Recumbency is a common event in buiatric practice which can occur in all stages of lactation. Consequently, there is a multitude of differential diagnoses that need to be considered in the clinical examination of recumbent cows. Categorisation of affected animals into three groups, where one contains cattle with undisturbed sensorium, another animals with disturbed sensorium, and the last those with disturbed general condition may help to confine potential causes of the disease (5). The first category includes cows with musculoskeletal as well as neural disorders caused by traumatic injuries (
Therefore the goal of this study was to compare clinical chemistry findings in different cases of recumbency in German Holstein cows at > 5 d in milk (DIM) and thus out of the typical risk period for hypocalcaemic paresis. We investigated serum concentration of standard parameters in blood including minerals, metabolites, and enzymes to show differences between recumbent and healthy animals and between various causes of recumbency, and to examine the occurrence of hypocalcaemia outside the risk period of hypocalcaemic paresis.
Serum concentrations of different minerals (magnesium, calcium, iron, sodium, potassium, and inorganic phosphorus (Pi)), metabolites (β-hydroxybutyrate (BHB), total bilirubin, NEFA, urea, and creatinine), and total protein (TP) were determined. Activities of alkaline phosphatase (AP), aspartate aminotransferase (AST), creatine kinase (CK) and γ-glutamyl transferase (GGT) were also measured. Table 1 shows the detection methods for each parameter.
Detection methods for the serum parameters
Parameter | Methoda |
---|---|
Mg, Ca, Pi | photometric determination |
Na, K | ion-sensitive electrode |
Fe | identification with ferrozine (without deproteinisation) |
BHB | UV method |
Bilirubin | according to Jendrassik and Grof (10) |
NEFA, urea | kinetic UV assay |
Creatinine | Jaffe reaction |
AST | optimised standard method of DGKC |
AP, CK | NAC-activated, optimised standard method of DGKC |
γ-GT | according to Szasz (29) |
TP | biuret method |
DGKC – German Society of Clinical Chemistry and Laboratory Medicine
a performed at 37°C with Cobas C311 analyser, Roche Diagnostics GmbH, Germany
For statistical data analysis, IBM SPSS Statistics 25 (IBM, USA) and WinSTAT (R. Fitch Software, Germany) software was used. Continuous data comprised count, mean, standard deviation, median, and minimum and maximum, and were checked for normal distribution using histograms and the Shapiro–Wilk test.
If necessary, data were transformed logarithmically (decadic logarithm) to gain normality. Homogeneity of variances was investigated by the Levene test (27).
In the first step, clinical chemistry values of RDC and CDC were compared by Student’s
Subsequently, the numbers of animals exceeding or falling short of the reference ranges were compared between the groups using cross tabulations and McNemar’s test. The odds ratios (OR) were calculated as described in Table 2 (25).
Example cross table used for the calculation of odds ratios and McNemar’s test
RDC – recumbent dairy cows; CDC – control dairy cows; OR – odds ratio
≠ depending on clinical significance of deflection in recumbent dairy cows
Finally, animals within the RDC group were classified according to diagnosis (1 – adductor muscle injury, 2 – fractures, 3 – other mechanical traumatic lesions of locomotor system, 4 – coliform mastitis, and 5 – miscellaneous (ruminal tympany, cachexia, spinal abscess, tarsitis)). The means of clinical chemistry parameters were compared by using a univariate analysis of variance (ANOVA) for independent samples.
Data were analysed from 64 animals enrolled in this trial. Descriptive statistics of the RDC and CDC groups regarding lactation number, DIM, proportion of gravid cows, as well as day of gestation are presented in Table 3.
Descriptive statistics of study groups
Item | Study groups |
||
---|---|---|---|
RDC group (n = 32) | CDC group (n = 32) | ||
1 (n/%) | 5 (15.6) | 5 (15.6) | |
Number of lactations | 2 (n/%) | 7 (21.9) | 7 (21.9) |
3 (n/%) | 8 (25.0) | 8 (25.0) | |
> 3 (n/%) | 12 (37.5) | 12 (37.5) | |
mean ± SD | 144 ± 110 | 146 ± 111 | |
DIM | min–max | 7–383 | 6–384 |
Proportion of gravid cows | (n/%) | 14 (43.8) | 14 (43.8) |
Day of gestationa | mean ± SD | 95 ± 72 | 89 ± 69 |
RDC – recumbent dairy cows; CDC – control dairy cows; DIM – days in milk
a only gravid animals
A comparison of mean blood serum concentrations between RDC and CDC is shown in Table 4. All mean serum concentrations of minerals except Na were decreased in RDC, whereas K and Pi as well as Fe concentrations in RDC were significantly lower than those in the healthy group. However, we could find hypocalcaemia in three cows of the RDC group (mmol/L: 0.85; 1.70; and 0.97). RDC had significantly higher concentrations of NEFA and bilirubin compared to CDC, while other blood metabolic markers (creatinine, urea) did not differ statistically. Regarding enzymes, in RDC increased AP, AST, CK, and GGT activities were detected, although only CK concentration was significantly different between the two groups. In addition, the mean concentration of BHB was lower and serum TP was non-significantly higher in recumbent than in healthy cows.
Serum concentrations of examined parameters of the study groups
RDC group (n = 32) | CDC group (n = 32) | |||
---|---|---|---|---|
Parameter | Physiological rangea | Mean ± SD | Mean ± SD | P¥ |
Mg (mmol/L) | 0.90–1.32 | 0.94 ± 0.16 | 1.03 ± 0.09 | 0.008 |
Ca (mmol/L) | 2.00–2.54 | 2.32 ± 0.51 | 2.57 ± 0.16 | 0.012 |
Pi (mmol/L) | 1.55–2.29 | 1.50 ± 0.61 | 1.91 ± 0.24 | < 0.001 |
Na (mmol/L) | 135.00–157.00 | 147.31 ± 8.21 | 142.59 ± 5.71 | 0.008 |
K (mmol/L) | 3.90–5.20 | 4.43 ± 0.75 | 5.10 ± 0.77 | < 0.001 |
Fe (μmol/L) | 13.00–33.00 | 11.31 ± 8.15 | 25.06 ± 4.48 | < 0.001 |
BHB (mmol/L) | < 0.62 | 0.47 ± 0.27 | 0.61 ± 0.24 | 0.003 |
Bilirubin (μmol/L) | 3.30–5.30 | 4.24 ± 3.16 | 1.03 ± 0.55 | < 0.001 |
NEFA (μmol/L) | < 400.00 | 509.72 ± 330.15 | 136.88 ± 76.36 | < 0.001 |
Urea (mmol/L) | 2.00–6.80 | 5.06 ± 2.07 | 3.91 ± 0.96 | 0.025 |
Creatinine (μmol/L) | 50.00–150.00 | 70.34 ± 19.67 | 71.05 ± 15.90 | 0.0625 |
AP (U/L) | 40.00–300.00 | 54.19 ± 24.96 | 45.94 ± 13.98 | 0.062 |
CK (U/L) | < 150.00 | 5011.28 ± 13386.53 | 666.44 ± 1645.44 | < 0.001 |
AST (U/L) | < 80.00 | 231.07 ± 207.75 | 92.32 ± 14.96 | 0.020 |
GGT (U/L) | < 50.00 | 36.43 ± 20.45 | 31.14 ± 12.36 | 0.0625 |
TP (g/L) | 68.00–82.00 | 84.36 ± 8.01 | 80.72 ± 6.04 | 0.029 |
RDC – recumbent dairy cows; CDC – control dairy cows
a according to Fürll (9) and Van Winden
¥ Student’s
OR for exceedance or lower deviation of different parameter threshold values are displayed in Table 5. Significantly more recumbent cows exceeded the upper reference value compared to the CDC group only with regard to CK activity.
Odds ratios for exceedance or lower deviation of serum parameter threshold values of the experimental group
RDC group | ||||
---|---|---|---|---|
Parameter deviation | Physiological threshold valuea | OR | 95% CI | P¥ |
Mg ↓ | < 0.90 mmol/L | 3.667 | 1.023–13.143 | 0.092 |
Ca ↓ | < 2.00 mmol/L | 4.429 | 0.467–42.021 | 0.250 |
Pi ↓ | < 1.55 mmol/L | x | ||
Na ↓ | < 135.00 mmol/L | 0.484 | 0.042–5.621 | 1.000 |
K ↓ | < 3.90 mmol/L | x | ||
Fe ↓ | < 13.00 μmol/L | x | ||
BHB ↑ | > 0.62 mmol/L | 0.407 | 0.121–1.369 | 0.227 |
Bilirubin ↑ | > 5.30 μmol/L | x | ||
NEFA ↑ | > 400.00 μmol/L | x | ||
Urea ↑ | > 2.00 mmol/L | x | ||
Creatinine ↑ | > 150.00 μmol/L | xx | ||
AP ↓ | < 40.00 U/L | 0.866 | 0.302–2.481 | 1.000 |
CK ↑ | > 150.00 U/L | 23.059 | 2.778–191.410 | < 0.001 |
AST ↑ | > 80.00 U/L | 5.357 | 0.555–51.709 | 1.000 |
GGT ↑ | > 50.00 U/L | 1.554 | 0.241–10.010 | 1.000 |
TP ↑ | > 82.00 g/L | 2.341 | 0.843–6.353 | 0.210 |
RDC – recumbent dairy cows; OR – odds ratio; CI – confidence interval; x – reference value was not exceeded but was attained in healthy cows; xx – reference value was exceeded in none of the study groups
a according to Fürll (9) and Van Winden
¥ McNemar’s test
When comparing results for blood analysis between the diagnoses within the RDC group, some descriptive differences could be detected (Fig. 1), but they were proven not to be statistically significant. Serum urea concentration was higher in cows suffering from coliform mastitis (group 4), while AST and CK were increased in animals with adductor muscle rupture, in which CK activity reached values above 68,000 U/L, as well as mechanical traumatic lesions of the locomotor system (groups 1 and 3). Cows with adductor muscle injury (group 1) had increased bilirubin as well as NEFA values, and those suffering from coliform mastitis (group 4) also showed an increase in NEFA in their blood. In respect of minerals, Fe was depleted in cases of adductor muscle lesion and coliform mastitis (groups 1 and 4) and there was hypokalaemia in cases of fractures and coliform mastitis (groups 2 and 4). Moreover, adductor muscle injury (group 1) caused lower Pi values. Serum concentrations of selected parameters within the diagnosis groups are displayed in Fig. 1, for which parameters returning means differing significantly between the RCD and CDC groups were chosen for plotting (Table 4).
The main aim of this study was to check the clinical background of blood serum parameter changes in recumbent lactating German Holstein cows outside the risk period for hypocalcaemic parturient paresis in comparison with parameters of healthy control cows. Mean serum concentrations and activities of NEFA, bilirubin, and CK were statistically higher in RDC than in healthy cows, while RDC showed significantly lower concentrations of Fe, K, Pi, and BHB compared to CDC. Significant differences regarding other parameters were not observed.
The degree of mineral deficiencies in RDC was pronounced in the present study, as animals were sampled before the first treatment (
Ca homeostasis is stable for more than 3 d postpartum and thus, primary hypocalcaemia in recumbent dairy cows should be insignificant after this period. Serum Ca concentrations of three cows observed in the RDC group show the presence of subclinical (1.40–2.00 mmol/L) and clinical (< 1.40 mmol/L) hypocalcaemia after the periparturient period (19). The Ca threshold value of 2.00 mmol/L was selected to exclude cows with a decreased serum concentration that is normal in the periparturient period (21). Besides being regulated by 1,25-dihydroxyvitamin D, blood Ca is also regulated by parathyroid hormone (PTH) controlling bone Ca mobilisation as well as renal and intestinal Ca reabsorption (14, 18). Both metabolic alkalosis and hypomagnesaemia are able to impair PTH receptor function. At higher pH values as a consequence of a diet that supplies more cations, the receptor protein may alter its conformation, rendering the tissues less sensitive to PTH. Mg as a ligand of adenylate cyclase and phospholipase C plays a key role in second messenger cascades after PTH has bound to its receptor and is crucial for their full activity. Maintenance of physiological blood Mg concentration is almost exclusively dependent on a constant influx from nutrition. Thus, partial or total anorexia in the cow is frequently accompanied by lower blood Mg values. Clinical signs of hypomagnesaemia such as recumbency combined with convulsions are only seen if blood Mg falls below 0.5 mmol/L (13, 20, 22). The hypocalcaemia observed in the cows of this study most likely arose secondarily, due to a lower feed intake as the reason for hypomagnesaemia, and this can usually be cured by an intravenous Ca infusion. Despite Mg or Ca deficiencies being impossible to allocate to certain diagnoses within the RDC group and seeming not to be the main cause of recumbency in this study, hypocalcaemia merits consideration as a possible cause of recumbency in later lactation. Therefore, attention should be focused on sufficient supply of Mg.
It should be noted that hypocalcaemic cows tend to become hypophosphataemic, because PTH, secreted in response to low blood Ca, increases urinary and salivary loss of Pi (14). Otherwise, foetal Pi demand (especially in twin pregnancies) in late gestation, decreased absorption of salivary Pi due to reduced ruminal motility, an elevated tumour necrosis factor (TNF)-α level, and excessive cortisol secretion can induce hypophosphataemia (2, 14). This might explain the Pi concentrations detected in the RDC group, since it included heavily pregnant cows possibly secreting more cortisol and digesting less by reason of their impaired welfare as recumbent animals.
If recumbent cows are normocalcaemic or are not able to rise 24–48 h after Ca treatment, this condition is termed downer cow syndrome (DCS). Its development varies (13). In the following, various disorders which were detected in this study and which may cause DCS are discussed.
Hypokalaemia observed in RDC in our study can occasionally co-occur with muscle weakness and recumbency. Most cases occur secondarily due to prolonged inappetence, diarrhoea, or gastrointestinal passage disorders; however, this hypokalaemia is unlikely to be severe enough to cause clinical signs of weakness. Renal K excretion is regulated by aldosterone, which is in turn controlled by adrenocorticotropic hormone (ACTH). Aldosterone enhances secretion of K and causes retention of Na. Hypovolaemia and low plasma Na are the most important triggers for its secretion. This might be the reason for the more frequent K loss in cows with fractures as they usually develop circulatory insufficiency. If glucocorticoids, which are sometimes administered to cows as antiphlogistics or to stimulate gluconeogenesis, also have some mineralocorticoid effects they will lead to hypokalaemia by increasing K excretion in the kidneys (14, 23). Previous treatments of the glucocorticoid kind could be ruled out in the present study. Another important point is that there can be disturbances of the hypothalamic-pituitary-adrenal axis and therefore changes in plasma electrolytes during sepsis or endotoxin exposure. While ACTH concentrations were found to be reduced after experimentally induced sepsis in rats, aldosterone was continuously elevated, resulting in a significant K reduction (7). This could be an explanation for the higher occurrence of hypokalaemia in cattle suffering from coliform mastitis, as this disease commonly induces endotoxaemia in these animals.
The majority of blood Fe is bound to transferrin, a negative acute phase protein in cattle. While animals with acute infections have normal transferrin concentrations, chronic infectious diseases were characterised by relatively low transferrin values, which may also induce low Fe concentrations (30). This observation could not be shown in our study, since low serum Fe values were associated with adductor muscle injury and coliform mastitis as diseases of primarily acute character. However, evidence suggests that transport capacity for Fe is reduced in recumbent cows during their acute phase reaction (19). Deficiency of Fe due to continuing inappetence is additionally possible in the present cases. In the same way as for K and Pi deficiency, cows will rapidly restore blood Fe to normal levels if they are consuming feed. Therefore, in tending these animals, treatment should be given for any concomitant or pre-existing problems (23).
Hyperbilirubinaemia detected in RDC results secondarily from elevated NEFA concentrations because NEFA occupy the binding site of transport proteins for bilirubin. In these cows, lipomobilisation is most likely initiated by anorexia (inanition icterus), increased stress caused by the recumbent position, and the inhibiting effects of TNF-α secreted in inflammatory processes on insulin-mediated reduction of lipolysis (8, 15). Concentrations of BHB were within the physiological range for the large part of the RDC group, indicating that these cows did not show subclinical or clinical ketosis. This suggests that ketosis was not important for recumbency in this study.
Prerenal uraemia caused by hypovolaemia and circulatory insufficiency might explain the higher urea concentrations of cows suffering from coliform mastitis within the RDC group. This is consistent with the observation that the endotoxin from
Levels of CK are known to rise 12–24 h after the start of recumbency and can be subject to large fluctuations beforehand (3, 26). Mean CK activity was high in RDC, as expected in our study. The reason for this is affected animals’ long-lasting inability to rise. High CK and AST values are associated with a poor prognosis in recumbent cattle (26). Due to low statistical power, conclusions about cure rates of RDC cannot be drawn in the present work (data not shown). In contrast to the case with CK, the significant rise in the AST activity in cows with muscle injury could be explained by a slower increase and longer half-life of AST after trauma than of CK, reaching maximum concentration in blood after 36–48 h (24).
Other diagnostic measures such as determination of acute phase proteins (haptoglobin and serum amyloid A) or cerebrospinal fluid analysis to identify central nervous system lesions could have helped to find further correlations between RDC and healthy cows (1, 30). It would also have been desirable to include a larger number of cattle in the two groups. However, the comparison of RDC and CDC exposed to equal nutritional and housing conditions should be of interest.
In conclusion, the present study provides information about the clinical background as well as blood serum concentrations of different clinical chemistry parameters in recumbent and healthy lactating dairy cattle outside the risk period for hypocalcaemic paresis. Recumbency > 5 DIM has mainly traumatic and inflammatory causes and we could only show additional diagnostic (