Downloaded from http://veterinaryrecord.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Paper
Paper Enteral administration of monosodium phosphate, monopotassium phosphate and monocalcium phosphate for the treatment of hypophosphataemia in lactating dairy cattle M. J. Idink, W. Grünberg Hypohosphataemia is a frequent finding in early lactating and anorectic dairy cows. Sodium phosphate is commonly used for oral phosphorus (P) supplementation, although other phosphate salts may present useful treatment alternatives. Objectives of this study were to compare the efficacy of monopotassium phosphate (KH2PO4) and monocalcium phosphate (Ca(H2PO4)2) to monosodium phosphate (NaH2PO4) in P-depleted cows. Furthermore, the effect of concentrated NaH2PO4 on the reticular groove reflex was studied. Six healthy but P-depleted dairy cows underwent four treatments in randomised order. Treatments consisted of intraruminal administration of NaH2PO4, KH2PO4 and Ca(H2PO4)2 providing the equivalent of 60 g P. A fourth treatment consisting of concentrated NaH2PO4 combined with acetaminophen as a marker substance was administered orally to determine whether the reticular groove reflex could be induced. Intraruminal administration of NaH2PO4 and KH2PO4 resulted in similar increases in plasma Pi concentrations ([Pi]) while intraruminal Ca(H2PO4)2 resulted in lower increases in plasma [Pi]. Oral and intraruminal administration of NaH2PO4 resulted in similar times to peak plasma [Pi] and acetaminophen concentration, indicating that concentrated NaH2PO4 administered orally did not trigger the reticular groove reflex. These results suggest that oral administration of KH2PO4 is equally effective as NaH2PO4. Oral administration of Ca(H2PO4)2 in contrast has a less pronounced effect on the plasma [Pi]. Introduction Concerns about pollution of soils and surface water with phosphorus (P) of agricultural origin through manure led to incentives targeting at reducing the dietary P content of ruminant rations in order to limit faecal P excretion (Satter 2002). Recommendations for daily dietary P requirements of dairy cows have been reduced over the past decades based on numerous studies showing that the digestibility of dietary P in ruminants was underestimated in the past (NRC 2001). The revised recommendations were found to be adequate for most of the lactational cycle, but the onset of lactation that is associated with sudden changes in P homeostasis presents a particular challenge for the mechanisms regulating the P balance in dairy cows (Grünberg 2008). Hypohosphataemia is a common finding in early lactating dairy cows and sick cows with reduced feed intake Veterinary Record (2015) M. J. Idink, DVM, W. Grünberg, Dr. med vet, MS, PhD, Dip ECAR & ECBHM, assoc. Dip ACVIM, Department of Farm Animal Health, Utrecht University, Utrecht 3507 TD, the Netherlands W. Grünberg, Dr. med vet, MS, PhD, Dip ECAR & ECBHM, assoc. Dip ACVIM,
doi: 10.1136/vr.102847 Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Hanover, Germany E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed. Accepted February 1, 2015
(Grünberg 2014). The clinical relevance of hypophosphataemia in sick and periparturient cows is still under contentious debate, but empirical associations between hypophosphataemia and recumbency, feed intake depression and intravascular haemolysis have led to the common practice of supplementing hypophosphataemic cows with P in the field (Goff 1999, Constable 2003). Treatment of hypophosphataemia can consist of either oral or intravenous administration of phosphate salts. For both treatment approaches, sodium phosphate salts have been studied extensively and were found to be suitable for P supplementation (Cheng and others 1998, Braun and others 2007, Grünberg and others 2013). Because hypophosphataemia is frequently associated with hypocalcaemia in early lactating cows and with hypokalaemia in cows with pronounced or prolonged feed intake depression, the use of phosphate salts also providing calcium or potassium could present useful alternatives to sodium phosphate salts (Constable and others 2013). Oral dicalcium phosphate (CaHPO4) has been previously studied in cattle and was found to be insufficiently effective for the rapid substitution of P, but neither the oral use of monocalcium phosphate (Ca(H2PO4)2) nor monopotassium phosphate (KH2PO4) has been studied in P-depleted cattle (Cheng and others 1998, Grünberg and others 2013). The solubility of a phosphate salt is an important criterion for treatment efficacy after oral administration as only dissolved phosphate is available for intestinal absorption (Grünberg and others 2013). The poor solubility of CaHPO4 provides a sound explanation for the poor efficacy of this salt for rapid correction of hypophosphataemia in cattle. May 9, 2015 | Veterinary Record
Downloaded from http://veterinaryrecord.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Paper Accordingly, the considerably higher solubility of Ca(H2PO4)2 than CaHPO4 implies that Ca(H2PO4)2 might be more suitable for the treatment of hypophosphataemia. Similarly, differences in solubility between sodium phosphate and potassium phosphate salts indicate that the efficacy of potassium phosphate salts cannot be simply extrapolated from the efficacy of sodium phosphate. Another unresolved question relevant for the understanding of the absorption kinetics of phosphate salts is the function of the reticular groove reflex after oral administration of these salts. Most studies investigating the efficacy of oral sodium phosphate for P supplementation in cattle reported peak plasma phosphate concentrations ([Pi]) three to four hours after treatment (Braun and others 2007, Geishauser and others 2010, Grünberg and others 2013). In contrast, peak [Pi] in plasma within one hour were reported in one study using a concentrated sodium phosphate solution, suggesting that the higher concentration may have increased the absorption kinetics of P from the gut (Cheng and others 1998). Assuming that only negligible amounts of P are absorbed through the rumen epithelium, a peak plasma [Pi] within one hour after oral treatment is plausible only if the liquid bypasses the reticulorumen, as this occurs with closure of the reticular groove (Grünberg and others 2013). The reticular groove reflex could not be induced by oral administration of a more dilute 1M sodium phosphate solution (Grünberg and others 2013). The main objective of the study presented here was to compare the suitability of oral treatment with KH2PO4 and Ca (H2PO4)2 to the currently best established treatment, which is oral administration of monosodium phosphate (NaH2PO4) for correction of hypophosphataemia in cattle. We hypothesised that KH2PO4 would be similarly effective as NaH2PO4 in raising the plasma [Pi] in P-depleted dairy cows and would furthermore significantly increase the plasma potassium concentration ([K]). A second objective was to evaluate whether the closure of the reticular groove can be induced by oral administration of NaH2PO4 as a 3.2M solution. Our hypothesis was that the reticular groove reflex cannot be triggered by oral administration of a concentrated NaH2PO4 solution.
Materials and methods Animals, housing and feeding The national and institutional guidelines for the care and use of experimental animals were followed, and all experimental procedures were approved by the Utrecht University Institutional Animal Care and Use Committee (DEC; permit no 2013. iii.03.033). A total of six healthy, lactating, non-pregnant Holstein-Friesian cows were used for this study. Cows were between five and seven years old and between 100 and 200 days in lactation. The mean body weight was 611±68.6 kg (mean±sd) and the mean 305 day milk yield of the previous lactation was 9850±1490 kg. All cows were healthy based on complete physical examination and haematological and blood biochemical examination. Cows were housed in individual tie stalls with rubber bedding, covered with sawdust, in a temperature-controlled facility. The cows received a diet that was markedly P deficient from four weeks before administering the first experimental treatment until the end of the study. The diet offered as total mixed ration was based on corn silage, grass seed straw and beet pulp and was formulated to meet the National Research Council recommendations, except for the P content (NRC 2001). The P content in the ration was 2.0 g/kg dry matter, which is at least 40 per cent below current recommendations (NRC 2001). Feed was offered twice daily ad libitum between 06.00 and 07.00 hours and between 18.00 and 19.00 hours. Cows were milked twice daily between 06.00 and 07.00 hours and between 18.00 and 19.00 hours.
Experimental study All cows received four treatments in randomised order with a washout period of 48 hours between treatments. None of the Veterinary Record | May 9, 2015
cows received the four experimental treatments in the same treatment order. Treatments consisted of either 302 g NaH2PO4 dihydrate dissolved in 1.5 litre warm water (38°C) and administered by orogastric tube (1.3 mol/l, NaRu), 263 g KH2PO4 dissolved in 1.5 litre warm water administered by orgastric tube (KRu), 244 g Ca(H2PO4)2 monohydrate dissolved in 1.5 litre warm water administered by orgastric tube (CaRu) and 302 g NaH2PO4 dihydrate dissolved in 600 ml warm water to obtain a salt solution with a theoretical osmolarity of 6400 mOsmol/l (3.2 mol/l), administered via drench gun (NaOr). Acetaminophen (AP) was added as a marker substance to the test solution in groups NaRu and NaOr at a dose of 50 mg/kg BW to evaluate the function of the reticular groove. AP ( paracetamol) is an analgesic and antipyretic agent, which is mainly absorbed in the proximal small intestine but not in the stomach. Therefore, in monogastric species and preruminating calves the Acetaminophen Absorption Test (APAT), consisting of measuring the plasma AP concentration in short time intervals after oral administration of an AP-containing solution, is considered a reliable diagnostic tool to determine the gastric-emptying rate (Clements and others 1978, Marshall and others 2005). In ruminants, APAT has been used to study the function of the reticular groove reflex in lambs and calves, and adult cows (Schaer and others 2005, Sharifi and others 2009, Grünberg and others 2013). Since the objective was to compare the effect of NaH2PO4 solutions with different osmolarites on the reticular groove, AP was only added to the two NaH2PO4 treatments. All treatments contained the equivalent of 60 g P. No attempt was made to measure the amount of fluid lost due to spillage and drooling from the mouth when administering the test solution via drench gun. Cows were fitted aseptically with a 16 G jugular venous catheter (Angiocath; Becton-Dickinson, Heidelberg, Germany) with an extension set (Discofix C-3, 10 cm; Braun Melsungen AG, Melsungen, Germany) for blood collection in the evening before the first experimental treatment. Treatments were administered in the morning of each treatment day between 8.00 and 8.30 hours and at least one hour after the morning feeding. Blood samples were collected immediately before treatment (T0) and at 30, 60, 90, 120, 180, 240, 300, 420, 720 and 1440 minutes after each treatment. Blood was collected in tubes containing lithium heparin as anticoagulant and kept at room temperature until they were centrifuged at 1600 g for 10 minutes. Harvested plasma was stored at −20°C until analysed.
Biochemical analysis The plasma inorganic phosphate concentration ([Pi], ammonium molybdate method), magnesium concentration ([Mg], calmigate method) and AP concentration ([AP], turbidimetric inhibition immunoassay) were determined spectrophotometrically. Total plasma calcium ([Ca]), potassium ([K]) and sodium ([Na]) concentrations were determined by indirect potentiometry (ion-selective electrodes). An automated analyser (DXC-600, Beckman Coulter, Brea, California, USA) was used for the biochemical analysis. Total protein was determined by refractometry.
Data analysis The increment in plasma [Pi] ([Pincr]) was calculated by subtracting plasma [Pi] at T0 from the plasma [Pi] determined at the different sampling times. Similarly, the increment in plasma [AP] ([APincr]) and [K] ([Kincr]) was calculated. Maximal plasma [Pi] (CPmax) and maximal plasma [Pincr] (Pincr max) as well as time to maximal plasma [Pi] (TPmax) and time to maximal [Pincr] (TPincr max) for the time interval 0–420 minutes were obtained from the plasma concentration time curve of each treatment. The time interval from immediately before treatment until 420 minutes post-treatment was chosen to circumvent a potentially confounding effect of the evening feeding that was provided two hours before blood sampling at 720 minutes post-treatment.
Downloaded from http://veterinaryrecord.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Paper Areas under the plasma [Pincr] and [APincr] time curve for the first 240 (AUCPincr240) and 420 minutes (AUCPincr420) were calculated using the trapezoidal rule. Plasma volume changes were crudely estimated on the basis of total protein concentration ([TP]) changes in plasma relative to T0 (Fielding and Magdesian 2011). Plasma volume changes for each sampling time ‘i’ were calculated using the equation Voldiffi ð%Þ ¼ ½TPi =½TP0 100%: Electrolyte concentrations as well as increments relative to T0 were corrected for plasma volume changes to identify a possible effect of plasma volume changes on the electrolyte concentrations using the equation Cvolelectrolyte ¼ Celectrolyte =Voldiff 100:
Statistical analysis Results are expressed as mean and sd or median and IQR for parameters that were not normally distributed. Normality of distribution was tested by Shapiro-Wilk’s test for normality. Repeated measures analysis of variance was used to determine time effects, treatment effects, as well as treatment time interaction effects using an autoregressive(1) covariance matrix with animal ID as repeated variable. Terms in the used model were
treatment, time and the interaction of treatment and time. Post hoc tests were conducted to compare treatment, time and treatment–time interaction effects. Significance was assumed at P
Paper
Paper Enteral administration of monosodium phosphate, monopotassium phosphate and monocalcium phosphate for the treatment of hypophosphataemia in lactating dairy cattle M. J. Idink, W. Grünberg Hypohosphataemia is a frequent finding in early lactating and anorectic dairy cows. Sodium phosphate is commonly used for oral phosphorus (P) supplementation, although other phosphate salts may present useful treatment alternatives. Objectives of this study were to compare the efficacy of monopotassium phosphate (KH2PO4) and monocalcium phosphate (Ca(H2PO4)2) to monosodium phosphate (NaH2PO4) in P-depleted cows. Furthermore, the effect of concentrated NaH2PO4 on the reticular groove reflex was studied. Six healthy but P-depleted dairy cows underwent four treatments in randomised order. Treatments consisted of intraruminal administration of NaH2PO4, KH2PO4 and Ca(H2PO4)2 providing the equivalent of 60 g P. A fourth treatment consisting of concentrated NaH2PO4 combined with acetaminophen as a marker substance was administered orally to determine whether the reticular groove reflex could be induced. Intraruminal administration of NaH2PO4 and KH2PO4 resulted in similar increases in plasma Pi concentrations ([Pi]) while intraruminal Ca(H2PO4)2 resulted in lower increases in plasma [Pi]. Oral and intraruminal administration of NaH2PO4 resulted in similar times to peak plasma [Pi] and acetaminophen concentration, indicating that concentrated NaH2PO4 administered orally did not trigger the reticular groove reflex. These results suggest that oral administration of KH2PO4 is equally effective as NaH2PO4. Oral administration of Ca(H2PO4)2 in contrast has a less pronounced effect on the plasma [Pi]. Introduction Concerns about pollution of soils and surface water with phosphorus (P) of agricultural origin through manure led to incentives targeting at reducing the dietary P content of ruminant rations in order to limit faecal P excretion (Satter 2002). Recommendations for daily dietary P requirements of dairy cows have been reduced over the past decades based on numerous studies showing that the digestibility of dietary P in ruminants was underestimated in the past (NRC 2001). The revised recommendations were found to be adequate for most of the lactational cycle, but the onset of lactation that is associated with sudden changes in P homeostasis presents a particular challenge for the mechanisms regulating the P balance in dairy cows (Grünberg 2008). Hypohosphataemia is a common finding in early lactating dairy cows and sick cows with reduced feed intake Veterinary Record (2015) M. J. Idink, DVM, W. Grünberg, Dr. med vet, MS, PhD, Dip ECAR & ECBHM, assoc. Dip ACVIM, Department of Farm Animal Health, Utrecht University, Utrecht 3507 TD, the Netherlands W. Grünberg, Dr. med vet, MS, PhD, Dip ECAR & ECBHM, assoc. Dip ACVIM,
doi: 10.1136/vr.102847 Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Hanover, Germany E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed. Accepted February 1, 2015
(Grünberg 2014). The clinical relevance of hypophosphataemia in sick and periparturient cows is still under contentious debate, but empirical associations between hypophosphataemia and recumbency, feed intake depression and intravascular haemolysis have led to the common practice of supplementing hypophosphataemic cows with P in the field (Goff 1999, Constable 2003). Treatment of hypophosphataemia can consist of either oral or intravenous administration of phosphate salts. For both treatment approaches, sodium phosphate salts have been studied extensively and were found to be suitable for P supplementation (Cheng and others 1998, Braun and others 2007, Grünberg and others 2013). Because hypophosphataemia is frequently associated with hypocalcaemia in early lactating cows and with hypokalaemia in cows with pronounced or prolonged feed intake depression, the use of phosphate salts also providing calcium or potassium could present useful alternatives to sodium phosphate salts (Constable and others 2013). Oral dicalcium phosphate (CaHPO4) has been previously studied in cattle and was found to be insufficiently effective for the rapid substitution of P, but neither the oral use of monocalcium phosphate (Ca(H2PO4)2) nor monopotassium phosphate (KH2PO4) has been studied in P-depleted cattle (Cheng and others 1998, Grünberg and others 2013). The solubility of a phosphate salt is an important criterion for treatment efficacy after oral administration as only dissolved phosphate is available for intestinal absorption (Grünberg and others 2013). The poor solubility of CaHPO4 provides a sound explanation for the poor efficacy of this salt for rapid correction of hypophosphataemia in cattle. May 9, 2015 | Veterinary Record
Downloaded from http://veterinaryrecord.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Paper Accordingly, the considerably higher solubility of Ca(H2PO4)2 than CaHPO4 implies that Ca(H2PO4)2 might be more suitable for the treatment of hypophosphataemia. Similarly, differences in solubility between sodium phosphate and potassium phosphate salts indicate that the efficacy of potassium phosphate salts cannot be simply extrapolated from the efficacy of sodium phosphate. Another unresolved question relevant for the understanding of the absorption kinetics of phosphate salts is the function of the reticular groove reflex after oral administration of these salts. Most studies investigating the efficacy of oral sodium phosphate for P supplementation in cattle reported peak plasma phosphate concentrations ([Pi]) three to four hours after treatment (Braun and others 2007, Geishauser and others 2010, Grünberg and others 2013). In contrast, peak [Pi] in plasma within one hour were reported in one study using a concentrated sodium phosphate solution, suggesting that the higher concentration may have increased the absorption kinetics of P from the gut (Cheng and others 1998). Assuming that only negligible amounts of P are absorbed through the rumen epithelium, a peak plasma [Pi] within one hour after oral treatment is plausible only if the liquid bypasses the reticulorumen, as this occurs with closure of the reticular groove (Grünberg and others 2013). The reticular groove reflex could not be induced by oral administration of a more dilute 1M sodium phosphate solution (Grünberg and others 2013). The main objective of the study presented here was to compare the suitability of oral treatment with KH2PO4 and Ca (H2PO4)2 to the currently best established treatment, which is oral administration of monosodium phosphate (NaH2PO4) for correction of hypophosphataemia in cattle. We hypothesised that KH2PO4 would be similarly effective as NaH2PO4 in raising the plasma [Pi] in P-depleted dairy cows and would furthermore significantly increase the plasma potassium concentration ([K]). A second objective was to evaluate whether the closure of the reticular groove can be induced by oral administration of NaH2PO4 as a 3.2M solution. Our hypothesis was that the reticular groove reflex cannot be triggered by oral administration of a concentrated NaH2PO4 solution.
Materials and methods Animals, housing and feeding The national and institutional guidelines for the care and use of experimental animals were followed, and all experimental procedures were approved by the Utrecht University Institutional Animal Care and Use Committee (DEC; permit no 2013. iii.03.033). A total of six healthy, lactating, non-pregnant Holstein-Friesian cows were used for this study. Cows were between five and seven years old and between 100 and 200 days in lactation. The mean body weight was 611±68.6 kg (mean±sd) and the mean 305 day milk yield of the previous lactation was 9850±1490 kg. All cows were healthy based on complete physical examination and haematological and blood biochemical examination. Cows were housed in individual tie stalls with rubber bedding, covered with sawdust, in a temperature-controlled facility. The cows received a diet that was markedly P deficient from four weeks before administering the first experimental treatment until the end of the study. The diet offered as total mixed ration was based on corn silage, grass seed straw and beet pulp and was formulated to meet the National Research Council recommendations, except for the P content (NRC 2001). The P content in the ration was 2.0 g/kg dry matter, which is at least 40 per cent below current recommendations (NRC 2001). Feed was offered twice daily ad libitum between 06.00 and 07.00 hours and between 18.00 and 19.00 hours. Cows were milked twice daily between 06.00 and 07.00 hours and between 18.00 and 19.00 hours.
Experimental study All cows received four treatments in randomised order with a washout period of 48 hours between treatments. None of the Veterinary Record | May 9, 2015
cows received the four experimental treatments in the same treatment order. Treatments consisted of either 302 g NaH2PO4 dihydrate dissolved in 1.5 litre warm water (38°C) and administered by orogastric tube (1.3 mol/l, NaRu), 263 g KH2PO4 dissolved in 1.5 litre warm water administered by orgastric tube (KRu), 244 g Ca(H2PO4)2 monohydrate dissolved in 1.5 litre warm water administered by orgastric tube (CaRu) and 302 g NaH2PO4 dihydrate dissolved in 600 ml warm water to obtain a salt solution with a theoretical osmolarity of 6400 mOsmol/l (3.2 mol/l), administered via drench gun (NaOr). Acetaminophen (AP) was added as a marker substance to the test solution in groups NaRu and NaOr at a dose of 50 mg/kg BW to evaluate the function of the reticular groove. AP ( paracetamol) is an analgesic and antipyretic agent, which is mainly absorbed in the proximal small intestine but not in the stomach. Therefore, in monogastric species and preruminating calves the Acetaminophen Absorption Test (APAT), consisting of measuring the plasma AP concentration in short time intervals after oral administration of an AP-containing solution, is considered a reliable diagnostic tool to determine the gastric-emptying rate (Clements and others 1978, Marshall and others 2005). In ruminants, APAT has been used to study the function of the reticular groove reflex in lambs and calves, and adult cows (Schaer and others 2005, Sharifi and others 2009, Grünberg and others 2013). Since the objective was to compare the effect of NaH2PO4 solutions with different osmolarites on the reticular groove, AP was only added to the two NaH2PO4 treatments. All treatments contained the equivalent of 60 g P. No attempt was made to measure the amount of fluid lost due to spillage and drooling from the mouth when administering the test solution via drench gun. Cows were fitted aseptically with a 16 G jugular venous catheter (Angiocath; Becton-Dickinson, Heidelberg, Germany) with an extension set (Discofix C-3, 10 cm; Braun Melsungen AG, Melsungen, Germany) for blood collection in the evening before the first experimental treatment. Treatments were administered in the morning of each treatment day between 8.00 and 8.30 hours and at least one hour after the morning feeding. Blood samples were collected immediately before treatment (T0) and at 30, 60, 90, 120, 180, 240, 300, 420, 720 and 1440 minutes after each treatment. Blood was collected in tubes containing lithium heparin as anticoagulant and kept at room temperature until they were centrifuged at 1600 g for 10 minutes. Harvested plasma was stored at −20°C until analysed.
Biochemical analysis The plasma inorganic phosphate concentration ([Pi], ammonium molybdate method), magnesium concentration ([Mg], calmigate method) and AP concentration ([AP], turbidimetric inhibition immunoassay) were determined spectrophotometrically. Total plasma calcium ([Ca]), potassium ([K]) and sodium ([Na]) concentrations were determined by indirect potentiometry (ion-selective electrodes). An automated analyser (DXC-600, Beckman Coulter, Brea, California, USA) was used for the biochemical analysis. Total protein was determined by refractometry.
Data analysis The increment in plasma [Pi] ([Pincr]) was calculated by subtracting plasma [Pi] at T0 from the plasma [Pi] determined at the different sampling times. Similarly, the increment in plasma [AP] ([APincr]) and [K] ([Kincr]) was calculated. Maximal plasma [Pi] (CPmax) and maximal plasma [Pincr] (Pincr max) as well as time to maximal plasma [Pi] (TPmax) and time to maximal [Pincr] (TPincr max) for the time interval 0–420 minutes were obtained from the plasma concentration time curve of each treatment. The time interval from immediately before treatment until 420 minutes post-treatment was chosen to circumvent a potentially confounding effect of the evening feeding that was provided two hours before blood sampling at 720 minutes post-treatment.
Downloaded from http://veterinaryrecord.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Paper Areas under the plasma [Pincr] and [APincr] time curve for the first 240 (AUCPincr240) and 420 minutes (AUCPincr420) were calculated using the trapezoidal rule. Plasma volume changes were crudely estimated on the basis of total protein concentration ([TP]) changes in plasma relative to T0 (Fielding and Magdesian 2011). Plasma volume changes for each sampling time ‘i’ were calculated using the equation Voldiffi ð%Þ ¼ ½TPi =½TP0 100%: Electrolyte concentrations as well as increments relative to T0 were corrected for plasma volume changes to identify a possible effect of plasma volume changes on the electrolyte concentrations using the equation Cvolelectrolyte ¼ Celectrolyte =Voldiff 100:
Statistical analysis Results are expressed as mean and sd or median and IQR for parameters that were not normally distributed. Normality of distribution was tested by Shapiro-Wilk’s test for normality. Repeated measures analysis of variance was used to determine time effects, treatment effects, as well as treatment time interaction effects using an autoregressive(1) covariance matrix with animal ID as repeated variable. Terms in the used model were
treatment, time and the interaction of treatment and time. Post hoc tests were conducted to compare treatment, time and treatment–time interaction effects. Significance was assumed at P
