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ANIMAL NUTRITION |
,2
* Department of Animal and Poultry Science, and
and
Department of Human Health and Nutritional Sciences, University of Guelph, Ontario, Canada, N1G 2W1; and and
Department of Animal and Food Sciences, University of Kentucky, Lexington 40546
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: acid-base homeostasis • anionic supplement • physicochemical approach • sheep
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Additionally, low or negative dietary cation-anion difference (DCAD) diets also have an acidifying effect and alter the acid-base status of an animal (Oetzel et al., 1991; Hu and Murphy, 2004; Waller et al., 2005). A nutritional intervention that has been established to produce a low DCAD diet and induce systemic acidosis is the commercial anionic dairy supplement NutriChlor (Mutsvangwa et al., 2004) used to control low plasma Ca2+ at calving in dairy cows.
The Henderson-Hasselbalch equation has traditionally been used in clinical assessment and management of acid-base disturbances in ruminants (Constable, 1999, 2000). However, the Henderson-Hasselbalch equation is more descriptive than mechanistic and therefore does not adequately explain the underlying origins of acid-base disturbances (Lindinger, 2004). Consequently, clinical evaluation of acid-base disturbances (Carlson, 1997; Lindinger, 2004) is better accomplished using the physicochemical approach (Stewart, 1981, 1983), which identifies changes in the dependent variables (H ion concentration, [H+], total CO2, (TCO2), and the concentration of bicarbonate, [HCO3–]) in response to changes in the concentration of 1 or more of the independent variables [partial pressure of carbon dioxide (pCO2), strong ion difference ([SID]), and weak anions and cations ([Atot])].
The objective of the current study was to develop and characterize an animal model of acid-base disturbance caused by reducing DCAD. We tested the hypothesis that a decrease in plasma pH and the resultant acid-base perturbation in sheep supplemented with an anionic supplement (AS) were primarily due to reduced plasma [SID].
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Ten fully fleeced yearling wethers (Rideau-Arcott, 54.3 ± 6.7 kg of BW) of similar nutritional and environmental background obtained from Ponsonby Research Station, University of Guelph, Ontario, Canada, were used in the study. The sheep were housed in individual pens (1.5 x 2.0 m), blocked by BW, and randomly assigned to 1 of 2 treatments (n = 5) in a randomized complete block design. The sheep were fed individually, and water was available at all times.
Experimental Treatments
The sheep were maintained on a basal diet of dehydrated alfalfa pellets (1.2 kg/d; Langs Dehy Ltd, Palmerston, Ontario, Canada) containing 90% DM, 22% CP, and 1.2 Mcal of NEg/kg of DM, divided into 2 equal portions and offered twice daily at 1000 and 1300. From d 0 to 10, the sheep were supplemented with either one) canola meal (control supplement, CS) or 2) HCl-treated canola meal (NutriChlor, Nutritech Solutions, Abbotsford, British Columbia, Canada; AS) at 0700 and 1100 to induce an acid-base disturbance, as described by Mutsvangwa et al. (2004) and as modified by Odongo et al. (2006) for sheep. NutriChlor consists of HCl-treated canola meal [DCAD = –1,985 mEq/kg of DM, calculated as (Na+ + K+) – (Cl– + S2–)]. For this reason, canola meal was used as the control. The daily allocations of canola meal or NutriChlor were adapted to the sheep as 0 g (d 0), 50 g (d 1), 100 g (d 2), 150 g (d 3), and 200 g/d (d 4 to 10). The daily allocation was divided into 2 equal portions and mixed thoroughly by hand with 30 mL of molasses each as a carrier.
The feeding schedule was as follows: 1) at 0700, the sheep were offered one-half of their canola meal or HCl-treated canola meal allocation for the day; 2) at 1000, the sheep were offered half of their alfalfa pellets allowance for the day, which was thoroughly mixed with the canola meal or HCl-treated canola meal that had not been consumed; 3) at 1100, the mixture of canola meal or HCl-treated canola meal and the alfalfa pellets that had not been consumed was removed, and the sheep were offered the second half of their canola meal or HCl-treated canola meal allocation; 4) at 1500, the sheep were offered the second half of their alfalfa pellets allowance, which was thoroughly mixed with the canola meal or HCl-treated canola meal that had not been consumed; and 5) at 1700, the sheep were offered all feed weigh-backs for the day. The feed was left with the sheep overnight, and the cycle was repeated the following day. Feed intake of the individual sheep was recorded daily. The DCAD of the control and NutriChlor treatments were 184 and –206 mEq/kg of DM, respectively. Ingredient and chemical compositions of the diets are presented in Table 1.
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). On d 0, blood samples were withdrawn via the catheters using sterile 3-mL nonventing blood gas syringes, with lithium heparin as the anticoagulant (Gaslyte, Vital Signs Inc., Englewood, CO). The blood was immediately (within 3 min) analyzed for hematocrit, plasma pH, pCO2, partial pressure of O2, and the plasma concentrations of Na+, K+, Cl, Ca2+, glucose, and lactate using ion-selective electrodes (StatProfile 9+ analyzer, Nova Biomedical Corp., Waltham, MA). From the directly measured results, the analyzer calculated O2 saturation, base excess of plasma, base excess of extracellular fluid, HCO3–, standard HCO3–, TCO2, O2 content, normalized Ca, anion gap {[(Na+ + K+) – (Cl– + HCO3–)]; Emmet and Narins, 1977}, and osmolarity. Blood was then centrifuged for 15 min (2,500 x g) using a Beckman tabletop centrifuge (model TJ-6RS, Beckman Coulter Inc., Palo Alto, CA), and the plasma was analyzed for total protein (PP) using a clinical refractometer (Atago, SPR-T2 refractometer, Atago, Tokyo, Japan). Urine samples were collected in plastic centrifuge tubes using plastic funnels placed over the sheath of the sheep and fastened around the body of the animal using rubber tie-down cords as described by Odongo et al. (2006), and urine pH determined was using an Accumet pH meter (Fisher Scientific, Ottawa, Ontario, Canada). The d 0 plasma and urine sample measurements were used for the baseline acid-base status of the sheep and were used as covariates in the statistical analysis. Jugular venous blood and urine samples were then obtained daily from each sheep from 1100 to 1130 from d 1 to 10, and plasma and urine indicators of acid-base status (i.e., pH, HCO3–, and TCO2) were determined as already described. On d 10, blood and urine samples were also obtained at 0700, 1000, 1300, 1600, and 1900 to determine the within-day time course of the responses. On d 11, the experimental treatments were discontinued, and the sheep were slaughtered by captive bolt stunning and exsanguination.
Calculations and Statistical Analysis
The physicochemical analysis of acid-base physiology requires the application of 2 basic principles. The first is electroneutrality, which dictates that, in aqueous solutions, the sum of all positively charged ions must equal the sum of all negatively charged ions. The second is conservation of mass, which means that the amount of a substance remains constant unless it is added to or generated or removed or destroyed (Stewart, 1981, 1983).
Plasma [H+] was calculated using the measured pH, such that:
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Plasma [SID] was calculated as the sum of the plasma concentrations of the strong cations minus the strong anions (Stewart, 1983), such that:
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Plasma [Atot] was calculated by multiplying the [PP] (in g/dL) by 2.9 (Constable, 1999; Waller and Lindinger, 2005).
Calculations of the dependent acid-base parameters ([H+], [HCO3–], and [TCO2]) were made using the Acid-Basics II software (Watson, 1999), with the following equation (Stewart, 1983):
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where KW, KA, K3, and KC = equilibrium constants for the dissociation of water, weak acids, carbonic acid, and bicarbonate, respectively, and KW = 4.4 x 10–14 (eq/L)2, KA = 2.22 x 10–7 eq/L (Constable, 1997; Stämpfli et al., 1999), K3 = 5.76 x 10–11 eq/L, and KC = 2.45 x 10–11 (eq/ L)2/mmHg.
Applying this equation to the experimental data can provide a way to determine the contribution of each independent variable alone to the changes in the dependent variables, as detailed previously (Lindinger et al., 1992; Lindinger, 2004; Miller et al., 2005; Waller and Lindinger, 2005). Briefly, to determine the contributions of plasma [SID] alone, values for the 2 independent variables pCO2 and plasma [Atot] were held constant at the time that the 0700 values and the values for the dependent variables were computed, with the changes in plasma [SID]. This process was repeated for determining the contributions of pCO2 alone and [Atot] alone. This procedure allowed the time-course response of [H+], [HCO3–], and [TCO2] to be separately determined for changes in each of the independent variables.
Two-way (treatment and time) repeated measures ANOVA were performed on DMI (d 4 to 10), acid-base parameters collected at 1100 (d 4 to 10), and time-course data on d 10 using PROC MIXED (SAS Inst. Inc., Cary, NC) and the model Yij = µ + 
i + ßj + ßij + 
ij, in which Yij = the dependent variable; µ = the overall mean; i = the effect of treatment (i = 1, 2); ßj = the effect of block (j = 1, 2); ßij = the effect of the treatment x block interaction (ij = 1, 2, 3, 4); and ij = the random residual error. To determine time-dependent changes, and interactions between time and treatment, the effects of CS vs. AS over time were evaluated using orthogonal contrasts. Treatment means were compared using Tukey’s multiple comparison procedure, and the effects were considered significant at a probability of P < 0.05. Data are expressed as means plus or minus the SEM, which represents the pooled SE for the model.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. The DCAD did not (P > 0.05) affect DMI of the sheep (Table 2). However, lowering DCAD increased (P < 0.05) [Ca2+] in plasma by 13%. The AS was associated with reduced (P < 0.05) plasma pH, plasma [SID], and urine pH (Table 2). This is consistent with previous studies (Waller et al., 2005) showing that reduced DCAD (increased dietary Cl– and S2–) contributes to the reduced pH of body fluids. The AS did not (P > 0.05) alter pCO2, but plasma pH was reduced as a result of reduced plasma [SID]. The AS treatment also reduced (P < 0.05) the concentration of plasma glucose, base excess, and bicarbonate and increased (P < 0.05) the concentration of electrolytes K+ and Cl– (Table 2). Day had no (P > 0.05) effect on any of the parameters measured.
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; Odongo et al., 2006). The acidifying effect of a ration on urine pH first becomes apparent within 24 h of introduction (Goff and Horst, 1998). Horst et al. (1997) proposed that urine pH of 5.5 to 6.2 was indicative of compensated metabolic acidosis. When urine pH is reduced to between 6.0 and 7.0 through the acidification of the ration, the animal responds to the subacute metabolic acidosis by excreting H+ ions in the urine to compensate for the acidosis (Jardon, 1995; Pehrson et al., 1999). Horst et al. (1997) suggested that a pH < 5.5 should be avoided, because it might indicate that the metabolic acidosis was close to being uncompensated.
The concentration of H+ in plasma and various other body solutions is well regulated and, although it is affected by numerous activities of the body, it is highly buffered to help keep free H+ within physiological limits (Houpt, 2004). In the current study, traditional acid-base variables (pH, pCO2, HCO3–) using plasma sampled at 1100 on d 4 through 10 (Table 2
) or repeatedly on d 10 (Figures 1 to 4) indicated only a minimal plasma pH disturbance; regulatory mechanisms, including increased renal acid excretion, effectively maintained plasma pH within normal ranges (7.31 to 7.53; Duncan and Prasse, 1994; Vagnoni and Oetzel, 1998; Houpt, 2004) throughout the experiment. Using the time-course data from d 10, a deeper examination of the contributions of each of the independent physicochemical variables that contributed to acid-base balance showed that changes in some independent variables contributed more to the acidifying effect on plasma than others (Figures 1, 2, 3, and 4).
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). Dietary treatments had no effect (P > 0.05) on plasma pCO2 and [Atot] (Table 2, Figure 1). Changes in plasma [Atot] are linked to the net flux of water between vascular and nonvascular compartments or the addition of protein with other extracellular fluids via the lymphatic system. The pCO2 of the AS group decreased between 1000 and 1300 (Figure 1, panel B), suggesting a respiratory attempt to reduce [H+]. This is in agreement with Waller et al. (2005), who also reported that a decrease in pCO2 is a respiratory response that will partially offset acid-base disturbance. It can therefore be concluded that [Atot] is not a major contributing factor to acid-base disturbance associated with DCAD. The lowering of plasma pH and the resultant acid-base perturbation was primarily due to reduced [SID].
The major contributor to the reduced plasma [SID] was the elevated plasma [Cl] resulting from the ingestion of NutriChlor. Plasma pH or [H+] is ultimately determined by the concentrations of strong and weak cations and anions and the CO2 concentration within plasma (Stewart, 1983; Goff et al., 2004). Plasma [H+] was greater (P < 0.05) in the AS sheep compared with the CS sheep (Figure 2). The primary contributor to the increase in plasma [H+], and decreased plasma [HCO3–] and [TCO2], was the decrease in the independent variable plasma [SID] (Figures 2, 3, 4). Compared with the acidifying contributions of decreased plasma [SID], the contributions of pCO2 and [Atot] were minor and variable, in agreement with the absence of significant change in these 2 variables over time (Figure 1).
Furthermore, plasma [SID] showed a significant circadian pattern, with time points 1000 and 1300 being lower (P < 0.05) than time points 0700, 1600, and 1900 (Figure 1, panel A). Figures 2, 3, and 4 present similar circadian patterns, in which the measured [H+], [HCO3–], and [TCO2] are lower (P < 0.05) at 1000 and 1300 than at 0700, 1600, and 1900. Characteristically, profiles were biphasic, with a drop in plasma pH after feeding NutriChlor, a gradual reduction to nadir post-feeding, then either a sustaining of nadir or a gradual increase until the next feeding. Similar ruminal and plasma pH diurnal patterns have been reported in cows (Keunen et al., 2002; Rustomo et al., 2006).
Plasma [HCO3–] (Figure 3) and [TCO2] (Figure 4) were greater (P < 0.05) in the CS sheep compared with the AS sheep. Within the CS sheep, the calculated values were lower than measured (P < 0.05), a systematic effect that is likely due to the application of a human plasma acid-base-related constant to ovine plasma. The differences are physiologically minor and do not affect the interpretation of how the independent acid-base variables determined plasma [H+] (Figure 2), [HCO3–] (Figure 3), and [TCO2] (Figure 4). The influence of the [SID] at 1000 and 1300 in the AS sheep was lower (P < 0.05) than at 0700, 1600, and 1900. Although the measured values in the AS sheep at 1000 and 1300 were lower (P < 0.05) than those at 0700, only the measured values at 1300 were lower (P < 0.05) than those at 1600 and 1900 (Figures 3 and 4). Figure 4, panel B, shows a pronounced reduced [TCO2] in the AS group, indicating animals experienced a severe acid-base disturbance, with the largest effect at 1300. Constable (2000) stated that [TCO2] was a useful measurement for determining acid-base disturbances in domestic animals that do not display clinical evidence of a respiratory disease. In such cases, a decrease in [TCO2] was indicative of severe acid-base disturbance, whereas an increase in [TCO2] was evidence of systemic alkalosis.
Low DCAD prepartum can mitigate hypocalcemia in dairy cows via increased ionized Ca in plasma. Adding anions (specifically Cl–) to the diet increases Ca mobilization and absorption and elevates plasma Ca levels. Although the acidifying effects of negative DCAD in the diet may have short-term prophylactic effects of elevating ionized Ca in plasma, negative DCAD may have detrimental effects on acid-base balance.
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Footnotes |
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2 Corresponding author: nodongo{at}uoguelph.ca
Received for publication January 17, 2007. Accepted for publication May 2, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, whereas those receiving the anionic supplement are represented by 
. The bars indicate the SEM, and the up arrow indicates the time at which the canola meal or NutriChlor (Nutritech Solutions, Abbotsford, British Columbia, Canada) was supplemented. Means bearing * were significantly different from the mean at 0700.
) to the dependent acid-base variable plasma [H+] of sheep receiving the control (panel A) and the anionic (panel B) supplement on d 10 at 0700, 1000, 1300, 1600, and 1900. Measured [H+] (
) and [H+] calculated from the changes in all independent variables (&utrif). The bars indicate the SEM, and the up arrow indicates the time at which the canola meal or NutriChlor (Nutritech Solutions, Abbotsford, British Columbia, Canada) was supplemented.
). The bars indicate the SEM, and the up arrow indicates the time at which the canola meal or NutriChlor (Nutritech Solutions, Abbotsford, British Columbia, Canada) was supplemented.