Effects of anionic salts supplementation on blood pH and mineral status, energy metabolism, reproduction and production in transition dairy cows

Research in Veterinary Science 89 (2010) 72–77

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Effects of anionic salts supplementation on blood pH and mineral status, energy metabolism, reproduction and production in transition dairy cows Hesam A. Seifi a,b,*, Mehrdad Mohri a,b, Nima Farzaneh a, Hadi Nemati a, Shima Vahidi Nejhad a a b

Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran Center of Excellence in Ruminant Abortion and Neonatal Mortality, Ferdowsi University of Mashhad, Mashhad, Iran

a r t i c l e

i n f o

Article history: Accepted 21 January 2010

Keywords: Dairy cows Dietary cation–anion difference Negative energy balance Milk fever Culling

a b s t r a c t The objectives of this study were to determine the effects of a pre-partum diet with lower than recommended (DCAD = 82 mEq/kg of dietary DM) amounts of anionic salts on metabolism, health, reproductive performance and milk production in dairy cows. Sixty Holstein multiparous cows were enrolled 21 days prior to expected calving date. The animals were randomly assigned to receive one of two rations: 30 cows received anionic ration [ 82 mEq (NA + K Cl S)/kg of DM] for 21 d to parturition and the other group (n = 30) were fed a usual dry period ration (+192 mEq/kg of DM). Serum samples obtained at days 21, +3 and +21 relative to calving were analyzed for b-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA), glucose, calcium (Ca), inorganic phosphorus, magnesium, chloride, sodium, potassium, cholesterol, urea, creatinine, total protein, albumin, and aspartate aminotransferase (AST). Urine pH declined from 8.4 at 21 d before calving (pre-treatment) to 6.2 at day 7 pre-partum in the treatment group. Repeated-measure mixed model analysis indicated that the concentrations of Ca were significantly increased and creatinine, and AST were significantly decreased by lowering DCAD. The concentrations of BHBA, NEFA and glucose were not affected by treatment. The incidence of milk fever and culling were 5 and 11 times higher in the control group in comparison with the treatment group, respectively. The intervals from calving to first breeding and to pregnancy were not influenced by treatment. There was no group effect on average daily milk yield or fat percentage. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Decreasing the dietary cation–anion difference (DCAD: milliequivalents [(Na + K) (Cl + S)]/kg of DM) during the last 3– 4 weeks before calving is a popular method to prevent milk fever in dairy cows. Diets with a relative excess of anions prevented milk fever, and diets with a relative excess of cations predisposed cows to milk fever (Block, 1984). Low dietary cation–anion difference (DCAD) diets induce a mild, compensated metabolic acidosis (Fredeen et al., 1988; Tucker et al., 1988a). Improvements in Ca homeostasis by giving dietary anionic salts are thought to be mediated by this compensated acidosis. Increased plasma hydroxyproline concentrations (Block, 1984; Leclerc and Block, 1989) suggest that bone resorption may be stimulated because of the role of osseous tissues in buffering systemic acidosis (Bushinsky, 1989). Anionic salts are unpalatable, and a high inclusion rate can decrease feed intake before calving (Horst et al., 1994; Oetzel and Barmore, 1993). Decreased feed intake might increase * Corresponding author. Address: Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, P.O. Box 1793-91775, Mashhad, Iran. Tel.: +98 511 8788944; fax: +98 511 8763852. E-mail address: haseifi@um.ac.ir (H.A. Seifi). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.01.013

serum NEFA concentrations pre-calving and BHBA concentrations post-calving. The common recommendation is to add anionic salts until the DCAD value is 100 to 150 mEq/kg of dietary DM (Moore et al., 2000). The objectives of this study were to determine the effects of a pre-partum diet lower than recommended (DCAD = 82 mEq/kg of dietary DM) amounts of anionic salts on serum concentrations of minerals, and the incidence of milk fever and further to evaluate the effect of this ration on energy predictors of blood serum, milk production, disease occurrence and fertility post-partum.

2. Animals, materials and methods 2.1. Study design The study was conducted on a commercial dairy farm with 693 Holstein cows, with a rolling average milk production of 8300 kg. The farm was selected based on the willingness of the herd operators to participate in the study. Sixty multiparous cows averaging 595 kg body weight were selected and randomly divided into two groups during a 9 month period. Mean and median parity of cows were 3.92 and 3 years, respectively. The average duration of

H.A. Seifi et al. / Research in Veterinary Science 89 (2010) 72–77

dry period was 80 days for the animals of the study. All the cows were dried off by abrupt cessation of milking and application of blanket dry cow therapy with a commercial dry cow preparation (Albadry plus, Pfizer Animal Health, US). Cows were enrolled at 21 days before expected calving date. At enrolment, animals were randomly assigned to receive an anionic ration (n = 30; treatment group) or the usual dry period ration (n = 30; control group) for at least 21 days prior to parturition. The feed was offered as a total mixed ration (TMR). The predominant forages used in this farm were alfalfa haylage, and corn silage and the main concentrates were corn, mixed grain, soybean meal, canola meal, cotton seed meal, wheat bran, and sugarcane bagasse. Table 1 displays components and chemical composition of diets during the experimental period. The ration of the treatment group was supplemented with anionic salts daily offering ammonium chloride (104 g), magnesium sulphate (60 g), and calcium chloride (36 g) per cow. The diet of the treatment group had a DCAD of 82 mEq/kg DM using the equation DCAD (mEq/kg DM) = (Na + K) (Cl + S) (Goff and Horst, 1998). The control cows received a diet with DCAD of +192 mEq/ kg DM. The experiment was conducted blind, such that both farm and research staffs were unaware of which cows received each treatment. Lists of the cows that were due to calve were generated, and the randomization scheme was followed according to the order of calving. The animals were observed for disease occurrence during 2 months after parturition. Definitions used for periparturient disease events are based on Duffield et al. (1999). Subclinical ketosis was defined as serum BHBA concentration of P1400 lmol/l (Duffield, 2000). Reproductive performance was measured by the intervals from calving to first breeding, and from calving to pregnancy, using survival analysis. Cows were followed for 9 months until diagnosed pregnant or culled. Cows that remained in the herd non-pregnant after 9 months were retained in the analysis. Individual average of daily milk production and fat percentage in 3 months after parturition were collected for all cows. The average daily milk production and fat percentage in 1st, 2nd and 3rd

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month after parturition were used for analysis. Production data were used to assess the effect of treatment on average daily milk production and fat percentage. 2.2. Sample handling and laboratory procedures Blood samples were collected three times: at enrolment (21 days pre-partum), and again at both 3 and 21 days after parturition. Because of the difficulty in predicting the calving day, samples taken prior to calving were grouped as day 21 prior to calving (SD = 3). Two samples were taken at each sampling. One sample from the coccygeal vein was taken using a 2-ml heparinized syringe. Any excess air was immediately removed, and the syringe was stoppered and placed on ice. This aliquot was used to determine blood pH and HCO3 within 60 min of sampling, using blood gas analyzer (ABL50, Denmark). The second blood sample was collected from the coccygeal vein into 10-ml vacuum tube and was chilled immediately after sampling and transported to the laboratory within 1 h after collection. Serum was harvested after centrifugation, frozen and stored at 20 °C until analysis. Urine samples were taken twice (day 21 and 7 relative to calving) using a urine catheter to monitor optimal acidification. The concentrations of calcium (Ca), inorganic phosphorus, magnesium, chloride, cholesterol, urea, creatinine, total protein, albumin, glucose, and aspartate aminotransferase (AST) were determined by an automated biochemical analyzer (Biotecnica, Targa 3000, Rome, Italy) using commercial kits (Parsazmoon, Tehran, Iran). b-Hydroxybutyrate (BHBA) and non-esterified fatty acid (NEFA) were determined by a D-3-hydroxybutyrate kit and a NEFA Kit (Randox Laboratories Ltd., Ardmore, UK). Sodium and potassium concentrations were measured with flame photometry (Seac, Model FP20, Italy). The urine pH was measured using Digital pHmeter (Jenway, Model3040, England). All methods were in compliance with the guide for the care and use of agricultural animals in agricultural research and teaching. 2.3. Data management and statistical analysis

Table 1 Raw material and chemical composition of the experimental diets. Treatment

Control

Ingredient (% DM) Alfalfa hay Corn silage Barely grain Corn grain Soya bean meal Canola meal Cotton seed meal Wheat bran Wheat straw Sugarcane bagasse Fat supplement Calcium carbonate Salt DCAD

24.31 31.41 15.15 3.56 4.55 4.10 2.53 2.70 6.00 2.76 0.10 1.32 0.16 1.35

24.84 32.10 15.50 3.64 4.64 4.18 2.60 2.76 6.13 2.82 0.10 0.52 0.17 0

Chemical composition NE Mcal/kg CP (% DM) ADF NDF Ca P Mg K Na Cl S

1.56 14.1 25.4 40.3 1.17 0.37 0.26 1.30 0.11 0.80 0.38

1.56 14.1 25.4 40.3 0.78 0.38 0.26 1.30 0.11 0.27 0.18

82

+192

DCAD (mEq/kg)

The data were analyzed by SAS 8.2. Because serum metabolites were measured over time, a repeated measures approach using ANOVA with Mixed linear models in SAS (SAS Inst. Inc., Cary, NC) was used (fixed effects of treatment and covariates, random effect of cow). All outcome variables were screened for normality by visual assessment of the distributions and calculation of kurtosis and skewness. The distributions of BHBA, NEFA, phosphorus, potassium, cholesterol, creatinine and AST were not normal and thus were converted using a natural logarithmic transformation to achieve a normal distribution. Several covariance structures were evaluated for each analyzed metabolite. The covariance structure that resulted in Akaike’s information criterion (AIC) closest to zero was used (Wang and Goonewardene, 2004). Cow variables were considered for entry into the models. Cow variables included parity, treatment assignment, and sampling period. Cows were classified into two parity groups (2 and 3, group 1; and P4, group 2). Cow was considered as random effects to account for the correlation between observations of the same cow. All variables were offered to each model and then removed in a backward stepwise elimination approach. Interactions between treatment and the remaining significant variables were tested and included in the final model if significant. The interaction time (days 21, +3 and +21 relating to calving) and treatment was tested. If there was a significant interaction, data were reanalyzed after stratification by sample. Because there were three samples for each cow, a Bonferroni correction of the probability value was used (P < 0.016 = 0.05 divided by 3).

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Table 2 Logistic regression model for disease in Holstein cows that received a ration without anionic salts (DCAD +192 mEq/kg DM) in comparison to a ration with anionic salts (DCAD 82 mEq/kg DM) during the dry period within 3 weeks pre-partum. Health variable

Milk fever Retained placenta Lameness Subclinical ketosis Cull <61 DIM

Ratio of affected cows to total number of cows in each group Anionic salts supplementation

Without anionic salts supplementation

1/29 3/28 4/29 6/30 1/30

5/29 4/25 6/26 4/30 8/30

All disease outcomes and the variables, died and sold (culled), were analyzed by multivariable logistic regression (LOGISTIC procedure in SAS) model that included parity of the cows. The effects of treatment on time to first breeding and pregnancy were analyzed with multivariable survival analysis using Cox’s proportional hazards regression (the PHREG procedure in SAS). The models used included treatment, parity, disease (cows that suffered from one or more illnesses after calving were considered diseased, otherwise healthy). Milk production and fat percentage data were analyzed using repeated measures ANOVA (Mixed procedure in SAS). Variables considered in each model included group, parity group and disease.

Odds ratio

95% Confidence interval

(P value)

5 1.4 2 1.6 11

0.64–53 0.3–8 0.49–8 0.4–6 1.3–91

0.12 0.69 0.34 0.49 0.03

Table 4 The effects of time, group DCAD 82, dairy cows received anionic salts, group DCAD +192, or diet without anionic salts, parity, and disease on milk production and fat percentage using repeated measures ANOVA. There was no treatment  time interaction. Variable

3. Results Monitoring urine pH as an indicator of acidifying capacity of the ration revealed pH of treatment group declined from 8.4 at 21 days before calving (pre-treatment) to 6.2 at day 7 pre-partum (two weeks after anionic salts feeding). Table 2 presents the incidence of diseases during 60 days after calving. In the present study five control cows developed milk fever, while only one treated animal suffered from this disease. Nine cows were culled (eight controls and one treated cow). Reasons for culling were: parturient paresis (4), lameness (3), metritis and lameness (1), while for one cow the reason of culling was not recorded. The cows in the control group were 5 times more at risk [odd ratio (OR): 5; CI = 0.64–53; P = 0.12;] to suffer from milk fever in comparison with the animals in the treated group. Anionic salts supplementation had a significant protective effect on culling. The cows in the control group were 11 times more at risk [odd ratio (OR): 11; CI = 1.3–91.76, P = 0.03, Logistic Regression Test] of culling in comparison with the animals in the treated group. We observed in our study furthermore, that a significantly higher risk to be culled for cows suffering from milk fever and for cows with the parity P4 (P < 0.05). Furthermore, a significantly higher risk to become lame was observed in parity P4 cows (P < 0.05). Table 3 presents a summary of reproductive indices by treatment. The intervals from calving to first breeding and to pregnancy were not influenced by group of treatment. There were no group effects on average day milk yield (P = 0.23), or fat percentage (P = 0.72) (Table 4).

Outcomes Average day yield (kg)

Average day fat (%)

LSM

SE

P

LSM

SE

P

Group DCAD 82 DCAD +192

36.65 38.70

1.15 1.31

0.23

3.03 3.05

0.06 0.07

0.76

Average day 1st Month 2nd Month 3rd Month

35.71 39.49 37.82

1.13 1.14 1.15

0.004

3.22 2.93 2.97

0.09 0.09 0.09

0.06

Parity 2 and 3 P4

38.18 37.16

1.14 1.36

0.56

3.02 3.06

0.06 0.07

0.71

Clinical disease Yes No

35.06 40.29

1.10 1.39

0.004

2.92 3.16

0.08 0.06

0.01

The concentrations of Ca, creatinine, and AST were significantly influenced by treatment (Table 5). Treatment by time interactions was significant for Ca (P < 0.0001), albumin (P < 0.05), creatinine (P < 0.05) and AST (P < 0.001). No significant treatment effects were found for phosphorus, potassium, sodium, or chloride, magnesium, cholesterol, urea, total protein, glucose, BHBA and NEFA. The effect of parity and the random effect of cow were accounted for the significant differences of Ca, creatinine, albumin and AST between treatment and control groups. Ca concentrations at day 3 post-partum were significantly higher (P < 0.0001) for cows that had been fed with anionic salts in comparison with controls (Fig. 1). The treatment significantly decreased creatinine concentrations (P = 0.013) at day 21 after calving (Fig. 2). Albumin concentrations were significantly higher (P = 0.025) at day 3 after calving for cows that had received anionic salts in comparison with controls (Fig. 3). The treatment significantly lowered AST activities in cows that had received anionic salts (P < 0.001) at day 3 after calving in comparison with controls (Fig. 4).

Table 3 Summary of the reproductive performance in dairy cows that received anionic salts (DCAD 82 mEq/kg DM) or ration without anionic salts (DCAD +192 mEq/kg DM) during the dry period. Cows were followed for 9 months until pregnancy or culling and the data were analyzed using Kaplan–Meier and Cox’s proportional hazards survival analysis, accounting for the effects of parity and clinical disease. Treatment

n

Outcomes Time to first insemination

DCAD 82 mEq/kg DCAD +192 mEq/kg

29 22

Time to pregnancy

Median (days)

Means (days)

HR

P

Median (days)

Means (days)

HR

P

60 58

67.8 60.1

0.69

0. 23

108 79

100.4 91.5

0.98

0.93

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H.A. Seifi et al. / Research in Veterinary Science 89 (2010) 72–77 Table 5 Comparison of least square of means (LSM) and standard errors (SE) of serum constituents and blood pH and bicarbonate between cows received anionic salts (DCAD DM) or ration without anionic salts (DCAD +192 mEq/kg DM) dry period at days 21, +3 and +21 relating parturition. 21 days prior to calving

Calcium (mmol/l) Phosphorus (mmol/l) Magnesium (mmol/l) Chloride (mEq/l) Sodium (mEq/l) Potassium (mEq/l) Cholesterol (mmol/l) Glucose (mmol/l) BHBA (mmol/l) NEFA (mmol/l) Urea (mmol/l) Creatinine (lmol/l) Albumin (g/l) Total protein (g/l) AST (u/l) Blood pH Blood bicarbonate (mEq/l) *

3 days after calving

21 days after calving

Cows fed anionic salts diet LSM (SE)

Cows fed control diet LSM (SE)

Cows fed anionic salts diet LSM (SE)

Cows fed control diet LSM (SE)

Cows fed anionic salts diet LSM (SE)

Cows fed control diet LSM (SE)

2.6 (0.032) 2.20 (0.30) 0.89 (0.037) 102.36 (0.59) 145.5 (0.79) 4.69 (0.08) 2.35 (0.079) 3.34 (0.103) 0.52 (0.06) 0.41 (0.08) 3.50 (0.213) 144.09 (4.42) 28.4 (0.6) 72.3 (1.3) 51.55 (4.2) 7.37 (0.01) 21.92 (0.61)

2.6 (0.032) 2.27 (0.30) 1.06 (0.037) 102.37 (0.59) 146.1 (0.79) 4.73 (0.08) 2.49 (0.09) 3.42 (0.104) 0.49 (0.08) 0.38 (0.08) 3.64 (0.216) 147.63 (4.42) 28.5 (0.6) 70.6 (1.2) 52.33 (4.2) 7.34 (0.01) 21.50 (0.61)

2.56 (0.0035) 2.14 (0.30) 0.81 (0.029) 100.63 (0.47) 151.57 (0.84) 4.88 (0.10) 1.99 (0.069) 2.83 (0.108) 0.89 (.08) 1.17 (0.104) 4.65 (0.381) 128.18 (5.30) 28.3 (0.61) 67.5 (0.86) 74.26 (6.4) 7.33 (0.01) 22.61 (0.56)

2.28 (0.035) 1.95 (0.30) 0.83 (0.029) 101.89 (0.47) 151.53 (0.84) 5.14 (0.10) 1.92 (0.07) 3.10 (0.108) 0.82 (0.08) 1.12 (0.104) 5.26 (0.381) 137.02 (3.30) 26.3 (0.61) 67.6 (0.85) 104.94 (6.4) 7.33 (0.01) 21.49 (0.56)

2.50 (0.035) 2.85 (0.30) 0.97 (0.037) 97.25 (0.45) 148.45 (0.84) 4.83 (0.08) 2.99 (0.103) 2.77 (0.105) 0.57 (0.07) 0.74 (0.08) 4.88 (0.256) 107.85 (4.42) 25.6 (0.7) 74.5 (1.1) 74.3 (4.3) 7.29 (0.009) 21.60 (0.61)

2.50 (0.04) 2.37 (0.34) 0.87 (0.041) 98.03 (0.50) 147.42 (0.93) 4.83 (0.09) 2.89 (0.112) 2.54 (0.116) 0.51 (0.07) 0.69 (0.09) 5.22 (0.273) 120.22 (4.42) 26.8 (0.7) 73.8 (1.1) 71.47 (4.7) 7.32 (0.01) 22.15 (0.66)

82 mEq/kg

P Value*

0.002 0.81 0.29 0.82 0.82 0.15 0.84 0.64 0.63 0.66 0.15 0.04 0.56 0.44 0.01 0.89 0.89

Significant difference was considered at the level of P < 0.05.

2.7

160

*

150

Creatinine µmol/l

Ca mmol/l

2.6

2.5

2.4

2.3

140

130

* 120

110

2.2

100

Day -21

Day +3

Day +21

Days relating to calving Fig. 1. Least square of means values and (accounting for parity) standard errors for serum calcium (mmol/L) concentrations at days 21, +3 and +21 in dairy cows receiving anionic salts supplemented ration (d), and ordinary ration without anionic salts (s). Significant time  treatment interactions (P < 0.0001) are shown by star.

Significant time related changes ( 21 to +21 d relative to calving) were observed on blood Ca, magnesium, sodium, chloride, cholesterol, urea, creatinine, total protein, albumin, glucose, BHB, NEFA, AST and pH.

4. Discussion Based on the present study we demonstrated that by implementing anionic salts in the dry period starting at day 21 before parturition and reducing the DCAD to 82 mEq/kg DM, the number of milk fever cases could be reduced (numerically, but not statistically significant) and furthermore, the number of culled cows were significantly reduced in comparison with the control group which received a ration with a DCAD level of +192 mEq/ kg DM over the same time period. Further evidence that this implementation was successful, could be demonstrated by the

Day -21

Day +3

Day +21

Days relating to calving Fig. 2. Least square of means values and (accounting for parity) standard errors for serum creatinine (lmol/L) concentrations at days 21, +3 and +21 in dairy cows receiving anionic salts supplemented ration (d), and ordinary ration without anionic salts (s). Significant time  treatment interactions (P = 0.013) are shown by star.

fact that in the treated group there was a decrease in the urine pH of the cows from 8.4 at day 21 to 6.2 at day 7 pre-partum. With only 30 cows per group the power of this study to detect significant effects on the occurrence of clinical disease was limited. However, the incidence of milk fever tended to be significantly lower in the cows fed anionic salts. Culling rate as a dependent variable was of particular interest in this study. Culling risk were 4. 57% (one case out of 22) and 27.6% (eight cases out of 29) for cows fed anionic and control diets, respectively (P = 0.03, Fisher’s exact test, two sided). The effect of DCAD on culling is difficult to assess because multiple factors are involved. However, eight out of nine culled cows were due to milk fever and lameness. It is reasonable to hypothesize that feeding rations with a decreased DCAD during dry period indirectly decreases the culling rate. On the other hand, we were not able to show any affect of this strategy on the reproductive performance and the amount of milk and fat produced during the first 3 months in lactation.

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H.A. Seifi et al. / Research in Veterinary Science 89 (2010) 72–77 30

*

29

Albumin g/l

28

27

26

25

24 Day -21

Day +3

Day +21

Days relating to calving Fig. 3. Least square of means values and (accounting for parity) standard errors for serum albumin (g/L) concentrations at days 21, +3 and +21 in dairy cows receiving anionic salts supplemented ration (d), and ordinary ration without anionic salts (s). Significant time  treatment interactions (P = 0.025) are shown by star.

120

*

110 100

AST u/l

90 80 70 60 50 40 Day -21

Day +3

Day +21

Days relating to calving Fig. 4. Least square of means values and (accounting for parity) standard errors for serum AST (u/L) concentrations at days 21, +3 and +21 in dairy cows receiving anionic salts supplemented ration (d), and ordinary ration without anionic salts (s). Significant time  treatment interactions (P = 0.001) are shown by star.

There was no effect of treatment on serum concentrations of BHBA, NEFA and glucose. Since BHBA, NEFA and glucose are considered as indictors of the negative energy balance, we can argue that feeding anionic salts during the last 3 weeks before calving has a limited or no effect on the energy balance during the first weeks of lactation. These results are consistent with those from Moore et al. (2000). In the latter paper energy balance was evaluated by measuring feed intake and milk production, while also concentrations of NEFA and IGF-I were determined in the blood plasma and triglyceride in liver. The inclusion of anionic salts in diets fed to dry cows has caused nonsignificant (Oetzel et al., 1988, 1991) and significant decreases in DMI (Phillippo et al., 1994; Vagnoni and Oetzel, 1998; Hu and Murphy, 2004; Hu et al., 2007). According to Moore et al. (2000) the reduced dry matter intake noticed in the heifers given the 150 mEq/kg DM ration may have contributed to increased concentrations of triglycerides in the liver of these animals at calving. Since DMI was not measured in this study, effect of anionic salts on DMI was not concluded; however, the indicators of negative

energy balance and fat mobilization like concentrations of NEFA and BHBA were not significantly different between treated and control animals. In this study adding an anionic salt supplement and a DCAD of 82 mEq/kg DM did not affect blood pH values. Tucker et al. (1988b) however did observe significant decreases in blood pH when adding anionic salts to DCAD levels of 168 or 268 mEq of DCAD/kg of DM. The difference in results when comparing our study with the one of Tucker et al. (1988b) might be related to the different amounts of anionic salts supplemented and the difference in moment of blood sampling in relation to feeding. Vagnoni and Oetzel (1998) could demonstrate numerical though not significant differences between controls and supplemented animals. Serum Ca was significantly higher in cows fed anionic salts than cows fed control diet at 3 days after calving. Dietary anionic salts pre-partum increased total Ca (Block, 1984; Goff et al., 1991; Leclerc and Block, 1989; Oetzel et al., 1988; Joyce et al., 1997; Charbonneau et al., 2006) in serum on the day of parturition but had no effect in dry cows (Oetzel et al., 1991; Vagnoni and Oetzel, 1998). Effects of anionic salts on serum Ca may be evident only during times of extreme Ca stress. Jugular venous blood Ca2+ was not altered by DCAD, in dairy cows averaging 44 days in milk (Hu et al., 2007). No significant effect on serum concentrations of other minerals (inorganic phosphorus and magnesium) could be found in this study. Other studies showing effects of supplementation of anionic salts on inorganic phosphorus and magnesium have been inconsistent (Block, 1984; Goff et al., 1991; Leclerc and Block, 1989; Oetzel et al., 1988; Phillippo et al., 1994; Vagnoni and Oetzel, 1998). If bone hydroxyapatite was the source of increased Ca concentrations in blood at parturition, phosphorus concentrations in serum might have been expected to increase also (Joyce et al., 1997). However, reports in the literature with respect to changes in phosphorus levels in serum and plasma caused by DCAD are contradictory (Block, 1984; Gaynor et al., 1989; Goff et al., 1991; Leclerc and Block, 1989; Tucker et al., 1992; Wang and Beede, 1992). Tucker et al. (1988b) concluded that phosphorus and magnesium were much less responsive than urinary mineral excretion to DCAD manipulation. Blood content of Na, K and Cl were not affected by DCAD in our study. The increased activity of AST in control cows might be attributed to the higher incidence of milk fever in this group. A variety of tissues have high AST activity. Notably striated muscle and hepatocytes are noteworthy tissues (Meyer and Harvey, 2004). A significant increase in serum AST activity usually is associated with liver or muscle injury (Bain, 2003). Since we are not aware of any biologically relevant explanation for the significant increase of albumin levels in treated cows at day 3 after parturition and for the significant difference in creatinine concentrations at day 21, both might be caused by a random effect. Decreased trend of creatinine in both groups may be related to loss of muscle mass (Gregory, 2003), that is a physiologic process due to body reserves mobilization for compensating negative energy balance during the transition period. In conclusion, the results support this fact that the DCAD at the level of 82 mEq/kg DM, which is higher than recommended (Moore et al., 2000; Oetzel, 2000) could prevent milk fever without a significant effect on energy metabolism, production and reproduction indices.

Acknowledgement This study was supported by research fund of Ferdowsi University of Mashhad (Project No.: 9/3698, 83/10/27).

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References Bain, P.J., 2003. Liver. In: Latimer, K.S., Mahaffey, E.A., Prasse, K.W. (Eds.), Veterinary Laboratory Medicine Clinical Pathology, fourth ed. Blackwell Publishing, Ames, pp. 193–214. Block, E., 1984. Manipulation of dietary anions and cations for prepartum dairy cows to reduce incidence of milk fever. Journal of Dairy Science 67, 2939–2948. Bushinsky, D.A., 1989. Internal exchanges of hydrogen ions: bone. In: Seldin, D.W., Giebisch, G. (Eds.), The Regulation of Acid–Base Balance. Raven Press, New York, NY, pp. 69–88. Charbonneau, E., Pellerin, D., Oetzel, G.R., 2006. Impact of lowering dietary cation– anion difference in nonlactating dairy cows: a meta-analysis. Journal of Dairy Science 89, 537–548. Duffield, T.F., 2000. Subclinical ketosis in lactating dairy cattle. Veterinary Clinics of North America: Food Animal Practice 16, 231–253. Duffield, T.F., Leslie, K.E., Sandals, D., Lissemore, K., McBride, B.W., Lumsden, J.H., Dick, P., Bagg, R., 1999. Effect of a Monensin-controlled release capsule on cow health and reproductive performance. Journal of Dairy Science 82, 2377–2384. Fredeen, A.H., DePeters, E.J., Baldwin, R.L., 1988. Effects of acid–base disturbances caused by differences in dietary fixed ion balance on kinetics of calcium metabolism in ruminants with high calcium demand. Journal of Animal Science 66, 174–184. Gaynor, P.J., Mueller, F.J., Miller, J.K., Ramsey, N., Goff, J.P., Horst, R.L., 1989. Parturient hypocalcemia in Jersey cows fed alfalfa haylage-based diets with different cation to anion ratios. Journal of Dairy Science 72, 2525–2531. Goff, J.P., Horst, R.L., 1998. Factors to concentrate on to prevent periparturient disease in dairy cow with special emphasis on milk fever. In: Proceedings 31st Conference of American Association of Bovine Practitioners, Spokane, WA, pp. 154–163. Goff, J.P., Horst, R.L., Mueller, F.J., Miller, J.K., Kiess, G.A., Dowlen, H.H., 1991. Addition of chloride to a prepartal diet high in cations increase 1,25dihydroxyvitamin d response to hypocalcemia preventing milk fever. Journal of Dairy Science 74, 3863–3871. Gregory, C.R., 2003. Urinary system. In: Latimer, K.S., Mahaffey, E.A., Prasse, K.W. (Eds.), Veterinary Laboratory Medicine Clinical Pathology, fourth ed. Blackwell Publishing, Ames, pp. 231–259. Horst, R.L., Goff, J.P., Reinhardt, T.A., 1994. Calcium and vitamin D metabolism in the dairy cow. Journal of Dairy Science 77, 1936–1951. Hu, W., Murphy, M.R., 2004. Dietary cation–anion difference effects on performance and acid–base status of lactating dairy cows: meta-analysis. Journal of Dairy Science 87, 2222–2229. Hu, W., Murphy, M.R., Constable, P.D., Block, E., 2007. Dietary cation–anion difference and dietary protein effects on performance and acid–base status of dairy cows in early lactation. Journal of Dairy Science 90, 3355–3365.

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Joyce, P.W., Sanchez, W.K., Goff, J.P., 1997. Effect of anionic salts in prepartum diets based on alfalfa. Journal of Dairy Science 80, 2866–2875. Leclerc, H., Block, E., 1989. Effects of reducing dietary cation–anion balance for prepartum dairy cows with specific reference to hypocalcemic parturient paresis. Canadian Journal of Animal Science 69, 411–423. Meyer, D.J., Harvey, J.W., 2004. Veterinary Laboratory Medicine: Interpretation and Diagnosis, third ed. W.B. Saunders. p. 174. Moore, S.J., VandeHaar, M.J., Sharma, B.K., Pilbeam, T.E., Beede, D.K., Bucholtz, H.F., Liesman, J.S., Horst, R.L., Goff, J.P., 2000. Effects of altering dietary difference on calcium and energy metabolism in peripartum cows. Journal of Dairy Science 83, 2095–2104. Oetzel, G.R., 2000. Management of dry cows for the prevention of milk fever and other mineral disorders. Veterinary Clinics of North America: Food Animal Practice 16, 369–386. Oetzel, G.R., Barmore, J.A., 1993. Intake of a concentrate mixture containing various anionic salts fed to pregnant, non-lactating cows. Journal of Dairy Science 76, 1617–1623. Oetzel, G.R., Fettman, M.J., Hamar, D.W., Olson, J.D., 1988. Screening anionic salts for palatability, effects on acid–base status, and urinary calcium excretion in dairy cows. Journal of Dairy Science 74, 965–971. Oetzel, G.R., Olson, J.D., Curtis, C.R., Fettman, M.J., 1991. Ammonium chloride and ammonium sulfate for prevention of parturient paresis in dairy cows. Journal of Dairy Science 71, 3302–3309. Phillippo, M., Reid, G.W., Nevison, L.M., 1994. Parturient hypocalcemia in dairy cows: effects of dietary acidity on plasma minerals and calciotrophic hormones. Research in Veterinary Science 56, 303–309. Tucker, W.B., Harrison, G.A., Hemken, R.W., 1988a. Influence of dietary cation–anion balance on milk, blood, urine, and rumen fluid in lactating dairy cattle. Journal of Dairy Science 71, 346–354. Tucker, W.B., Xin, Z., Hemken, R.W., 1988b. Influence of dietary calcium chloride on adaptive changes in acid–base status and mineral metabolism in lactating dairy cows fed a diet high in sodium bicarbonate. Journal of Dairy Science 71, 1587– 1597. Tucker, W.B., Hogue, J.F., Adams, G.D., Aslam, M., Shin, I.S., Morgan, G., 1992. Influence of dietary cation–anion balance during the dry period on the occurrence of parturient paresis in cows fed excess calcium. Journal of Animal Science 70, 1238–1250. Vagnoni, D.B., Oetzel, G.R., 1998. Effects of dietary cation–anion difference on the acid–base status of dry cows. Journal of Dairy Science 81, 1643–1652. Wang, C., Beede, D.K., 1992. Effects of ammonium chloride and sulfate on acid–base status and calcium metabolism of dry Jersey cows. Journal of Dairy Science 75, 820–828. Wang, Z., Goonewardene, L.A., 2004. The use of mixed models in the analysis of animal experiments with repeated measure data. Canadian Journal of Animal Science 84, 1–11.