carbonate and tertiary calcium phosphate under various conditions. J. biol. Chem. 71, 797-846. SIGGAARD-ANDERSm'¢O. (1965) The Acid-Base Status of the Blood. Third edn. Munksgaard, Copenhagen. SIMKISSK. (1961) Calcium metabolism and avian reproduction. Biol, Rev. 36, 321-367. SVLLXVANW. J. & DGRMANP. J. (1955) The renal response to chronic respiratory acidosis. jT. din. Invest. 34, 268-276. TAYLORT. G. (1962) Calcium absorption and metabolism in the laying hen. In Nutrition of Pigs and Poultry (Edited by MORGANJ. T. & Lswls D.) pp. 148-157. Butterworth, London. WINGEr C. M., ]VIEPHAMC. A. & AVERKINE. G. (1965) Variations in intrauterine pH within a circadian rhythm (Gallus domesticus). A m . J . Physiol. 208, 1031-1035. WOLBACHR. A. (1955) Renal regulation of acid-base balance in the chicken. Arn.~. Physiol. 181, 149-156.