Electrolyte Concentrations and Blood Gas Values in Neonatal Calves With Diarrhea

Portable blood analyzers, which recently have been introduced to veterinary medicine, can facilitate immediate identication of sick calves in livestock farms. However, no appropriate standard values exist for neonatal calves; therefore, reference values for adult cattle guide diagnosis and treatment of newborn calves. Our goal was to determine electrolyte, blood chemistry, and blood gas values from healthy calves and compare them to those for diarrheic calves, thus providing useful information for diagnosis and prognosis. We evaluated 193 calves (£1 month old), including those with (n = 88) and without diarrhea (n = 105), using two-tailed, independent t tests after determining normality (Shapiro−Wilk test). Electrolyte measurements in the diarrheic calves included signicant decreases in sodium and signicant increases in potassium, chloride, and blood urea nitrogen. Strong ion difference (SID), pH, bicarbonate, partial pressure of carbon dioxide, and base excess (BE) were signicantly lower in the diarrheic calves (p < 0.001); the anion gap (AG) was signicantly higher among diarrheic calves aged 1-10 days (p < 0.001) compared to healthy calves. Our results demonstrate that SID, pH, bicarbonate, and BE correlated strongly with metabolic acidosis, suggesting that these indicators, including AG, can be important tools for evaluating calves’ health status and for providing useful information to diagnose diarrhea.


Background
Currently, studies on calves focus primarily on epidemiological surveys of disease outbreaks. Among these outbreaks, neonatal calf diarrhea is associated with high mortality rates worldwide, resulting in severe economic losses to cattle producers due to the costs associated with medical treatment as well as to animal weakness and retarded growth [1][2][3][4][5]. Most diarrheic calves become ill before weaning. Many studies performed in the Republic of Korea have focused mainly on identifying pathogens associated with diarrheal illness [3,[6][7][8]. However, etiologies associated with diarrheal illness in calves are diverse and complex; disease may be secondary to infection, malnutrition, or environmental factors [5].
Diarrhea can lead to either the loss or the accumulation of water and major electrolytes; these ndings have speci c impact in newborn calves (≤ 1 month of age), as their dysregulation may ultimately be fatal [9][10][11]. Secretory, exudative diarrhea is associated with release of various ions in the plasma; of note, loss of HCO 3 − results in profound acid − base disturbances [12]. If this situation persists, a decrease in strong ion difference (SID) develops, notably caused by acidosis and acidemia, resulting in physical deterioration. Therefore, it is imperative to rapidly and accurately analyze the changes in SID that re ect the overall severity of the disease [13].
Moreover, to treat and manage diseased calves with maximum e ciency, biometric information that can accurately determine animal health status is needed, together with a way to diagnose any infectioncausing pathogens. However, given the weight and volume of test equipment, these methods are currently underutilized in industrial (i.e., large) animal elds. In recent years, portable blood analyzers have become available; this equipment (e.g., i-STATs) can provide test results from blood samples within a few minutes. Widespread use of these devices has facilitated immediate identi cation of sick calves in livestock farms [14,15]. Thus, the objectives of this study were to analyze the changes in the SID in evaluations of electrolytes, blood chemistry, and blood gas pro les in both healthy and diarrheic calves so as to provide useful information for diagnosing sick calves, which ultimately may direct current clinical practices in the livestock industry.

Animal ethics
All animal procedures were conducted according to ethical guidelines for the use of animal samples, and were approved by the Jeonbuk National University (Institutional Animal Care and Use Committee Decision No. CBNU 2020-052). All procedures and possible consequences were explained to the managers of the surveyed farm, and written consent was obtained.

Sample collection
This study included 193 Korean beef calves (≤ 30 days old) raised in the Republic of Korea. The calves were divided into three groups by age: those 1−10 days of age (n = 53), 11−20 days (n = 65), and 21− 30 days (n = 75; Table 3). Blood samples were collected from all animals through the jugular vein and were placed on a bottle roller and continuously agitated for at least 20 seconds to prevent formation of microclots. Fecal samples were collected after rectal stimulation to induce bowel movements; these samples were transported to the laboratory under refrigeration for examination. We examined the samples and subdivided them into solid, semisolid, loose, and watery; the solid and semisolid vs. loose and watery samples were classi ed as normal feces and as diarrhea, respectively. We also conducted a physical examination using the skin-tenting test and determining the degree of dehydration. Diarrheic calves in this study tested positive to one or more of the pathogens associated with diarrhea; none of these pathogens were detected in fecal samples from healthy calves. In this study, cases of diarrhea were not subdivided into bacterial, viral, and parasitic aetiologies.

Electrolyte, chemistry, and blood gas analyses
Blood parameters were measured in the eld immediately after obtaining fecal samples using the EC8 + i-STAT cartridge (Abbott, Princeton, NJ, USA). Anion gap (AG) and base excess (BE) values were calculated from the concentrations of the major electrolytes Na + , K + , Cl − , and HCO 3 − . The SID was calculated based on the combined electrolyte concentrations; SID = ([Na + ] + [K + ]) -[Cl − ]. Blood urea nitrogen (BUN) and glucose levels were evaluated in serum; pH, and partial pressure of CO 2 (pCO 2 ) were also measured.

Statistical analysis
Statistical analysis was performed using the SPSS Statistics 25 software package for Windows (SPSS Inc, Chicago, IL, USA). Results are presented as mean ± standard error of the mean (SEM). Differences in blood test results were compared using two-tailed independent t-tests after documenting normality (Shapiro-Wilk test). Multiple linear regression analysis with stepwise exclusion was used to test the association between the age of calves and clinicopathological variables. In addition, the differences in the clinicopathological variables between the two groups were determined using multivariate logistic regression analysis. Odds ratio (OR) and 95% con dence intervals (CI) were calculated to determine the likelihood of diarrhea association. A p-value of less than 0.05 was considered statistically signi cant.

Results
We clinically determined the presence of diarrhea by physical examination, including the skin-tenting test, stool status, body temperature, and degree of dehydration. Also, CBC (Hct, RBC, Hb) and chemistry data (e.g., serum protein) tests were also performed to determine grouping of the healthy and diarrheic calves (data not shown). In particular, the values of Hct and serum protein in the diarrheic calves were signi cantly increased compared with those of the healthy calves.
Seven pathogens were detected from the 88 diarrheic calves (   p values were obtained using two-tailed, independent t-tests comparing results from healthy and diarrheic calves ( a p < 0.05, b p < 0.01, c p < 0.001). Na + : sodium; K + : potassium; Cl − : chloride; BUN: blood urea nitrogen; AG: anion gap SID was signi cantly lower among the diarrheic calves at all ages compared with the healthy calves (p < 0.001); this difference was most signi cant in the diarrheic calves aged 21−30 days (p = 0.0001; Fig. 1). A decrease in SID was among the most signi cant contributors to the changes observed in pH and bicarbonate (HCO 3 − ) concentration. The mean pH value in blood differed little among the healthy calves of all ages; however, blood pH was signi cantly lower among all diarrheic calves when compared to their healthy counterparts (p < 0.001; Fig. 1). Furthermore, HCO 3 − concentrations were also signi cantly lower in all age groups of the diarrheic calves compared to the healthy calves (p < 0.001; Fig. 1). Interestingly, the values calculated for AG decreased with age in both the healthy and diarrheic calves. The values of AG were generally high in the diarrheic calves and were signi cantly higher among those aged 1−10 days (p < 0.01;  Fig. 1).

Discussion
Diarrhea is a major problem in calves less than one month of age and is of tremendous economic importance. Diarrhea results in electrolyte imbalance, dehydration, and metabolic acidosis [16][17][18][19][20]; however, only a few studies available have focused on acid−base balance, serum chemistries, and blood gas parameters associated with diarrhea caused by infectious agents in neonatal calves [21,22]. Unfortunately, because the information currently available for healthy young Korean native calves is not well documented, reference values for adult cattle have commonly been used to direct therapy. In our study, we examined electrolytes, serum chemistries, and blood gas parameters in both healthy and diarrheic calves through rapid and accurate eld testing. The results showed that SID, pH, bicarbonate, and BE were signi cantly decreased, whereas BUN and AG were signi cantly increased in diarrheic calves compared with healthy calves. As such, these parameters provide both critical and accurate information for treatment and prognosis.
Our ndings revealed signi cant changes in plasma electrolyte concentrations (Na + , K + , and Cl − ) that varied according to diarrheic calf age. Our results documented an overall decrease in sodium and overall increase in potassium, chloride, and BUN concentrations in diarrheic calves when compared with healthy calves. The decrease in sodium concentration can be explained by increased intestinal loss [23]; this situation results in a concomitant decrease in plasma sodium concentration accompanied by an increase in chloride concentration.
Moreover, the signi cant decrease in SID values was identi ed in the diarrheic calves; this nding can be explained by the combination of hyponatremia and hyperchloremia [11]. The decrease in SID represents the key role played by hyponatremia in this setting, which can be attributed to excessive loss of sodium into the gastrointestinal tract as well as decreased milk intake due to anorexia; the combination of these factors results in acidemia and strong ion acidosis [24,25]. As such, this study highlights the role of strong ions in the acid−base status in diarrheic calves.
In addition, we detected increases in potassium concentration in diarrheic calves. Hyperkalemia has clinically been associated with acidemia and decreased intracellular pH due to electrolyte imbalance [20,26]. While we did not include the results of packed red cell volume in these neonatal calves, we did note signi cant dehydration among the diarrheic calves aged 1−10 days, a nding that explains the hyperkalemia observed among diarrheic calves. Interestingly, BUN levels were signi cantly higher in the diarrheic calves when compared with the healthy calves, ndings that are consistent with previous results [27][28][29]. The signi cant increase in BUN at all age groups was associated with a decreased glomerular ltration rate due to dehydration; this also contributed to the acidemia and metabolic acidosis observed [30]. Taken together, our results suggest that potassium and BUN can be used as appropriate measurements for evaluating the severity of diarrhea in calves.
Blood gas analysis also provides valuable information for diagnosis, prognosis, and identi cation of therapeutic options [31]. In this study, we observed signi cant alterations in acid-base homeostasis. Of particular note, bicarbonate levels were signi cantly lower among diarrheic calves compared to their healthy counterparts. This nding is closely associated with reductions in sodium and increases in potassium concentrations. The signi cant decline in bicarbonate in diarrheic calves is indicative of severe metabolic acidosis; these ndings were associated with a marked decrease in blood pH as would be anticipated for acidotic calves [32]. Our results reveal a correlation between pH and animal health and suggest that blood pH may be a useful indicator of clinical status of diarrheic calves.
We found that blood gas analysis is an important indicator for determining the prognosis of and setting treatment policy for calves. In particular, calves with severe diarrhea showed a pH of less than 7.1, a Likewise, values calculated for AG were higher in the diarrheic calves than in the healthy ones. Changes in AG may be attributed to variations in unidenti ed strong anions that include D -lactate, ketones, phosphate, and inorganic anions [25,33]. Although we cannot draw speci c conclusions because concentrations of these biological acids were not measured directly in this study, we speculate that their levels may be increased in diarrheic calves [33]. In addition, acidemia in hyperkalemic diarrheic calves was associated with a decrease of sodium and increase of AG [34].
Finally, BE values were signi cantly reduced in the diarrheic calves, which may contribute to the estimation of the degree of metabolic acidosis and clinical status. Of note, all calves evaluated in this study were able to stand and suck at feeding time but preferred to remain in sternal recumbency. This may explain the signi cant correlation among BE values, levels of dehydration, and metabolic acidosis. Consequently, on the basis of our results, we propose that calf health may be determined by blood pH, bicarbonate levels, and BE as opposed to the other parameters measured.
A limitation of our research is that we did not perform continuous follow-up studies with the diarrheic calves, and we could not exactly predict the factors associated with death in those with severe diarrhea.
However, we believe it is meaningful to establish the biochemical and blood gas values of neonatal Korean native calves according to age and to compare these values with healthy and severe diarrheic calves, as we did in this study.

Conclusion
In conclusion, our ndings reveal that metabolic acidosis in diarrheic calves is directly associated with profound decreases in pH, bicarbonate, and BE; these values are accompanied by hyponatremia, hyperkalemia, hyperchloremia, and increases in BUN and AG. As such, our results provide useful information for the accurate diagnosis, treatment, and management of calves with diarrhea.  Figure 1