Postpartum retention of the placenta is an important problem in the dairy industry because of the direct and indirect economic losses it can cause and because it predisposes cows to other diseases.1,2 The several adverse reproductive effects of RP include longer intervals to first luteal activity, first estrus, and first breeding; lower probability of conception at first breeding; and longer interval to conception.3
In the United States, surveys4 conducted as part of the National Animal Health Monitoring System indicate that between 1995 and 2001, RP was the third most common health disorder in dairy cows, affecting 7.8% of lactating cows. Several factors that predispose cows to RP have been identified, with the most common clinical events being stillbirth, dystocia, twinning, and parturient paresis.5 In addition, RP is associated with impaired immune function in the 1 to 2 weeks before parturition.6–8
Some routinely measured biochemical and hematologic analytes may be useful in the preparturition period to predict the development of RP in dairy cows. In 1 study,9 a low serum concentration of cholesterol and high concentration of FAs in the preparturition period were identified in cows that subsequently developed RP. Other researchers detected a greater decrease in serum cholesterol concentration and increase in FAs concentration during the transition period (ie, the period between 3 weeks before parturition and 3 weeks after parturition) in cows that developed RP.10 The purpose of the study reported here was to identify biochemical and hematologic variables, as measured in the week before parturition, that predict development of RP in dairy cows.
Materials and Methods
Animals—Data used in the study were obtained as the result of a clinical trial11 of the effects of vitamin E on the health status of dairy cows in the transition period. Briefly, 1,038 randomly selected Holstein cows from 20 herds in southern Ontario were visited weekly by a technician from September 1998 through October 1999, at the same time on the same day each week, within 2 hours after the morning feeding. Postpartum retention of the placenta was defined as failure of a cow to expel the fetal membranes by 24 hours after parturition and was recorded by the same attendant in each herd. Owner consent for participation in the clinical trial was obtained, and the protocol of that study adhered to the Animal Care Policy and Procedures of the University of Guelph.
Sample collection and analysis—When cows were within 10 days before the expected parturition date, a 10-mL blood sample was collected from the coccygeal vein of each cow into an empty tube and a 5-mL blood sample was collected into a tube containing EDTA.a The 10-mL blood samples were allowed to clot and then centrifuged for 10 minutes at 1,300 X g. Serum was harvested and stored at −20°C (–4°F) pending analysis. The EDTA-treated blood samples were kept chilled until analysis, which was performed within 8 hours after collection.
After the exact day of parturition was known, only the set of samples collected between 7 and 1 days before parturition was used as the preparturition sample for each cow. Serum concentrations of FAs, β-hydroxybutyrate, cholesterol, glucose, urea, and calcium were determined by means of colorimetric methods with a chemistry analyzer.b Leukocyte counts (total leukocytes, neutrophils, lymphocytes, monocytes, and eosinophils) were measured with an automated hematologic analyzer.c
Statistical analysis—Data were analyzed with statistical software.d For the analysis, the unit of concern was the cow. The mean value of each biochemical and hematologic analyte in cows with and without RP was compared by means of a general linear model.e Associations of the following variables with the development of RP were assessed with the Fisher exact test: parity group (first, second, and third or greater lactation), season of parturition (September to November, December to February, March to May, or June to August), twins, dystocia (ie, veterinarian-assisted parturition), and body condition score (based on a 5-point scale: thin, ≤ 3.0; moderate, ≥ 3.25 and ≤ 3.75; and fat, ≥ 4.0). Variables that achieved a value of P ≤ 0.1 were then included in a multivariate logistic regression model,12,f with herd as a random effect. Interactions between all included variables were determined. Results of the multivariate model with a value of P < 0.05 were considered significant. Results are expressed as mean ± SD.
Results
Results of serum biochemical analysis of 1,038 cows were included, of which 161 (15.5%) developed RP. Blood samples were obtained from cows with and without RP within a mean ± SD interval of 3.86 ± 1.91 days and 3.88 ± 2.02 days prior to parturition, respectively, and these intervals were not significantly (P = 0.84) different.
Complete data sets were available for 990 cows for inclusion in the multivariate logistic regression model. Results of univariate ANOVA for continuous variables did not reveal any significant differences between cows with and without RP with respect to hematologic and serum biochemical values, with the exception of serum cholesterol concentration (P = 0.01; Table 1). The categoric variables dystocia and body condition score were also not associated with development of RP (Table 2).
Results of univariate ANOVA to assess differences in serum biochemical values and WBC counts in dairy cows with and without RP from 20 herds, as measured in the week before parturition.
| Variable | Cows without RP | Cows with RP | P value* | ||||||
|---|---|---|---|---|---|---|---|---|---|
| No. | Mean | SD | Median | No. | Mean | SD | Median | ||
| BHB (μmol/L) | 844 | 601 | 256 | 555 | 154 | 618 | 252 | 549 | 0.46 |
| FAs (mmol/L) | 844 | 0.4 | 0.3 | 0.3 | 154 | 0.4 | 0.3 | 0.3 | 0.08 |
| Cholesterol (mmol/L) | 844 | 2.1 | 0.4 | 2.1 | 154 | 2.2 | 0.4 | 2.2 | 0.01 |
| Urea (mmol/L) | 844 | 5.0 | 1.5 | 4.9 | 154 | 5.5 | 1.4 | 5.1 | 0.26 |
| Glucose (mmol/L) | 844 | 3.7 | 0.6 | 3.7 | 154 | 3.6 | 0.5 | 3.7 | 0.13 |
| Calcium (mmol/L) | 843 | 2.39 | 0.14 | 2.41 | 154 | 2.38 | 0.11 | 2.38 | 0.32 |
| Total leukocytes (× 109/L) | 826 | 8.9 | 3.2 | 8.4 | 155 | 9.0 | 3.4 | 8.3 | 0.80 |
| Neutrophils (× 109/L) | 826 | 4.1 | 1.4 | 3.9 | 153 | 4.1 | 1.7 | 3.8 | 0.66 |
| Lymphocytes (× 109/L) | 826 | 4.3 | 2.5 | 3.7 | 153 | 4.5 | 2.5 | 3.8 | 0.46 |
| Monocytes (× 109/L) | 826 | 0.1 | 0.2 | 0.1 | 153 | 0.1 | 0.1 | 0.1 | 0.25 |
| Eosinophils (× 109/L) | 826 | 0.3 | 0.3 | 0.2 | 153 | 0.3 | 0.3 | 0.3 | 0.88 |
Variables with a value of P < 0.10 were included in a multivariate logistic regression model.
BHB = β-Hydroxybutyrate.
Results of the Fisher exact test to assess differences in the distributions of characteristics of dairy cows with and without RPfrom 20 herds.
| Variable | Cows without RP | Cows with RP | |
|---|---|---|---|
| No. | Percentage | No. | |
| Parity | 0.05 | ||
| First | 267 | 30.4 | 38 |
| Second | 218 | 24.8 | 33 |
| Third or greater | 394 | 44.8 | 88 |
| Body condition score† | 0.68 | ||
| ≤ 3.0 | 132 | 15.2 | 26 |
| 3.25 to 3.75 | 535 | 61.5 | 100 |
| ≥ 4.0 | 203 | 23.3 | 32 |
| Twins | < 0.001 | ||
| No | 866 | 98.5 | 144 |
| Yes | 13 | 1.5 | 15 |
| Dystocia | 0.86 | ||
| No | 849 | 96.6 | 154 |
| Yes | 30 | 3.4 | 5 |
Body condition was evaluated by means of a 5-point scale, with 1 = thin and 5 = fat.
See Table 1 for remainder of key.
Results of the multivariate model indicated that serum cholesterol and FAs concentrations were associated with a higher odds of dairy cows developing RP (Table 3). For each increase of 0.1 mmol/L in the serum concentration of cholesterol or FAs in the week before parturition, the odds of RP in cows increased 5% for both analytes. An interaction between serum FAs concentration and serum cholesterol concentration was not evident (P = 0.99), nor was an interaction evident between any other variables.
Results of a multivariate logistic regression model to assess differences in serum biochemica values and distributions of characteristics of 990 cows with and without RP from 20 herds.
| Variable | Estimate | SEM | Odds ratio | 95% CI | P value* |
|---|---|---|---|---|---|
| Cholesterol (per 0.1 mmol/L increase)† | 0.052 | 0.210 | 1.05 | 1.01–1.09 | 0.014 |
| FAs (per 0.1 mmol/L increase)† | 0.050 | 0.252 | 1.05 | 1.01–1.09 | 0.047 |
| Season of parturition | |||||
| June to August | Referent | NA | 1.00 | NA | NA |
| September to November‡ | 0.374 | 0.249 | 1.45 | 0.89–2.37 | 0.134 |
| December to February‡ | 0.722 | 0.251 | 2.05 | 1.25–2.16 | 0.004 |
| March to May‡ | 0.180 | 0.273 | 1.19 | 0.70–2.04 | 0.511 |
| Twins§ | 1.894 | 0.405 | 6.64 | 3.00–14.70 | < 0.001 |
| Vitamin E treatment§ | 0.039 | 0.177 | 1.04 | 0.73–1.47 | 0.826 |
A value of P < 0.05 was considered signifcant.
Value measured once per cow, 1 to 7 days before parturition.
The odds ratio represents the odds of RP in the indicated group, compared with the odds for the cows in the referent group.
The odds ratio represents the odds of RP in cows with the indicated factor, compared with the odds for cows without the factor.
CI = Confdence interval. NA = Not applicable.
For cows that gave birth between December and February, the odds of developing RP were twice as high as the odds for those that gave birth between June and August. The odds of developing RP were 6.6 times as high in cows that delivered twins, compared with the odds for cows with only 1 calf (P < 0.001). Vitamin E treatment was not associated with development of RP (P = 0.83); however, the variable was retained in the final model.
Discussion
Other researchers13 identified a decreased number of monocytes in blood samples collected in the 2 weeks prior to parturition from cows that subsequently developed RP (n = 45), compared with the number in those that did not (184); this difference was attributed to possible changes in polyunsaturated FAs in the cell membrane. However, in the present study, which had greater statistical power, no significant difference was detected. A possible explanation for lack of concordance between findings is that the other study did not include a random sample of their population. Instead, cows were included only when they had a nonlactating period of at least 30 days, delivered 1 calf, and subsequently had been lactating for at least 30 days.
In another study,14 the association between counts and functions of circulating leukocytes was evaluated in periparturient cows with various postparturition disorders including RP. Results indicated no association between total or differential leukocyte counts in the 1.5 weeks before parturition and subsequent development of RP. However, significant differences were evident in these counts after parturition. Degree of expression of CD62L adhesins by neutrophils is also reportedly decreased at parturition, compared with the value in the prepartum period, but the total number of circulating neutrophils is not affected.15 The lack of an association between leukocyte counts and subsequent development of RP in the study reported here did not refute the hypothesis of an association of neutrophil function with RP,6,8 but it did indicate that immune function, including neutrophil function, is not always reflected in counts of circulating leukocytes.
In the present study, the odds of a cow developing RP increased as the serum concentration of cholesterol increased in the week before parturition. These results are opposite to those of another study.9 One possible explanation is that the results of the other study may have been confounded by a difference in the timing of sample collection. In that study, samples were collected a mean of 8.4 days before parturition in the cows with RP and a mean of 15.4 days before parturition in cows without RP. The plasma cholesterol concentration in cows generally decreases as parturition approaches, whereas the plasma FAs concentration generally increases. After parturition, the plasma FAs concentration decreases and the plasma cholesterol concentration increases gradually.16–19 This pattern is a reflection of the lipid mobilization that occurs during the transition period.20,21
In cows with and without RP, serum concentrations of FAs and cholesterol reportedly do not differ significantly between 12 and 24 hours after parturition.22 It is possible that the natural increase in both analytes that occurs in the early postpartum period obscures differences at that time. On the other hand, cows with RP reportedly have a greater decrease in serum cholesterol concentration and a greater increase in serum FAs concentration, compared with respective values in cows without RP.10 Results regarding serum cholesterol concentrations differ among studies, depending upon the status of cows evaluated.23 High serum cholesterol values might be attributable to a reduced energy balance24 or to a ration high in dietary fat.25 The fat content in the rations was not determined in the present study; however, the possible influence of dietary fat content should be considered when interpreting serum cholesterol values.
Despite their significant associations with subsequent development of RP in the present study, serum concentration of neither FAs nor cholesterol was a strong diagnostic test for prediction of RP. To this end, test results may support a review of the energy or fat content in a cow's diet but should not be used to rule out an anomaly in energy or fat metabolism. In the present study, as serum concentration of cholesterol or FAs increased 0.1 mmol/L in the last week prior to parturition, the odds of developing RP increased by 5%. These associations suggested that prepartum energy metabolism is an element that contributes to the occurrence of RP.
Programs designed to monitor the health of dairy cows can include measurements of serum cholesterol and FAs concentrations in the last week prior to the expected parturition date to help detect an energy disturbance, which might increase the odds of a cow developing RP, particularly when compounded by the presence of other clinical risk factors such as twinning, dystocia, stillbirth, induced parturition, abortion, and hypocalcemia. In the present study, total or differential leukocyte counts were not useful for predicting RP in dairy cows.
Abbreviations
| FA | Fatty acid |
| RP | Postpartum retention of the placenta |
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911 Hitachi chemistry analyzer, Roche, Tokyo, Japan.
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Proc GENMOD in GLIMMIX, SAS, version 9.1, SAS Institute Inc, Cary, NC.
