Dynamic responses of blood metabolites to nutrient depletion and repletion in broiler chicken nutrition (2024)

Animal Care

All animal experiments were approved by the University of Arkansas Institutional Animal Care and Use Committee under protocol 21050 and were in accordance with the recommendations in NIH's Guide for the Care and Use of Laboratory Animals.

Birds and Diets

A total of 720 male Cobb 500 byproduct broiler chicks were obtained from a commercial hatchery (Cobb Vantress, Siloam Springs, AR) and transported to the University of Arkansas broiler research farm. Barn temperatures were 32°C at d1 and gradually decreased to 25°C by d18. Relative humidity ranged from 35-70%, with an average of 46% over the trial period. All birds were reared on top-dressed recycled pine shavings and offered food/water ad libitum. Birds were randomly allocated into 40 pens (18 birds/pen) and fed a common starter diet from d 1 to 14. To induce short-term nutritional stress, 5 experimental treatments were provided from 15 to 17 d posthatch: a nutrient adequate control diet, low dCP (10% reduction; -0.11 dLys), low AME (5% reduction; -160 kcal/kg AME), low digestible P (42% reduction; -0.18 dP), or low Ca (30% reduction; -0.26 Total Ca). These dietary treatments were selected to represent a range of commercially relevant nutrients and diet profiles that were experimentally robust but recognizable for commercial poultry producers. Birds were then returned to a common grower from d 17 to 19. Experimental diets are presented in Table 1. Birds were checked at least twice each day for any signs of distress (leg issues, lethargy, impaired appetite, dehydration, etc.), and those overtly exhibiting these signs were removed. All mortality was weighed and recorded. Extra pens were maintained for replacement of mortality and sampled birds, to maintain stocking density.

Measurements

Blood samples were collected from 8 birds on D14, prior to the introduction of the experimental diets (representing T0). Then, blood samples were collected from 1 bird/pen (n = 8/treatment) at 3, 6, 12, 24, and 48h after the introduction of the experimental grower diets. All birds were then returned to a common grower diet, and blood samples were collected from 1 bird/pen (n=8/treatment) at 3, 6, 12, 24, and 48h postdiet switch. All blood samples were collected via heart puncture using heparinized 3 mL syringes with 23G needles, and birds were immediately euthanized via cervical dislocation following collection. Replacement birds were not sampled.

As there is no previous published work exploring the dynamic changes in blood metabolite profiles following specific changes to diet nutrient density, a relatively short-term feeding regimen was selected. Future work may consider longer term sampling protocols to evaluate the effects of diet on blood biochemistry over several weeks or longer. For the purpose of the current experiment, the hypothesis that diet transitions would create more short- rather than long-term influence on blood metabolite profiles was explored.

Chemical Analysis

Heparinized whole blood was analyzed for pH, sodium (Na), potassium (K), chloride (Cl), total carbon dioxide (CO₂), anion gap, ionized calcium (iCa), glucose (Glu) and hematocrit using the i-STAT Alinity system with the i-STAT CHEM8+ cartridge (600-9008-25, Abaxis, Union City, CA, United States) according to manufacturer's recommendation. Creatine kinase (CK), AST, UA, glucose, total Ca, P, total protein, bicarbonate (HCO3) albumin and globulin were analyzed using the VetScan VS2 with the Avian/Reptilian Profile Plus cartridge (500-0041, Abaxis, Union City, CA) according to manufacturer's recommendation. Total carotenoid concentration in whole blood was measured using the iCheck Carotine Photometer and test kit (BioAnalyt, Berlin, Germany) according to manufacturer's recommendation. Diet samples were ground using a 0.75 to 1 mm sieve with an Ultra Centrifugal Mill (ZM 200, Retsch, Germany) before their analysis by near infrared reflectance spectroscopy. ISO FDIS 12099 standard guidelines were followed. A Bruker Tango FT-NIR machine (Bruker-Optics, Billerica, MA) was used for the analysis. The recorded spectra range was from 400 to 2,500 nm, every 0.5 nm. The manipulation of the spectra for the Bruker device was done with OPUS 7.2 software (Bruker-Optics, Billerica, MA).

Statistical Analysis

There were 2 major components to the statistical analysis. Firstly, and most importantly, regression analyses were performed to assess the dynamic changes in blood biomarkers over time and how these were influenced during the depletion and repletion phases. Secondly, a treatment comparison was made at each sampling time to establish differences between treatment groups. This second analytical step is presented for clarity in the Tables, but the primary aim of the work was to explore how various diet changes would influence blood metabolites over time, not to contrast for example, the low Ca diet with the low P diet etc.

The statistical analysis that was conducted to compare the concentration of each analyte across different treatment groups over the course of several hours was performed using JMP 16 software (SAS Institute, Cary, NC). This comparison was achieved using Analysis of Variance (ANOVA) to determine if there were any statistically significant differences between the treatments. The threshold for statistical significance was set at a p-value of less than 0.05. Following ANOVA, mean comparisons were performed using Tukey's HSD test to identify specific pairs of treatments between which the differences were statistically significant. Significance was indicated using superscripts, within columns, to clarify treatment effects. To further dissect the treatment effects over time (3, 6, 12, 24, and 48h for the depletion phase and 51, 54, 60, 72, and 96h for the repletion phase, all relative to T0), especially during the depletion and repletion phases, the data were analyzed for linear, quadratic, and cubic responses. By evaluating linear, quadratic, and cubic trends, we were able to capture a broad spectrum of potential responses, ranging from simple linear changes to more complex curvilinear patterns. This robust analytical strategy ensures a thorough understanding of how treatments influence analyte concentrations over time, with the p-value threshold of less than 0.05 consistently applied to all forms of statistical analysis. Visual representations of the statistical analyses and trend assessments were created using the matplotlib package in Python (Python Software Foundation, Wilmington, DE).

Table 2

Analyzed chemical composition (% as fed) of the experimental diets.

Dynamic Changes in the Blood Biomarker Profile for Birds Fed the Control Diet

Changes in the blood biomarker profile of birds that received the control diet are presented in Tables 3, ​,4,4, ​,5,5, and ​and6.6. There was no change in plasma pH, anion gap, total protein, globulin, hematocrit, AST, CK, UA, glucose, iCa, P or carotene for birds fed the control diet, either during the depletion or repletion phase. However, plasma HCO3 and CO₂ concentrations increased in a linear (P < 0.01), quadratic (P < 0.05) and cubic (P < 0.05) response during the nutrient depletion phase. This is explained by a short-term decrease in plasma HCO3 and CO₂ after 6 h (from 29.2 mmol/L to 26.6 mmol/L for HCO3) and a subsequent increase in concentration thereafter (HCO3 concentration 48h postfeeding was 31.3 mmol/L). Plasma Na, Cl, K and albumin increased during the nutrient depletion phase, with a peak around 12h, declining thereafter (cubic P < 0.05). Plasma total Ca increased during the nutrient depletion phase for birds fed the control diet (linear P < 0.05). There was no change in plasma metabolite concentrations for birds that were fed the control diet during the nutrient repletion phase.

Influence of Dietary Ca and Digestible P on Blood Biomarkers

There was no effect (all regression P > 0.05) of short-term feeding of diets depleted in Ca or digestible P to broilers for plasma Cl, total protein, globulin, hematocrit, AST, CK, or UA. Feeding the Ca depleted diet resulted in a decrease in plasma pH around 6 to 12h postintroduction of the diet, with a rise thereafter (cubic P < 0.01). Birds that received the diet with low digestible P had a short-term decrease in plasma HCO3 and CO_2, but concentrations of both metabolites rose thereafter (cubic P < 0.05). There was a linear (P < 0.05) decrease in plasma anion gap associated with feeding the diet with a low concentration of digestible P. Plasma Na peaked 12h after introduction of the diet with low digestible P, falling thereafter (cubic P < 0.05) whereas plasma K concentration peaked 6h after introduction of the same diet, also decreasing thereafter (cubic P < 0.01). A similar response pattern for plasma K was noted for the diet with low total Ca (cubic P < 0.05). Plasma albumin concentration increased linearly (P < 0.05) in birds that received the Ca depleted diet. Plasma glucose concentration initially increased (3–6h postfeeding) for birds fed the diet with low digestible P and then decreased thereafter (cubic P < 0.05). Birds that received the diet with low digestible P returned a linear increase in plasma iCa (1.39 mmol/L to 1.73 mmol/L; P < 0.001) and total Ca (11.36 mg/dL to 14.31 mg/dL; P < 0.001). Finally, feeding the low Ca diet resulted in an increase in plasma P that peaked around 12h postfeeding (rising from 6.19 mg/dL at T0 to 8.33 mg/dL at 12h), declining thereafter (cubic P < 0.05). There were few significant regression responses during the nutrient repletion phase. However, plasma P increased (linear P < 0.05) and plasma total and ionized Ca decreased, following reintroduction of the nutritionally replete diet for birds that had previously received the diet with low digestible P concentration. Birds that had previously received either the low Ca, or low digestible P diets, had increased hematocrit (linear P < 0.05) during the nutrient repletion phase.

Influence of Dietary Digestible AA and AME on Blood Biomarkers

There was no effect of introduction of a diet with low digestible AA or AME concentrations on plasma anion gap, Cl, K, globulin, hematocrit, CK or iCa. Plasma pH rose from 7.56 to 7.59 after 12h of feeding the diet with low digestible AA concentration, decreasing thereafter (cubic P < 0.05). Plasma HCO3 and CO2 concentrations dropped after 3h, following the introduction of both the low AME and the low digestible AA diets, rising thereafter to concentrations similar to T0 (cubic P < 0.05). Plasma Na concentration decreased at 3h after introduction of the diet with low AME, rising thereafter (cubic P < 0.05). Plasma total protein and albumin increased gradually for birds fed the diet with a low digestible AA concentration, peaking after 48h (2.15 g/dL to 2.41 g/dL for albumin; linear P < 0.01). Feeding the diet with low AME resulted in a gradual increase in plasma AST but this increase was not apparent until 12h postfeeding (from 167 U/L at T0 to 195.4 U/L at 48h; quadratic P < 0.001). Offering the low AME diet to birds generate a significant linear decrease in plasma glucose. Feeding the diet with a low concentration of digestible AA generated an increase in plasma Ca (from 11.36 mg/dL to 12.38 mg/dL after 48h; linear P < 0.01). There were few significant effects of returning birds that had been previously fed low digestible AA or AME concentration to the common nutritionally replete diet. Notable exceptions were for plasma Cl and Na where the re-introduction of the nutritionally replete diet created some perturbation in plasma levels. Specifically, plasma Na and Cl increased around 6-12h after the nutritionally replete diet was introduced.

Baselining Blood Metabolites in the Control Population

Birds that received the control diet experienced the most moderate change in diet composition compared with the diets with specific nutritional depletion (Table 1), with changes in nutrient density as per commercial broiler production norms. Specifically, the transition from the starter to the grower phase on d15 was associated with an increase in the concentration of corn and a reduction in the concentration of soybean meal, commensurate with the changing nutrient requirements of growing chicks. Interestingly, the transition from the starter to the grower phase resulted in a significant change in several blood metabolites including bicarbonate, total CO₂, Cl, K, Na, albumin and Ca. However, during the nutrient repletion phase, where no adjustment was made to the diet offered to the birds on the control treatment, no changes in blood metabolite profile was observed, suggesting the changes at d15 were associated with the altered chemical profile of the feed.

Transitioning the birds from the starter to the grower diet on d15 resulted in a temporary increase in the plasma concentration of Cl and Na, both of which peaked 12h after the grower diet was introduced, and a reduction in plasma K (Table 4; Figure 1, Figure 2). These effects may be related to the higher dietary Na and lower dietary K in the grower diet and the need for the birds to defend electroneutrality (sum of plasma Na and K must always equal the sum of plasma Cl, bicarbonate and the anion gap) in the plasma (Wolf, 2022). This rise in plasma Na and Cl was also associated with a spike in plasma albumin (from 2.15 g/dL at T0 to 2.38 g/dL at 12h, Table 5; Figure 2) suggesting that transitioning the chicks from the starter to the grower diet may have perturbed their feed and water intake behavior, resulting in short-term dehydration or interruption to osmotic balance in the blood. Hyperalbuminemia, hypernatremia and hyperchloremia are often associated with dehydration and although this has not been experimentally demonstrated via water restriction studies in poultry, there is evidence of this signature in water-deprived goats (Semaida and Abd El-Ghany, 2021).

The rise in plasma CO₂ and bicarbonate following the introduction of the grower diet on d15 appears to have not been directly associated with the grower feed per se, as similar trends were observed for birds fed alternative experimental feeds (except for the diet with a low Ca concentration; Table 3). A possible explanation for the rise in plasma CO₂ from around 26 to 27 mmol/L to around 29 to 30 mmol/L from 3 to 24h following introduction of the experimental feeds is that all experimental diets contained higher concentrations of corn relative to the common starter diet (an increase from around 58% to 62–69%). Corn contains a high concentration of digestible carbohydrate (Cowieson, 2005) which would increase glycolysis in the liver and muscle, a side-product of which is CO₂. The authors are unaware of any published work in poultry demonstrating an association between dietary digestible carbohydrate intake and blood gases but Alessandro et al. (2015) and Arsyad et al. (2020) observed higher blood TCO₂ in humans and rats, respectively, when a diet with a high digestible carbohydrate concentration was contrasted with one with a low carbohydrate load. Thus, transitioning birds from starter to grower, and presumably also from grower to finisher, diets, may be expected to generate both short-term and longer-term changes in blood metabolite composition, including shifts in osmotic equilibrium, HCO₃ and blood gases. The latter may have an influence on respiratory rate and could have implications for appropriateness of stocking density and ventilation programs. The anecdotal observation by poultry producers that diet phase changes may be associated with short-term disruption to feed efficiency or growth rate per se may be related to these changes in blood metabolite profiles as birds adapt to the change in chemical composition of the feed.

Effect of Feeding Low Concentrations of Digestible P and Total Ca on Blood Metabolite Profiles

Birds that received the diet with a low concentration of total Ca (0.60% vs. 0.86%) had a short-term (6–12h) rise in plasma anion gap, P, K, and a drop in pH. None of these effects were permanent and in most cases metabolite concentrations had returned to values not significantly different from the control population by 24 to 48h postintroduction of the nutritionally depleted diet. Furthermore, transitioning birds from the low Ca diet to the nutritionally complete repletion diet generated few significant differences in blood biochemistry, suggesting that birds had already adapted to the low Ca supply during the nutrient depletion phase. Indeed, a modest increase in plasma AST and hematocrit during the repletion phase of the experiment were the only significant effects for the low Ca treatment group. In contrast to the short-term flux in plasma metabolites in response to ingestion of the low Ca diet, chicks receiving the diet with a low concentration of digestible P experienced more long-term changes in plasma biochemistry (Figure 3). These rather different dynamics in adaption to dietary Ca and P may have an exaggerated effect on bird biology given the substantial resulting shift in blood Ca and P ratios. Plasma Na and K were the only metabolites where a transient change was observed, rising within 3-6h of the introduction of the diet with a low concentration of digestible P, but returning to baseline thereafter. Plasma HCO₃, CO₂, albumin, ionized Ca, total Ca, and carotene increased in concentration during the entire nutrient depletion phase of the experiment, returning to baseline only when the diet with adequate digestible P was reintroduced. In contrast, plasma glucose, anion gap and P, dropped significantly during the nutrient depletion phase, with plasma concentrations rapidly restored on introduction of the nutrient replete diet. These effects will be discussed in more detail hereafter but the possibility that dietary P supply may have sustained effects on blood biochemistry is relevant and of significant importance for optimization of feeding programs, especially involving inorganic P, Ca:P ratios, phytate and phytase, limestone characteristics and possibly vitamin D and drinking water quality.

Whilst plasma Ca and P concentrations are hormonally regulated (Wideman, 1987; Proszkowiec-Weglarz and Angel, 2013), a persistent influence of dietary Ca and P supply on plasma Ca and P concentrations has been previously reported. Manangi et al. (2018) noted that large particle size limestone, when fed to broiler breeder hens, generated a significantly higher plasma Ca concentration (0.5–1 mg/dL) compared with a low particle size limestone, effects that may be associated with different solubility or digestibility profiles of limestone of varying particle size. Marjina Akter et al. (2018) reported an increase in plasma Ca (0.7 mg/dL) and a reduction in plasma P (1.8 mg/dL) when dietary Ca was increased from 6 to 10 g/kg. These authors also observed an increase in plasma P concentration of 1.2 mg/dL when dietary non-phytate P content was increased from 3 to 4 g/kg. Fallah et al. (2018) also noted a decrease in plasma P when dietary Ca concentration was increased, especially above 8.5 g/kg, for Ross 308 broiler chickens, although these authors did not observe an influence of dietary Ca on plasma Ca. Similarly, Mansilla et al. (2020) found that increasing dietary Ca from 0.4 to 1.0%, in young broiler chicks, resulted in no change in plasma Ca (though a numerical rise was detected from around 10.5 to 11.5 mg/dL) but significantly reduced plasma P. It may be that plasma Ca is more tightly regulated than plasma P, which would be consistent with the results from the present experiment. Furthermore, altering dietary Ca supply may influence both plasma Ca and P (and more consistently so for P), whereas changing dietary P supply may have limited effect on plasma Ca, but a more long-term influence on plasma P.

Walk et al. (2024) noted that feeding a diet with a low concentration of both Ca and digestible P resulted in a reduction in plasma glucose concentration, effects that were exaggerated by the addition of phytase to the diet. The authors interpreted this to be associated with an increase in glucose transport from the blood into skeletal muscle and further involvement of glucose in ATP generation. These effects were supported by increases in gene and protein expression of several glucose transporters in the breast muscle. It is relevant that the drop in plasma glucose concentration was evident in the diet with a low concentration of both Ca and digestible P, although exogenous phytase addition created a further reduction (to a level significantly lower than the nutritionally complete diet). These effects, and those in the present experiment, may be associated with phytate degradation in the intestine and an increase in plasma myo-inositol concentration. Reducing dietary Ca and P increases phytate hydrolysis rate and increases plasma myo-inositol concentration (Cowieson et al., 2013), and myo-inositol has the capacity to influence various metabolic pathways, including glucose metabolism (Gonzalez-Uarquin et al., 2020). Therefore, it may be that the diets with reduced Ca and digestible P concentrations generated an increase in phytate solubility and hydrolysis in the lumen, increasing plasma myo-inositol and altering glucose uptake from blood.

Birds that received the diet with a low concentration of digestible P experienced a significant rise in plasma CO₂, HCO₃ and a reduction in the anion gap. There are 2 possible explanations for the rise in plasma CO₂, hypoventilation, or an increase in basal metabolic rate (glycolysis, Krebs cycle activity etc). The drop in anion gap is likely to be an indirect consequence of the increase in HCO₃, to maintain electroneutrality in blood. Hypoventilation may be unlikely given that all birds were reared under the same environmental conditions, no respiratory challenges were observed, and temperature was maintained in the thermoneutral zone. However, the drop in plasma glucose and increase in plasma CO₂ and HCO₃, does suggest an involvement of dietary P supply on carbohydrate metabolism. It is possible that the involvement of myo-inositol, as mentioned above, directly influences glucose metabolism in birds. Indeed, Schmeisser et al. (2017) observed an increase in differentially expressed genes in muscle of broilers when birds received a P deficient diet supplemented with phytase. Microarray analysis revealed that phytase, and putatively myo-inositol, may influence pathways downstream from insulin-like growth factor for example, PI3K. Further research may be needed to directly examine the role of dietary P and myo-inositol on carbohydrate metabolism, ATP synthesis, gluconeogenesis, and muscle accretion.

The effect of the low Ca diet on plasma K, anion gap and pH are inconsistent and difficult to explain. Walk et al. (2024) noted that feed a diet with a low concentration of Ca and avP (reduced relative to a standard diet by around 0.1–0.15%) generated a reduction in plasma K, which was ameliorated when the same diet was supplemented with phytase. Results presented herein suggest that feeding a low Ca diet to broilers does have the capacity to influence pH, K and anion gap but these effects are variable and temporary. It is possible that changes in plasma anion gap, K and pH associated with alterations in diet Ca and P concentration, are indirect and related to an attempt by the bird to maintain plasma electroneutrality, possibly involving the renal system.

Effect of Feeding Low Concentrations of Digestible AA and AME on Blood Metabolite Profiles

The diet with a low concentration of AME generated a rise in plasma albumin, AST, P, carotene, and a drop in glucose (Figure 4, Figure 5). These effects were rapidly reversed when the nutrient replete diet was reintroduced. The low AME diet was formulated to provide 160 kcal/kg less AME than the requirement of the birds. The relatively rapid rise in plasma AST and a drop in plasma glucose (Figure 4) suggests some metabolic compensation for the inadequate supply of AME, and specifically, a reduced dietary lipid concentration. AST is responsible for amino acid transamination, a reaction that involves vitamin B6 as a cofactor (Moran, 2017). It is possible that the reduced energy density generated an increase in gluconeogenesis from dietary amino acids resulting in an increase in plasma AST. The relatively low blood glucose concentration in the birds that received the low AME diet is further evidence of altered energy metabolism, with the chicks adapting to the low dietary lipid content and relatively dilute AME supply. There is a paucity of information in the literature on the influence of dietary AME or added fat on blood metabolites in poultry. Rozenboim et al. (2016) and Bona et al. (2018) explored the influence of diets with variable protein and AME ratios although both studies were conducted in laying hens and for the purpose of exploring fatty liver disease. However, contradictory effects were reported, with no effect of a low protein, energy rich, diet on AST in the study of Bona et al. (2018) whereas Rozenboim et al. (2016) noted an increase in AST in hens fed the low protein, high fat, diet, but only after 15 wk of feeding, suggesting these effects may not be acute. The current authors interpret the changes in blood metabolite concentrations in response to reduced dietary AME and added fat as an adaptation of the birds to generate additional energy from amino acids and carbohydrate. The reason for the rise in plasma P for the birds fed the diet with a low concentration of AME is not clear. However, it is possible that this is further evidence of a change in energy metabolism per se as P is required for the process of glycolysis and in the Krebs Cycle.

The diet with a low concentration of digestible AA resulted in a drop in plasma UA within 12h (Figure 6) and this was particularly acute by 48h. Following the reintroduction of the nutrient replete diet, a gradual rise in plasma UA was observed suggesting that plasma UA may react slowly to altered digestible protein intake. This contention is supported by Moss et al. (2019), who noted volatility in plasma ammonia associated with disruption of feeding behavior of birds during sampling whereasOspina-Rojas et al. (2014) found more stable plasma ammonia concentrations following 6h of fasting prior to sampling. It may be that plasma UA or ammonia may be biomarkers more suited to non-acute flux in dietary amino acid balance, digestible protein intake or the adequacy of amino acid supply for uric acid synthesis. In support of the present study, Selle et al. (2021) noted a reduction in the concentration of UA in the excreta of broilers fed a low protein (16.5 vs. 22.2%) diet. Helpfully, Namroud et al. (2008) previously reported a significant positive association between UA in blood and UA in excreta of broilers, suggesting that plasma UA concentration may be a useful biomarker of digestible protein intake, the adequacy of amino acid balance relative to the requirement of the bird (and so the extent of deamination) and plasma and excreta ammonia concentrations. Further work could consider more detailed analysis, perhaps transcriptomics, in the liver and in particular UA cycle intermediaries, ammonia or glutamine dehydrogenase activity.

Plasma Ca concentration rose in response to the ingestion of the diet with a low concentration of digestible AA (Figure 6), with no sign of this rise being regulated prior to the introduction of the nutrient replete diet. Plasma P reacted similarly to Ca, although evidence of self-regulation was apparent, with blood P beginning to drop prior to the nutrient repletion phase (Figure 6). An involvement of Ca and P in protein metabolism has been appreciated for several years. Hammoud et al. (2017) and Ragi et al. (2019) noted that increasing the supply of P to rodents that received a low protein diet resulted in improved growth rate and reduced plasma urea nitrogen. Cowieson et al. (2020b) noted that similar effects could be generated in broiler chickens and that feeding a low protein diet to broilers resulted in a reduction in plasma P and an increase in plasma Ca. A similar effect of dietary protein on plasma Ca was reported by Dao et al. (2022). It may be that protein has a calciuretic effect and axiomatically, reduced protein diets will result in an increase in plasma Ca concentration. These observations offer insight for further optimization of feeding strategies for poultry fed diets with varying protein concentration.

Total plasma protein and plasma albumin both rose in response to the ingestion of the diet with a low digestible protein concentration, as did plasma HCO₃ and CO₂ concentration. The reason for these changes is not clear and the most likely explanation is that as the low protein diet was inadequate to support maximal skeletal muscle accretion, some catabolism may have occurred, increasing the movement of amino acids in circulation. The inadequacy of amino acid supply via the diet may also have generated an increase in carbohydrate and lipid metabolism, temporarily increasing CO₂ production and transport, as HCO₃, in the blood.

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