This study evaluated the reliability of various blood parameters to assess the ruminal acidosis in cattle. Six whole heifers were fed three experimental rations in a 3 x 3 Latin square design. The diets had different starch levels: high (HS), medium (MS) or low (CT). Ruminal pH values were continuously measured using wireless sensors. To evaluate the severity of ruminal acidosis, the amount of time per day that the pH was below 5.8, 5.5 and 5.0 was recorded. Blood samples were analyzed for complete blood count, venous blood gas and biochemical profile at 8:00 and 12:00 h. The data were analyzed according to a mixed model. Feeding on CT, MS and HS led to significant differences in DMI (7.7 vs. 6.9 vs. 5.1 kg/d; P < 0.01) which modified the amount of time per day that the pH was below 5.0 (0 vs. 12 vs. 92 min; P < 0.10). Feeding MS and HS diets led to inflammation as indicated by the significant increment of white blood cells when compared to the CT ones and to blood concentration due to the osmotic pressure at ruminal level. Furthermore a significant decrease of bicarbonate level, CO2 partial pressure and oxyhemoglobin was observed as consequence of the activation of metabolic processes aimed to prevent metabolic acidosis. No differences were observed on blood sampling time, suggesting that one daily blood sample was enough to evaluate the metabolic variations related to ruminal acidosis

Barbara Contiero

Filomena Montemurro

Giorgio Marchesini

Igino Andrighetto

Matteo Gianesella

Rebecca Ricci

Roberta De Nardi

Severino Segato

English

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259Agriculturae Conspectus Scientifi cus . Vol. 78 (2013) No. 3 (259-262)ORIGINAL SCIENTIFIC PAPERSummaryTh is study evaluated the reliability of various blood parameters to assess the ruminal acidosis in cattle. Six whole heifers were fed three experimental rations in a 3 x 3 Latin square design. Th e diets had diff erent starch levels: high (HS), medium (MS) or low (CT). Ruminal pH values were continuously measured using wireless sensors. To evaluate the severity of ruminal acidosis, the amount of time per day that the pH was below 5.8, 5.5 and 5.0 was recorded. Blood samples were analyzed for complete blood count, venous blood gas and biochemical profi le at 8:00 and 12:00 h. Th e data were analyzed according to a mixed model. Feeding on CT, MS and HS led to signifi cant diff erences in DMI (7.7 vs. 6.9 vs. 5.1 kg/d; P < 0.01) which modifi ed the amount of time per day that the pH was below 5.0 (0 vs. 12 vs. 92 min; P < 0.10). Feeding MS and HS diets led to infl ammation as indicated by the signifi cant increment of white blood cells when compared to the CT ones and to blood concentration due to the osmotic pressure at ruminal level. Furthermore a signifi cant decrease of bicarbonate level, CO2 partial pressure and oxyhemoglobin was observed as consequence of the activation of metabolic processes aimed to prevent metabolic acidosis. No diff erences were observed on blood sampling time, suggesting that one daily blood sample was enough to evaluate the metabolic variations related to ruminal acidosis.Key wordsdairy cattle, starch level, ruminal acidosis, blood parametersBlood Parameters Modifi cation at Diff erent Ruminal Acidosis Conditions Roberta DE NARDIGiorgio MARCHESINIMatteo GIANESELLARebecca RICCIFilomena MONTEMURROBarbara CONTIEROIgino ANDRIGHETTOSeverino SEGATO ( )Dipartimento di Medicina Animale, Produzioni e Salute, Università degli Studi di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy e-mail: severino.segato@unipd.itReceived: April 9, 2013 | Accepted: June 17, 2013ACKNOWLEDGEMENTS The research was financially supported by FONDAZIONE CARIVERONA (call 2010) and the project n. 60A08-5402/13 of Padova University.Agric. conspec. sci. Vol. 78 (2013) No. 3260 Roberta DE NARDI, Giorgio MARCHESINI, Matteo GIANESELLA, Rebecca RICCI, Filomena MONTEMURRO, Barbara CONTIERO, Igino ANDRIGHETTO, Severino SEGATOAimRuminal acidosis is a metabolic disease in cattle fed high starchy concentrate diets and it has been defi ned as a disorder associated with low ruminal pH (Plaizier et al., 2009). Th e drop of ruminal pH is not only infl uenced by the amount of starch ingested, but also by the starch source (e.g., maize, wheat or barley) and its particle size (De Nardi et al., 2013). Although clinical signs are not always obvious, ruminal acidosis aff ects dry matter intake (DMI) and productions, can cause laminitis and other disorders resulting in substantial economic losses in dairy farming (Corato et al., 2005; Calsamiglia et al., 2012). Although several parameters were investigated to help in the di-agnosis of ruminal acidosis (Gozho et al., 2007; Enemark et al., 2008; Marchesini et al., 2011) only few authors have extensively investigated the eff ect of ruminal acidosis and sampling time on blood variables (Brown et al., 2000; Marchesini et al., 2013). Th e aims of this study were to identify the blood variables change in heifers aff ected by diff erent levels of ruminal acido-sis and to evaluate the variation of these parameters at diff er-ent sampling times.Materials and methods  Animals and Experimental Design - According to a 3 x 3 Latin square arrangement, six crossbred Valdostana x Belgian Blue non-pregnant heifers were randomly assigned to 1 of 3 di-etary treatments. Each experimental period lasted 5 days and it was followed by a 2 weeks washout period during which the animals were fed the control diet (CT) ad libitum. Dietary treatment - During the experimental period, heif-ers received one of three total mixed rations (TMR) charac-terized by diff erent starch levels (% DM): control (CT) as low starch diet (17.3%), medium starch (MS) for SARA (33.4%) and high starch (HS) to induce acute ruminal acidosis (42.8%). Th e animals were individually restricted and fed three times a day at 8:00, 12:00 and 18.00 h. Heifers were off ered 10 kg of their ration at each meal, and the feed that was not consumed was removed and weighed. Acidosis Challenge Model - Each experimental period was preceded by 3 baseline days (pre-challenge days d-3, -2, -1) in which the heifers had access to the CT TMR three times a day. On the day before the challenge (restricted feeding day, d0), the feed was restricted to two meals a day (8:00 and 12:00 h) with a consequent reduction of DMI. On d1, the HS, MS and CT diets were fed to induce acute acidosis or subacute acidosis or to main-tain the physiological ruminal pH, respectively. On the follow-ing three days (d2, d3 and d4), all the animals were fed the CT diet three times a day.Ruminal pH - Th e ruminal pH of each heifer  was measured every 10 min during the entire trial using KB1001 wireless sensors (Kahne Limited, Auckland, New Zealand). As reported by other authors (Dohme et al., 2008; Marchesini et al., 2013), to measure the severity of ruminal acidosis, the amount of time per day that the pH was below three ruminal pH thresholds (pH < 5.0; 5.0 ≤ pH < 5.5 and 5.5 ≤ pH < 5.8) was determined for each heifer. Blood collection and analysis - Blood samples (20 mL) were taken from the jugular vein from each animal at 8:00 and 12:00 h on each experimental day immediately before the meal. Th e blood was analyzed for complete blood cell count (Cell Dyn 3500, Abbott Laboratories. Abbott Park, Illinois, USA), blood gas analysis (Synthesis 15, IL Instrumentation Laboratory SpA, Milano, Italy), urea and glucose (Roche Diagnostics, Indianapolis, IN, USA).Statistical Analysis - Sample distribution normality was as-sessed with the Shapiro-Wilk test. Th ose variables that showed W < 0.95 were log-transformed (natural logarithm) to meet par-ametric assumptions. However, data of normalized variables are presented in the results section as raw LSmeans. Th e associated P-values were calculated from the transformed data with the fol-lowing model. Th e blood pH, count, gas analysis, and plasma he-matological profi le data were analyzed using a mixed procedure with a CS (compound symmetry) structure. Th e linear model included fi xed eff ect of dietary treatment, period, day, daily time, and their interactions. Heifer was considered a random eff ect and day a repeated measure. In the case of DMI, data were analyzed with the same model but without the daily time eff ect and its interactions. Th e degrees of freedom of D eff ect were used in the two orthogonal contrasts: D1, CT vs. (MS and HS)/2; D2, MS vs. HS. Th e average amount of time for each heifer with a pH below the three established pH thresholds (< 5.0; between 5.0 and 5.5; between 5.5 and 5.8) were not normally distributed, thus they were tested using the non-parametric Kruskal-Wallis criteria to discriminate among the dietary treatments. Signifi cance was declared at P < 0.05, however a trend was considered to exist if 0.05  P < 0.10. All of the statistical analyses were performed using SAS (2008; release 9.2). Results and discussionDMI was signifi cantly aff ected by the treatment resulting the highest in CT-fed heifers (7.7, 6.9 and 5.1 kg/d DM; P < 0.01 both in D1 and D2).  Figure 1 shows the ruminal pH drop caused by the rapid fermentation due to both the high amount of starch in-gested and the small (0.5 mm) mean maize particle size (Plaizier et al., 2009). As expected, the ruminal pH of the animals fed the CT diet tended to spend the lowest (P < 0.10) time between 5.0 and 5.5 and never registered values lower than 5.0 (Figure 1). Figure 1. Mean time spent daily below three ruminal pH thresholds (a, b Means of time per day below pH 5.0 with different superscripts are different (P < 0.05); α, β Means of time between pH 5.0 and 5.5 with different superscripts are different (P < 0.10))Agric. conspec. sci. Vol. 78 (2013) No. 3261Blood Parameters Modification at Different Ruminal Acidosis ConditionsCompared to CT, MS and HS treatments led to an increase of white blood cells (WBC) and platelets (Table 1). Among WBC, both neutrophils and eosinophils increased, whereas mono-cytes and basophils decreased. Dong et al. (2011) reported that the early hours following grain engorgement are characterized by the rapid growth of Gram-negative bacteria, which undergo cell lysis and lipopolysaccharides (LPS) release following a re-duction in the ruminal pH. Th e translocation of LPS from the digestive tract to the bloodstream leads to the activation of the systemic immune response that is responsible of the increase of neutrophils and eosinophils. Th e increase of platelets might rep-resent the response to the onset of ruminal lesions consequent to ruminal acidosis (Steele et al., 2009).Th e drop in HCO3- level in the MS and HS diet is part of the buff ering mechanism to contrast the incoming of metabolic acidosis as a result of ruminal acidosis (González et al., 2012), whereas the reduction in the partial pressure of carbon diox-ide (pCO2) was due to the compensation of the metabolic acid–base imbalance by the respiratory tract, which lowers the pCO2 to prevent the drop in blood pH through increased ventilation (Dehkordi and Dehkordi, 2011). Signifi cantly lower HCO3- and higher platelets values were observed in the HS treatment when compared to the MS one (Table 1), because in the former the ruminal acidosis resulted more severe (Figure 1). Th e HS diet led also to signifi cantly higher concentrations of red blood cells (RBC), hemoglobin (HGB) and hematocrit (HCT), due to the high ruminal osmotic pressure that pulls fl uid from plasma into the rumen and concentrates the blood components during ru-minal acidosis. Moreover, during the HS treatment, excess or-ganic acids that accumulated in the rumen were absorbed into the bloodstream at the risk of overwhelming the bicarbonate buff ering system (González et al., 2012). Th is caused a shift  in  Dietary treatment SEM Probability CT MS HS D1 D2 TDaily Time 8 12 8 12 8 12   pH 7.39 7.39 7.39 7.39 7.39 7.39 0.004 ns ns nsRed blood cells (1012/L) 8.0 7.9 7.6 7.7 8.2 8.0 0.16 ns * nsWhite blood cells (109/L) 8.5 9.0 9.8 10.1 9.5 9.7 0.42 ** ns ns§Neutrophils (109/L) 2.9 3.1 3.8 4.0 3.8 3.9 0.28 ** ns nsLymphocytes, (109/L) 4.6 4.8 5.0 5.2 4.8 4.9 0.34 ns ns nsMonocytes (109/L) 0.84 0.93 0.82 0.82 0.72 0.75 0.068 ** * nsBasophils (109/L) 0.09 0.10 0.08 0.09 0.07 0.07 0.010 * ns ns§Eosinophils (109/L) 0.04 0.04 0.06 0.06 0.13 0.11 0.028 † † nsHemoglobin (g/dL) 11.1 11.0 10.7 10.8 11.4 11.2 0.16 ns * nsHematocrit (%) 33.8 32.9 32.8 32.7 34.1 33.3 0.51 ns * nsPlatelets (K/μL) 503 519 477 516 601 607 42.8 † ** nspCO2 (mmHg) 52.0 51.0 50.4 50.8 50.4 49.6 0.42 * ns nspO2 (mmHg) 62.1 61.1 71.3 67.8 67.4 64.6 4.26 ns ns nsHCO3- (mmol/L) 32.1 31.4 31.2 31.0 30.6 30.3 0.27 ** † nsOxyhemoglobin (%) 87.2 85.5 87.7 88.1 85.4 83.6 1.24 ns * nsReduced hemoglobin (%) 10.9 11.8 11.1 10.2 13.7 13.7 0.92 ns * nssO2m (%) 89.4 88.3 90.0 90.9 87.0 85.8 1.12 ns * nsUrea (mmol/L) 5.32 5.57 4.28 4.24 3.98 3.93 0.122 ** ** nsGlucose (mmol/L) 4.34 4.34 4.37 4.41 4.32 4.36 0.08 ns ns nsCT, control diet; MS, medium starch diet; HS, high starch diet. D1: orthogonal contrast CT vs (MS + HS)/2; D2: orthogonal contrast MS vs HS. The interaction DxT was never significant. **: P < 0.01; *: P < 0.05; †: P < 0.10. §Data are presented as raw LSmeans, and the associated P-values are given by statistical analysis of the log-transformed data. pCO2, partial pressure of CO2; pO2, partial pressure of O2; HCO3−, bicarbonate level; sO2m, measured oxygen saturation. Table 1. Eff ect of dietary treatment (D, n = 18) and daily time (T, n = 36) on blood pH, count, gas and hematological profi leFigure 2. Mean ruminal pH trend at the time of blood collection (CT = control, black continuous; MS = medium starch, black dashed; HS = high starch, gray continuous. Arrows = time of blood collection and feeding (8:00 and 12:00 h))Agric. conspec. sci. Vol. 78 (2013) No. 3262 Roberta DE NARDI, Giorgio MARCHESINI, Matteo GIANESELLA, Rebecca RICCI, Filomena MONTEMURRO, Barbara CONTIERO, Igino ANDRIGHETTO, Severino SEGATOthe oxyhaemoglobin (O2Hb) dissociation curve (Jones, 2010) and the red blood cells released oxygen to the tissues more readily, which increased the reduced hemoglobin (RHb) and lowered the O2Hb and the measured oxygen saturation (sO2m). Th e urea levels decreased signifi cantly from CT to HS, in line with the crude protein intake.Sampling time had no eff ect on any considered blood vari-ables (Table 1). Th is result could be explained by the fact that the second blood sampling occurred 4 h aft er the main meal of the day and, as reported in Figure 2, at 12:00 h the ruminal pH showed a reduction of only 0.4 units and had not reached its lowest value yet. Th e time interval between the main daily meal and the moment in which the ruminal pH reaches its lowest value (nadir pH) is variable and it is related to the feed distribution management. In this study the nadir pH was reached at 21:00 h, 13 h aft er the main daily meal.     ConclusionsTh e severity of ruminal acidosis, measured as the amount of time spent daily below three pH thresholds, aff ected many of the blood variables that are the expression both of the infl ammation and the osmotic pressure at ruminal level and of the activation of metabolic processes aimed to prevent the metabolic acidosis. Variables like RBC, WBC, HCT, platelets, pCO2, HCO3-, O2Hb and sO2m should be taken into account to investigate on ruminal acidosis in cattle, even though additional studies are necessary to confi rm their reliability. Th e blood sampling time did not aff ect the results suggesting that one blood daily sample is enough to identify the metabolic condition related to the ruminal acidosis.References Brown M. S., Krehbiel C. R., Galyean M. L., Remmenga M. D., Peters J. P., Hibbard B., Robinson J., Moseley W. M. (2000). Evaluation of models of acute and subacute acidosis on dry mat-ter intake, ruminal fermentation, blood chemistry, and endo-crine profi les of beef steers. J Anim Sci 78: 3155-3168.Calsamiglia S., Blanch M., Ferret A., Moya D. (2012). Is subacute ruminal acidosis a pH related problem? Causes and tools for its control. Anim Feed Sci Technol 172: 42-50.Corato A., Segato S., Andrighetto I. (2005). Eff ects of extruded corn on milk yield and composition and blood parameters in lactat-ing dairy cow. Ital J Anim Sci 4(3): 166-168.Dehkordi A. J., Dehkordi Z. K. (2011). Occurrence of metabolic alkalosis in rumen lactic acidosis: A review article. Comp Clin Path 20: 1-3.De Nardi, R., Marchesini G., Stefani A-L., Barberio, A., Andrighetto, I., Segato., 2013. Eff ect of feeding fi ne maize par-ticles on the reticular pH, milk yield and composition of dairy cows. J Anim Physiol Anim Nutr in press (DOI: 10.1111/jpn.12099).Dohme F., DeVries T. J., Beauchemin K. A. (2008). Repeated rumi-nal acidosis challenges in lactating dairy cows at high and low risk for developing acidosis: Ruminal pH. J Dairy Sci 91: 3554-3567. Dong G., Liu S., Wu Y., Lei C., Zhou J., Zhang S. (2011). Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows: Impacts on immunity and metabolism. Acta Vet Scand 53(1): 48.Enemark J. M. D. (2008). Th e monitoring, prevention and treatment of subacute ruminal acidosis (SARA): A review. Vet J 176(1): 32-43.Gozho G. N., Krause D. O., Plaizier J.C. (2007). Ruminal lipopoly-saccharide concentration and infl ammatory response during grain-induced subacute ruminal acidosis in dairy cows. J Dairy Sci 90(2): 856-866.González L. A., Manteca X., Calsamiglia S., Schwartzkopf-Genswein K. S., Ferret A. (2012). Ruminal acidosis in feedlot cattle: Interplay between feed ingredients, rumen function and feeding behavior (a review). Anim Feed Sci Technol 172: 66-79.Jones M. B. (2010). Basic interpretation of metabolic acidosis. Crit Care Nurse 30(5): 63-69.Marchesini G., Segato S., Berzaghi, P., Andrighetto I. (2011). Eff ect of non-forage roughage replacement on feeding behaviour and milk production in dairy cows. Ital J Anim Sci 10(4): 171-175.Marchesini G., De Nardi R., Gianesella M., Stefani A-L., Morgante M., Barberio A., Andrighetto I., Segato S. (2013). Eff ect of induced ruminal acidosis on blood variables in heifers. BMC Vet Res 9: 98.Plaizier J. C., Krause D. O., Gozho G. N., McBride B. W. (2009). Subacute ruminal acidosis in dairy cows: Th e physiological causes, incidence and consequences. Vet J 176(1): 21-31. Steele M. A., AlZahal O., Hook S. E., Croom J., McBride B. W. (2009). Ruminal acidosis and the rapid onset of ruminal par-akeratosis in a mature dairy cow: A case report. Acta Vet Scand 51(1): 39.acs78_44

Blood Parameters Modification at Different Ruminal Acidosis Conditions

This study evaluated the reliability of various blood parameters to assess the ruminal acidosis in cattle. Six whole heifers were fed three experimental rations in a 3 x 3 Latin square design. The diets had different starch levels: high (HS), medium (MS) or low (CT). Ruminal pH values were continuously measured using wireless sensors. To evaluate the severity of ruminal acidosis, the amount of time per day that the pH was
below 5.8, 5.5 and 5.0 was recorded. Blood samples were analyzed for complete blood count, venous blood gas and biochemical profi le at 8:00 and 12:00 h. The data were analyzed according to a mixed model. Feeding on CT, MS and HS led to significant differences in DMI (7.7 vs. 6.9 vs. 5.1 kg/d; P < 0.01) which modifi ed the amount of time per day that the pH was below 5.0 (0 vs. 12 vs. 92 min; P < 0.10). Feeding MS and HS diets led to infl ammation as indicated by the signifi cant increment of white
blood cells when compared to the CT ones and to blood concentration due to the osmotic pressure at ruminal level. Furthermore a significant decrease of bicarbonate level, CO2 partial pressure and oxyhemoglobin was observed as consequence of the activation of metabolic processes aimed to prevent metabolic acidosis. No differences were observed on blood sampling time, suggesting that one daily blood sample was 
enough to evaluate the metabolic variations related to ruminal acidosis

Blood Parameters Modification at Different Ruminal Acidosis Conditions

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