Abstract

This study aimed to evaluate hemogasometric and metabolic indicators in the first postpartum hours of dairy cows that received different cation-anion diets in the prepartum period. Holstein cows (n=14), multiparous, were divided into two groups: (1) acidogenic diet (DA -27.13 mEq/100 g of DM) (n=7) and (2) neutral diet (DN -3.25 mEq/100 g of DM) (n=7), provided from 30 days before the expected calving. Urine samples were collected every three days from the beginning of supplementation until the day of delivery for pH verification. Blood samples were collected at 0, 6, 12, 24, 36, 48, 60 and 72 h postpartum for hemogasometric and biochemical analyses. The animals that received DA presented lower urinary pH. The serum concentration of total calcium, ionized calcium and the incidence of subclinical hypocalcemia did not differ between groups. Animals that received DA presented reduction in blood levels of total plasma proteins, globulins, bicarbonate and blood pH, in addition to increased activity of paraoxone-1 and reduction in the concentration of haptoglobin from animals of DN. In conclusion, we can infer that, anionic diets can alter blood pH, interfere with protein synthesis, and probably improve antioxidant capacity.

Keywords:
acid-base; subclinical hypocalcemia; hemogasometry; dairy cows

Resumo

O objetivo deste estudo foi avaliar indicadores hemogasométricos e metabólicos nas primeiras horas pós-parto de vacas leiteiras, que receberam diferentes dietas cátion-aniônica no pré-parto. Vacas da raça Holandesa Preta e Branca (HPB) (n=14), multíparas, foram divididas em dois grupos: dieta acidogênica (DA -27,13 mEq/100g de MS) (n=7) e dieta neutra (DN -3,25 mEq/100g de MS) (n=7), fornecidas a partir de 30 dias antes da previsão do parto. Amostras de urina foram coletadas a cada três dias após o início da suplementação até o dia do parto, para a verificação do pH. Amostras de sangue foram coletadas às 0, 6, 12, 24, 36, 48, 60 e 72 horas pós-parto, para análises hemogasométricas e bioquímicas. Os animais que receberam DA apresentaram pH urinário menor. A concentração sérica de cálcio total, cálcio ionizado e a incidência de hipocalcemia subclínica não diferiram entre os grupos. Animais que receberam DA apresentaram redução nos níveis sanguíneos de proteínas plasmáticas totais, globulinas, bicarbonato e pH sanguíneo, além de aumento na atividade de paraoxonase-1 (PON-1) e redução na concentração de haptoglobina em relação aos animais da DN. Como conclusão podemos inferir que, dietas acidogênicas podem alterar o pH sanguíneo, interferir na síntese de proteínas, e provavelmente melhorar a capacidade antioxidante.

Palavras-Chave:
ácido-base; hipocalcemia subclínica; hemogasometria; vacas leiteiras

Introduction

Diets with different cation-anionic balance (DCAB) are used at different periods during the productive life of dairy cows, such as the dry period, pre- and postpartum⁽¹1 Santos JEP, Lean IJ, Golder H, Block E. Meta-analysis of the effects of prepartum dietary cation-anion difference on performance and health of dairy cows. Journal of dairy science. 2019;102(3):2134-54.^{, 2}2 Hu W, Murphy MR. Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis. Journal of dairy research. 2004;87(7):2222-9.⁾. Currently, the acidogenic diet provided before delivery is used to prevent hypocalcemia, characterized by serum Ca_t<8.0mg/dL and/or Ca_i<4.0mg/dL⁽³3 Oetzel GR. Effect of calcium chloride gel treatment in dairy cows on incidence of periparturient diseases. J Am Vet Med Assoc. 1996;209(5):958-61.

4 Goff JP. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. The Veterinary Journal. 2008;176(1):50-7.^-55 Martinez N, Sinedino L, Bisinotto R, Ribeiro E, Gomes G, Lima F, et al. Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows. Journal of dairy science. 2014;97(2):874-87.⁾. Its action is based on the supply of anionic salts with the objective of reducing DCAB⁽⁶6 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.^,77 Eklund M. The dietary cation-anion difference and its impact on the milk production in dairy cows. 2016.⁾.

The DCAB represents the difference between the cations (Na⁺ sodium, K⁺ potassium) and the anions (Chlorine Cl^- and sulfur SO4^-) present in the diet, in milliequivalents (mEq) of (Na⁺+K⁺) - (Cl^- + SO4^-) per 100g of dry matter (DM). To obtain an adequate acidogenic diet it is necessary that the sum of its ingredients is approximately -10 to -20mEq/100g of DM⁽⁸8 Roche JR, Dalley DE, O'Mara FP. Effect of a metabolically created systemic acidosis on calcium homeostasis and the diurnal variation in urine pH in the non-lactating pregnant dairy cow. Journal of dairy research. 2007;74(1):34-9.⁾. Its main action is to decrease the blood pH and, consequently, activate the calcium homeostatic mechanism, which occurs due to the increased sensitivity of calcium receptors in the parathyroid, which increases the secretion of parathyroid hormone⁽⁹9 Campion KL, McCormick WD, Warwicker J, Khayat ME, Atkinson-Dell R, Steward MC, et al. Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism. Journal of the American Society of Nephrology. 2015;26(9):2163-71.⁾.

The effectiveness of blood acidification can be confirmed by the urinary pH, which can be verified from 48 hours after the change in diet, with values below 6.8 indicating the correct administration of anionic salts⁽¹⁰10 Chan PS, West JW, Bernard JK. Effect of prepartum dietary calcium on intake and serum and urinary mineral concentrations of cows. Journal of dairy science. 2006;89(2):704-13.⁾. The use of these salts for 11 days already promotes the homeostatic activation of calcium in the postpartum of dairy cows⁽¹¹11 Pizoni C, Feijó JO, Londero US, Pereira RA, Corrêa MN, Brauner CC, et al. Parâmetros clínicos, hematológicos e bioquímicos de novilhas com hipocalcemia subclínica pré-parto suplementadas com dieta aniônica. Arquivo brasileiro de medicina veterinária e zootecnia. 2017;69(5):1130-8.⁾. However, in production systems, the acidogenic diet is usually indicated for a minimum period of 21 days before the calving is expected⁽¹²12 Oetzel GR. Meta-analysis of nutritional risk factors for milk fever in dairy cattle. Journal of dairy science. 1991;74(11):3900-12.^,1313 DeGaris PJ, Lean IJ. Milk fever in dairy cows: A review of pathophysiology and control principles. The veterinary journal. 2008;176(1):58-69.⁾, due to the uncertainty of the calving date.

During the peripartum period of the dairy cow, the requirement for calcium varies: in the prepartum, about 30 to 50g/day are needed, directed to the fetus, mammary gland (colostrum in the prepartum and milk in the postpartum) and its maintenance; in the immediate postpartum period, daily requirement can reach 100g of calcium/day⁽⁶6 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.^,1414 Fox DG, Tylutki TP. Accounting for the effects of environment on the nutrient requirements of dairy cattle. Journal of dairy research. 1998;81(11):3085-95.^,1515 DeGaris PJ, Lean IJ, Rabiee AR, Heuer C. Effects of increasing days of exposure to prepartum transition diets on reproduction and health in dairy cows. Aust Vet J. 2010;88(3):84-92.⁾. Depending on the production of colostrum and milk, calcium secretion can reach up to 2.3g/L only in colostrum, and in a cow that produces 40L of milk/day, the secreted calcium can be 7 to 10 times higher than the levels of ionized calcium (Ca_i) available in the body, predisposing to hypocalcemia⁽¹⁶16 Caixeta LS, Ospina PA, Capel MB, Nydam DV. Association between subclinical hypocalcemia in the first 3 days of lactation and reproductive performance of dairy cows. Theriogenology. 2017;94:1-7.^,1717 Horst RL, Goff JP, Reinhardt TA. Adapting to the transition between gestation and lactation: differences between rat, human and dairy cow. Journal of mammary gland biology and neoplasia. 2005;10(2):141-56.⁾. To maintain the physiological levels of circulating total calcium (Ca_t) it is necessary for the body to activate homeostatic mechanisms, a process that can take up to 72 hours, predisposing the animal to subclinical hypocalcemia, which can progress to clinical conditions and even death⁽⁶6 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.^,1616 Caixeta LS, Ospina PA, Capel MB, Nydam DV. Association between subclinical hypocalcemia in the first 3 days of lactation and reproductive performance of dairy cows. Theriogenology. 2017;94:1-7.⁾.

Subclinical hypocalcemia can cause a cascade of events such as decreased DM intake (DMI), placental retention, uterine prolapse, abomasal displacement, ketosis, metritis and reduced reproductive performance⁽¹⁸18 Martinez N, Risco CA, Lima FS, Bisinotto RS, Greco LF, Ribeiro ES, et al. Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease. Journal of dairy science. 2012;95(12):7158-72.

19 LeBlanc SJ. Relationships between metabolism and neutrophil function in dairy cows in the peripartum period. Animal : an international journal of animal bioscience. 2020;14(S1):s44-s54.

20 Brozos C, Mavrogianni VS, Fthenakis GC. Treatment and control of peri-parturient metabolic diseases: pregnancy toxemia, hypocalcemia, hypomagnesemia. The veterinary clinics of North America: food animal practice. 2011;27(1):105-13.^-2121 Lean IJ, Santos JEP, Block E, Golder HM. Effects of prepartum dietary cation-anion difference intake on production and health of dairy cows: A meta-analysis. Journal of dairy science. 2019;102(3):2103-33.⁾, which harms the health and productivity of the animal. The decrease in blood calcium levels can also cause a reduction in your cellular reserves, which directly impairs the immune response, contributing to a state of immunosuppression⁽¹⁸18 Martinez N, Risco CA, Lima FS, Bisinotto RS, Greco LF, Ribeiro ES, et al. Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease. Journal of dairy science. 2012;95(12):7158-72.^,2222 Clemens RA, Lowell CA. Store-operated calcium signaling in neutrophils. Journal of leukocyte biology. 2015;98(4):497-502.⁾. For this reason, it is necessary to prevent this disorder, with the acidogenic diet being the most used strategy. Even with its supply, the prevalence of hypocalcemia is high, reaching more than half of the animals, depending on the number of lactations and the level of milk production⁽²³23 Reinhardt TA, Lippolis JD, McCluskey BJ, Goff JP, Horst RL. Prevalence of subclinical hypocalcemia in dairy herds. The Veterinary Journal. 2011;188(1):122-4.

24 Silva DC, Fernandes BD, Santos Lima JM, Rodrigues GP, Dias DLB, Oliveira Souza EJ, et al. Prevalence of subclinical hypocalcemia in dairy cows in the Sousa city micro-region, Paraíba state. Tropical animal health and production. 2019;51(1):221-7.^-2525 McArt J, Neves R. Association of transient, persistent, or delayed subclinical hypocalcemia with early lactation disease, removal, and milk yield in Holstein cows. Journal of dairy science. 2020;103(1):690-701.⁾.

PON-1 is a negative acute-phase protein produced by the liver, with the ability to prevent the oxidation of HDL and LDL, ability to hydrolyze xenobiotic organophosphates, protection from free radicals by limiting the oxidation of phospholipids and inhibition of peroxide production⁽²⁶26 Canales A, Sánchez-Muniz FJ. Paraoxonasa, ¿algo más que una enzima? Medicina Clínica. 2003;121(14):537-48.⁾. PON-1 is quite stable under normal conditions, but during inflammatory events or chronic liver disease, its levels decrease⁽²⁷27 Ferré N, Camps J, Prats E, Vilella E, Paul A, Figuera L, et al. Serum paraoxonase activity: a new additional test for the improved evaluation of chronic liver damage. Clinical chemistry. 2002;48(2):261-8.⁾. Haptoglobin, on the other hand, is one of the most sensitive positive acute-phase proteins in cattle, with anti-inflammatory and immunomodulatory properties⁽²⁸28 Jelena A, Mirjana M, Desanka B, Svetlana I-M, Aleksandra U, Goran P, et al. Haptoglobin and the inflammatory and oxidative status in experimental diabetic rats: antioxidant role of haptoglobin. Journal of physiology and biochemistry. 2013;69(1):45-58.⁾, it can increase its plasma concentration by more than 100 times during infections⁽²⁹29 Eckersall PD, Young FJ, McComb C, Hogarth CJ, Safi S, Weber A, et al. Acute phase proteins in serum and milk from dairy cows with clinical mastitis. The Veterinary record. 2001;148(2):35-41.⁾, in addition to having antioxidant properties, with the ability to bind to hemoglobin, inhibiting the oxidative activity of the heme group⁽³⁰30 Tseng CF, Lin CC, Huang HY, Liu HC, Mao SJT. Antioxidant role of human haptoglobin. Proteomics. 2004;4(8):2221-8.⁾.

In light of the above, the study hypothesis is that highly acidogenic diets in the prepartum period can change hemogasometric and biochemical parameters in the postpartum, and the objective of this study was to evaluate the effects of different diets on the hemogasometric, mineral, protein and energy profile of dairy cows in the first hours postpartum that received different cation-anionic diets in the prepartum.

Materials and methods

This experiment was approved by the Animal Experimentation Ethics Committee (CEEA) of the Federal University of Pelotas (UFPel) registered under code 2563.

This study was carried out in a dairy farm in the south of Rio Grande do Sul, in the city of Rio Grande (32º 16’ S, 52° 32’ L). Fourteen pregnant cows of the black and white Holstein breed, multiparous, between third and fourth lactation, with body condition score (BCS) between 3.0 and 3.5 were selected⁽³¹31 Hady PJ, Domecq JJ, Kaneene JB. Frequency and precision of body condition scoring in dairy cattle. Journal of dairy science. 1994;77(6):1543-7.⁾. The cows remained in a semiextensive management regime, fed in a collective trough being offered the diet twice a day, and were randomly distributed into two groups: acidogenic diet (DA, n=7) and neutral diet (DN, n=7). In the DA group, the feeding was balanced to -27.13mEq/100g MS, while in the DN group, the feeding was balanced to -3mEq/100g MS. Both diets were given 30 days before delivery (Table 1).

Urine pH was analyzed at prepartum using a bench pH meter (Tecnopon, PA-210-SP, Brazil) every three days from the beginning of supplementation, before supplementation. The bromatological and mineral analysis of the diet (pasture, silage and concentrate) provided for the different groups was carried out in a commercial laboratory (3rLab - Minas Gerais, Brazil) using the NIRS method (Near Infrared spectroscopy).

Blood samples were collected through puncture of the coccygeal artery complex in tubes without anticoagulant (BD Diagnostics, São Paulo, Brazil), at 21 ± 6 days before calving (collection -1), and at 0, 6, 12, 24, 36, 48, 60 and 72h postpartum. Blood samples were centrifuged at 1800 ×g for 15min to obtain serum, and frozen at -80ºC for further analysis.

Serum concentrations of total calcium (Cat), magnesium, albumin, urea, total plasmatic proteins (TPP) and creatinine were analyzed using commercial kits (Labtest Diagnóstica SA, Brazil) in automated biochemical equipment (Labmax Plenno- Labtest Diagnóstica SA, Brazil). Globulin levels were estimated using the formula (Globulins = TPP - albumin).

To check the serum concentration of ionized calcium (Ca_i), pH (ionic hydrogen potential), bicarbonate, pCO₂ (partial pressure of carbon dioxide), pO₂ (oxygen pressure), sO₂ (oxygen saturation), TCO₂ (carbon dioxide content), Na⁺ (sodium), K⁺ (potassium), hemoglobin, hematocrit and glucose, the portable biochemical meter I-Stat (Aboot -USA) using GC+8 cartridges was used. Immediately after blood collections via the coccygeal vein in heparinized vacutainer tubes (BD Diagnostics, São Paulo, Brazil), the CG8+ cartridge was filled with blood and the reading was verified in the I-Stat. Animals with Ca_i levels ≤ 4.0mg/dL at least three times postpartum, at any time of collection, were considered to have subclinical hypocalcemia⁽⁵5 Martinez N, Sinedino L, Bisinotto R, Ribeiro E, Gomes G, Lima F, et al. Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows. Journal of dairy science. 2014;97(2):874-87.^,66 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.⁾.

Haptoglobin concentration was analyzed by colorimetric technique as described by Jones and Mould⁽³²32 Jones GE, Mould DL. Adaptation of the guaiacol (peroxidase) test for haptoglobins to a microtitration plate system. Research in veterinary science. 1984;37(1):87.⁾ and adapted by Schneider et al.,⁽³³33 Schneider A, Corrêa MN, Butler WR. Short communication: acute phase proteins in Holstein cows diagnosed with uterine infection. Research in veterinary science. 2013;95(1):269-71.⁾ using a microplate reader (Thermo Plate® TP-Reader, São Paulo, Brazil). The activity of paraoxonase-1 (PON-1) was analyzed by kinetic method through a protocol previously described by Browne et al.,⁽³⁴34 Browne RW, Koury ST, Marion S, Wilding G, Muti P, Trevisan M. Accuracy and biological variation of human serum paraoxonase 1 activity and polymorphism (Q192R) by kinetic enzyme assay. Clin Chem. 2007;53(2):310-7.⁾, and the reading performed in an ultraviolet light spectrophotometer (FEMTO Cirrus 80MB, FEMTO Indústria e Comércio de Instrumentos, São Paulo, Brazil).

Statistical analysis was performed using SAS software version 9.1 (SAS ® Institute Inc., Cary, NC, USA, 2009). Blood concentrations of all metabolites and hemogasometric indices were analyzed by the method of variance (ANOVA) with the MIXED procedure to assess the effect of group and hours and their interactions, Fisher’s exact test to assess the frequency of subclinical hypocalcemia and descriptive statistics to analyze the urinary pH. Statistical difference was considered p<0.05. All results are presented in Mean ± Standard Error of Mean (SEM).

Results

The urinary pH of animals that received DA was 5.9 ± 0.13, while in animals that received DN the pH was 7.4 ± 0.24.

No animal had clinical hypocalcemia during the study. When evaluating the incidence of subclinical hypocalcemia (Cai ≤ 4.0 mg/dL) (5), 71.4% (5/7) of the animals that received DN and 42.9% (3/7) of those that received DA presented the subclinical picture, however, the concentrations of Ca_i (Figure 1A) and Ca_t (Figure 1B) did not differ between groups (p=0.46 and p=0.76, respectively, Table 2).

Figure 1
Means ± standard error of the mean concentration of ionized calcium (A) and total calcium (B) of dairy cows in the first hours after parturition, submitted to neutral or acidogenic diets in the prepartum period. DA -Acidogenic Diet; DN - Neutral Diet; G = Groups; H = hours; G*H = interaction between groups and hours)

Serum sodium levels were higher (144.76 ± 0.30 vs. 143.59 ± 0.30; p=0.01) and TPP concentrations were lower (7.41 ± 0.06 vs. 7. 74 ± 0.06; p<0.01) in animals in the DA group. There was a decrease in GLOB concentrations (4.44 ± 0.07 vs. 4.66 ± 0.07; p=0.01), in blood pH (7.37 ± 0.01 vs. 7.41 ± 0.01; p=0.01) and in the bicarbonate concentration (26.92 ± 0.47 vs. 28.62 ± 0.46; p=0.02) in the animals of the DA group. The PON-1 activity was higher in the animals of the DA group (83.14 ± 3.60 vs. 69.02 ± 3.34 p<0.01), while the haptoglobin levels tended to be lower in these animals (0 .32 ± 0.04 vs. 0.43 ± 0.04; p=0.07).

Discussion

The present study showed metabolic variations in the recent postpartum of dairy cows that received diets with different DCAB in the prepartum period. Santos et al.,⁽¹1 Santos JEP, Lean IJ, Golder H, Block E. Meta-analysis of the effects of prepartum dietary cation-anion difference on performance and health of dairy cows. Journal of dairy science. 2019;102(3):2134-54.⁾ stated that animals with reduced urinary pH have a lower risk of hypocalcemia, however, Goff⁽⁶6 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.⁾ stated that a reduction in urinary pH below 6.2 in Holstein cows for a prolonged period could cause severe metabolic acidosis, reducing the DMI.

Even though no difference was found in the incidence of subclinical hypocalcemia, the DA group had high rates similar to those reported by Seely et al.,⁽³⁵35 Seely C, Leno B, Kerwin A, Overton T, McArt J. Association of subclinical hypocalcemia dynamics with dry matter intake, milk yield, and blood minerals during the periparturient period. Journal of Dairy Science. 2021;104(4):4692-702.⁾, which presented more than half of the animals with the disorder, even with the administration of an acidogenic diet. Among the factors that can influence the prevalence of subclinical hypocalcemia are the number of lactations⁽²³23 Reinhardt TA, Lippolis JD, McCluskey BJ, Goff JP, Horst RL. Prevalence of subclinical hypocalcemia in dairy herds. The Veterinary Journal. 2011;188(1):122-4.⁾, milk yield, breed⁽³⁶36 González F, Corrêa M, Silva S. Transtornos metabólicos nos animais domésticos. Félix H Díaz Gonzalez, Marcío Nunes Corrêa (e) Sérgio Ceroni da Silva-2 Ed-Porto Alegre: UFRGS. 2014:344.⁾, body condition score⁽³⁷37 Roche JR, Friggens NC, Kay JK, Fisher MW, Stafford KJ, Berry DP. Invited review: Body condition score and its association with dairy cow productivity, health, and welfare. Journal of dairy science. 2009;92(12):5769-801.⁾, age and previous hypocalcemia problems⁽³⁸38 Hernández-Castellano LE, Hernandez LL, Weaver S, Bruckmaier RM. Increased serum serotonin improves parturient calcium homeostasis in dairy cows. Journal of dairy science. 2017;100(2):1580-7.⁾, in addition to the low palatability of the salts used, which end up reducing the DMI⁽³⁹39 Moore SJ, VandeHaar MJ, Sharma BK, Pilbeam TE, Beede DK, Bucholtz HF, et al. Effects of altering dietary cation-anion difference on calcium and energy metabolism in peripartum cows. Journal of dairy research. 2000;83(9):2095-104.⁾. The decrease in IMS can further aggravate the disease and also predispose to secondary disorders⁽⁴⁰40 van Knegsel ATM, Van den Brand H, Dijkstra J, Tamminga S, Kemp B. Effect of dietary energy source on energy balance, production, metabolic disorders and reproduction in lactating dairy cattle. Reproduction nutrition development. 2005;45(6):665-88.⁾, it is necessary that the diets are adequate during the period, since when unbalanced, acidogenic diets tend to lower the urinary and blood pH too much, causing decompensated acidosis, compromising the animal organism⁽⁶6 Goff JP. Calcium and magnesium disorders. The veterinary clinics of North America: food animal practice. 2014;30(2):359-81, vi.⁾.

Blood pH can vary from 7.35 to 7.45, and the concentration of bicarbonate, one of the main regulators of acid-base balance, is 19 to 24mmol/L in the serum of animals with balanced pH. When the pH decreases, the bicarbonate concentration tends to decrease as well, capturing H⁺ ions, forming carbonic acid, so that a respiratory compensation occurs, decreasing the pCO₂⁽²2 Hu W, Murphy MR. Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis. Journal of dairy research. 2004;87(7):2222-9.⁾. In the present study, animals in the DA group showed a reduction in blood pH and also in urinary pH, due to acidogenic diets having a high amount of chlorine and sulfur, and these elements tend to decrease the pH of the blood, and consequently of the urine⁽⁴¹41 Goff JP. Pathophysiology of calcium and phosphorus disorders. The veterinary clinics of North America: food animal practice. 2000;16(2):319-37.⁾. Furthermore, during the period evaluated, there was a reduction in the concentration of TPP in this experimental group, which may be due to the reduction in the prolonged extracellular pH that the animals went through, due to the high load of anions present in the diet. This strategy may not be a good option, as it decreases the production of bicarbonate-dependent H⁺ transport proteins, which may lead to a decrease in intracellular pH⁽⁴²42 Schreiber R. Ca 2+ signaling, intracellular pH and cell volume in cell proliferation. The Journal of membrane biology. 2005;205(3):129.⁾, and consequently reduction in protein synthesis (in the transcription process), as described in humans⁽⁴³43 Lecker SH, Goldberg AL, Mitch WE. Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. Journal of the American society of nephrology. 2006;17(7):1807-19.⁾.

When the pH is lowered, the tendency is for the Ca_i to increase in an attempt to help the body’s buffer system. This Cai can be obtained by bone resorption, by the action of parathormone or even by the uncoupling of Cai from proteins, mainly from albumin⁽⁹9 Campion KL, McCormick WD, Warwicker J, Khayat ME, Atkinson-Dell R, Steward MC, et al. Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism. Journal of the American Society of Nephrology. 2015;26(9):2163-71.^,4444 Jackson JA, Hemken RW. Calcium and Cation-Anion Balance Effects on Feed Intake, Body Weight Gain, and Humoral Response of Dairy Calves. Journal of dairy science. 1994;77(5):1430-6.⁾. Although this study did not demonstrate a reduction in the subclinical hypocalcemia incidence in animals from the DA group, nor a change in the plasma concentrations of Ca_i and Ca_t, the reduction in blood pH may help to better mobilize calcium for the homeostasis of the animal’s body.

In this study, animals that received DA showed a reduction in serum globulin levels, which may be associated with improved immunity, since most of the globulins are immunoglobulins⁽⁴⁵45 Russell KE, Roussel AJ. Evaluation of the ruminant serum chemistry profile. Vet Clin North Am Food Anim Pract. 2007;23(3):403-26.⁾. Furthermore, the higher PON-1 activity and lower haptoglobin concentration suggested that the animals in the DA group had a less severe inflammatory state than the DN group. Given the antioxidant potential of PON-1⁽²⁷27 Ferré N, Camps J, Prats E, Vilella E, Paul A, Figuera L, et al. Serum paraoxonase activity: a new additional test for the improved evaluation of chronic liver damage. Clinical chemistry. 2002;48(2):261-8.⁾, animals that received DA may be less susceptible to the deleterious effects of oxidizing substances, maintaining the balance between oxidizing agents and antioxidants. Correlating with the present study, it can be said that oxidative processes have direct damage to all cells, ranging from damage to DNA components, alteration in protein function and intracellular metabolic processes⁽⁴⁶46 Sordillo LM, Aitken SL. Impact of oxidative stress on the health and immune function of dairy cattle. Veterinary immunology and immunopathology. 2009;128(1-3):104-9.⁾, leaving animals more susceptible to disease. This is demonstrated by Schneider et al.,⁽³³33 Schneider A, Corrêa MN, Butler WR. Short communication: acute phase proteins in Holstein cows diagnosed with uterine infection. Research in veterinary science. 2013;95(1):269-71.⁾, who observed that these proteins can be early disease markers, since they show alterations even in the prepartum of animals which developed metritis in the postpartum period.

Although calcium levels were reduced in animals that received DN, no change in plasma levels of Ca_t or Ca_i was observed between groups, and lower serum sodium levels were found in postpartum animals in the DN group. Sodium is an important regulator of plasma calcium levels, which is carried out by the sodium-calcium antiport system, where calcium reabsorbed in the distal tubules of the kidneys and in the gastrointestinal tract is released into the bloodstream by exchange with sodium molecules⁽⁴⁷47 Eaton D, Pooler J. Fisiologia renal de Vander. Porto Alegre - RS - Brasil: Artmed Editora; 2015.⁾. This mechanism is capable of releasing up to 5000 calcium ions per second⁽⁴⁸48 Carafoli E, Santella L, Branca D, Brini M. Generation, control, and processing of cellular calcium signals. Critical reviews in biochemistry and molecular biology. 2001;36(2):107-260.⁾.

Conclusion

This study demonstrated that the use of a diet with high negative DCAB was not able to reduce the incidence of subclinical hypocalcemia, and diets with high DCAB should be further evaluated, however, there was a better antioxidant protection for the body. More studies should be conducted with a larger sample size in order to better evaluate the results.

Acknowledgment

The authors thank the Center for Research, Teaching and Extension in Livestock (NUPEEC/UFPel) for the opportunity; Fazenda 4 Irmãos, located in Rio Grande-RS for the supply of animals; and the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Scientific and Technological Development Council (CNPq) for the financial support of this study.

References

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