The Effect of Prepartum Injection of Vitamin E on Health in Transition Dairy Cows

J. Dairy Sci. 85:1416–1426  American Dairy Science Association, 2002.

The Effect of Prepartum Injection of Vitamin E on Health in Transition Dairy Cows Stephen J. LeBlanc,* Todd F. Duffield,* Ken E. Leslie,* Ken G. Bateman,* Jeromy TenHag, * John S. Walton,† and Walter H. Johnson* *Department of Population Medicine and †Department of Animal and Poultry Science University of Guelph, Guelph, Ontario, Canada N1G 2W1

ABSTRACT The objective of this study was to investigate parenteral vitamin E for the prevention of peripartum disease in dairy cows. A randomized clinical trial was conducted in 21 commercial dairy herds. Cows (n = 1142) were randomly assigned to receive either a single subcutaneous injection of 3000 IU of vitamin E, or placebo, 1 wk before expected calving. Serum αtocopherol was significantly increased in treated cows at 7 and 14 d, but not at 21 d after injection. Overall, there were no significant differences between treatment groups in the incidence of retained placenta, clinical mastitis, metritis, endometritis, ketosis, displaced abomasum, or lameness. However, there was a conditional benefit of treatment for reduction of the incidence of retained placenta. Cows with marginal pretreatment vitamin E status (serum α-tocopherol to cholesterol mass ratio < 2.5 × 10-3) that received an injection of vitamin E tended to have reduced risk of retained placenta. However, in cows with adequate serum vitamin E, there was no reduction in the incidence of any disease. For clinical application, primiparous animals were most likely to benefit from prepartum injection of vitamin E. (Key words: tocopherol, parenteral, retained placenta, peripartum) Abbreviation key: α-T = α-tocopherol, α-T:C = αtocopherol:cholesterol mass ratio, LS = linear score, OR = odds ratio, RP = retained placenta. INTRODUCTION Most infectious and metabolic disease in dairy cows occurs in early lactation and is related to physiologic and management changes that occur during the tran-

Received November 23, 2001. Accepted January 11, 2002. Corresponding author: S. Le Blanc; e-mail: sleblanc@ovc. uoguelph.ca.

sition period. One critical event is a significant suppression of immune function in the peripartum period (Goff and Horst, 1997; Mallard et al., 1998). Evidence suggests that retained placenta (RP) is attributable, at least in part, to failure of the immune system to break down the placentome promptly after delivery of the fetus (Gunnink, 1984a, 1984b; Kimura et al., 2000). Peripartum immunosuppression is multifactorial but is associated with endocrine changes and decreased intake of critical nutrients (Goff and Horst, 1997). In particular, decreased phagocytosis and intracellular killing by neutrophils occur in parallel with decreased DMI and decreased circulating vitamin E (α-tocopherol; α-T) concentration (Hogan et al., 1992). Neutrophils are the primary mechanism of uterine immune defense (Bondurant, 1999) and also play an important role in mammary defense (Mallard et al., 1998). Vitamin E is a fat-soluble membrane antioxidant that enhances the functional efficiency of neutrophils by protecting them from oxidative damage following intracellular killing of ingested bacteria (Herdt and Stowe, 1991). Several studies (Weiss et al., 1990, 1992) have shown that dietary supplementation with 1000 IU of vitamin E/cow per day in the late dry period mitigates the peripartum drop in circulating α-T, but this does not necessarily decrease the incidence of disease (Allison and Laven, 2000). Only administration of 3000 to 5000 IU of vitamin E by injection in the last week before calving enhanced α-T and neutrophil function at calving (Hogan et al., 1992; Weiss et al., 1992; Politis et al., 1995). A recent study demonstrated a 50% reduction in the incidence of RP after a single intramuscular (i.m.) injection of 3000 IU of vitamin E 8 to 14 d prepartum (Erskine et al., 1997). However, there appeared to be a difference in the effect of treatment between primiparous and multiparous cows. Questions remain about the benefit, optimum dose, route, and timing of administration of parenteral vitamin E in peripartum dairy cows. The objective of this study was to measure the efficacy of a single subcutaneous injection of vitamin E,

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administered 1 wk before calving, for reduction of the incidence of retained placenta, metritis and clinical mastitis in early lactation. MATERIALS AND METHODS Experimental Design The study was conducted in a convenience sample of 21 dairy herds in the area of Guelph, Ontario, Canada, that were typical of commercial dairy herds in central Canada. Based on detecting a reduction in the incidence of RP from 8 to 4% with a maximum type I error of 5% and type II error of 20%, the required sample size was 552 animals per group or a total of 1104 cows (Martin et al., 1987). A technician visited each herd weekly, at the same time on the same day each week. Every cow and heifer was enrolled between 4 and 10 d before the expected calving date. Lists of expected calving dates were generated with a herd management software program (DairyComp 305, Valley AG Software, Tulare, CA), based on a gestation length of 280 d. Cows were randomly assigned within herd to receive either 3000 IU of RRR-α-tocopheryl acetate (vitamin E) in 10 ml of emulsifiable base, or a placebo, by s.c. injection in one site behind the scapula. The placebo solution consisted of an equal volume of laboratorygrade propylene glycol with <1% by volume of an injectable vitamin-mineral solution without vitamin E or selenium (Vitamaster, PVU, Victoriaville, Quebec, Canada), titrated to match the color of the test product. Within each block of 10 random assignments within a herd, treatments were balanced between vitamin E and placebo. Each animal received only one injection, regardless of the actual interval from treatment to calving. Immediately before treatment, and at each weekly visit up to and including the first week after calving, a 10-ml blood sample was collected from the coccygeal vein into an evacuated sterile tube without anticoagulant (Vacutainer, Becton-Dixon, NJ). Samples were allowed to clot and kept chilled until processing. The blood was then centrifuged and the serum was separated and stored at −20 C until analysis. Cows were assigned a BCS at the time of enrollment (Ferguson et al., 1994). Diaries were provided to producers to record all disease events. Additionally, at each weekly visit, the technician actively questioned producers about the occurrence of disease in the trial animals. Finally, medical records for the study herds were also collected. For the purpose of this study, disease events were recorded until 30 DIM. The diseases of interest and the case definitions used are described

in Table 1. A sample of milk was also collected at the postpartum visit (between 0 and 7 DIM) for automated SCC, which were transformed to linear score (LS) using the formula (LS = [log2(SCC/100,000) + 3]). At the end of the study, all animals that had RP and that had serum available were paired with one randomly selected cow from the study that did not have RP. Sera from all available samples from these animals (one to three prepartum samples and one postpartum sample) were submitted for determination of serum α-T, cholesterol, and α-T: cholesterol (α-T:C) mass ratio. Cholesterol determinations were performed at the Animal Health Laboratory at the University of Guelph using an enzymatic colorimetric test (Cholesterol CHOD-PAP, Hoffman-LaRoche, Ayr, Ontario, Canada). The interassay coefficient of variability was 1.6%. Vitamin E measurements were performed at the Animal Health Diagnostic Laboratory at Michigan State University (AHDL-MSU) by HPLC using a modification of the method described by Arnaud et al. (1991). The intraassay and interassay coefficients of variability were 16.7 and 13.1%, respectively. In addition, a subset of these sera was submitted to the AHDL-MSU for determination of pretreatment selenium concentration, using a fluorometric technique as described by Reamer and Veillon (1983). The intraassay and interassay coefficients of variability were 12.1 and 7.1%, respectively. Data Management and Statistical Analysis Statistical analyses were performed with SAS (version 8.0, 1999, SAS Institute, Cary, NC) with the cow as the unit of concern. Initial screening for simple associations of treatment with disease outcomes was done with a chi-square test. Outcomes that were associated with treatment at P < 0.2 were submitted to multivariable logistic regression (the GENMOD procedure in SAS with binomial distribution and the logit link function). Correlation exists among cows within a herd through feeding, housing, and environment, genetics, and management policies or programs, which represent unmeasured sources of heterogeneity of effects. The effect of intraherd correlation (clustering) was accounted for with generalized estimating equations applied to the logistic regressions, specifying a compound symmetry correlation structure, to produce robust standard errors for the estimates of parameter effects (Shoukri and Pause, 1999). The main effect of interest was treatment with vitamin E. In addition to accounting for the random effect of herd, covariates including parity group (1, 2, or ≥3), season of calving (fall: September through November; winter: December through February; spring: March Journal of Dairy Science Vol. 85, No. 6, 2002

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LEBLANC ET AL.

Table 1. Case definitions for health events in peripartum dairy cows. Disease

Case definition

Dystocia Milk Fever Retained Placenta (RP) Metritis

Obstetrical difficulty requiring veterinary assistance Producer or veterinary diagnosis of stage II hypocalcemic parturient paresis; cow down Producer diagnosis of failure to pass the fetal membranes within 24 h after calving Veterinary diagnosis based on systemic illness (fever and inappetance/anorexia, with or without signs of cardiovascular compromise) attributable to fetid uterine infection prior to 20 DIM Veterinary diagnosis of primary clinical ketosis based a presenting complaint of inappetance and/or decreased milk production in cows less than 30 DIM, presence of a positive urine or milk ketone test, and absence of a displaced abomasum or other primary disease. Veterinary diagnosis of a left or right side displacement of the abomasum within 30 DIM based on auscultation of a characteristic “ping”; generally confirmed at surgery. Veterinary diagnosis based on a presenting complaint of inappetance or anorexia in cows less than 30 DIM, with the absence of clinical ketosis or DA; includes diagnoses of simple indigestion and sub-acute rumen acidosis. Producer diagnosis of clinical mastitis within 30 DIM based on grossly abnormal milk, and/or presence of a swollen or hard quarter of the udder; also includes cases with systemic signs of toxic mastitis. Producer diagnosis of undifferentiated lameness within 30 DIM based on an abnormal gait or lack of weight bearing on a limb; includes diagnoses of interdigital and digital dermatitis.

Ketosis Displaced abomasum (DA) Off feed Mastitis Lameness

though May; summer: June through August), interval from enrollment to calving (0 to 7 d or 8 to 16 d), BCS at enrolment, twins, dystocia, and any antecedant disease events were offered to the models. Covariates that were not significant at P < 0.1 were removed by manual backward stepwise elimination. Finally, biologically plausible interactions of treatment with significant covariates were tested. When significant inter-

actions with treatment were detected, the data were stratified on the variable that was causing effect modification, and reanalyzed. A second set of logistic regression models was built, identical to those above, but including various thresholds of the pretreatment α-T status of animals as a covariate of treatment; interactions of initial vitamin E status with treatment were also tested. A mixed

Table 2. Descriptive data on the study animals 1 wk before expected calving. Parity group Parameter

Overall

1

2

3

n % of total BCS % of animals ≤ 3.0 % of animals 3.25 to 3.75 % of animals ≥ 4.0 Pretreatment serum concentration n α-Tocopherol (µg/ml)1 Mean (SD) Median α-Tocopherol:cholesterol (Mass ratio × 10-3)1 Mean (SD) Median % of animals < 1.5 % of animals < 2.0 % of animals < 2.5 % of animals < 3.0 % of animals < 3.5 % of animals < 4.0 % of animals < 4.5 Selenium (ng/ml)2 n Mean (sd) Median

1142

330 28.9

282 24.7

530 46.4

3.1 66.2 30.8

24.7 64.2 11.1

18.0 59.6 22.4

90

68

1

15.4 62.6 22.0 311 2.69 (1.34) 2.52

2.58 (1.32) 2.48

2.62 (1.15) 2.53

2.79 (1.42) 2.54

3.22 (1.65) 2.88 10 20 34 55 67 74 84

3.07 (1.58) 2.77 11 30 40 61 69 74 84

3.26 (1.48) 2.94 10 22 32 54 72 75 90

3.34 (1.76) 2.94 9 13 30 52 64 74 81

87 69.1 (14.6) 69

18 69.8 (8.8) 69.5

26 68.5 (14.5) 69.5

43 69.1 (16.7) 69.0

From 145 cows with RP and 166 randomly selected cows without RP. From 42 randomly selected cows with RP and 45 randomly selected cows without RP.

2

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PREPARTUM INJECTION OF VITAMIN E Table 3. The crude association of one subcutaneous injection of 3000 IU of vitamin E, or placebo, 1 wk before expected calving with postpartum health. See Table 1 for case definitions. Breakdown by parity group is provided where differences were observed. Incidence rate (%) Health outcome

Vitamin E

Placebo

Milk Fever Retained Placenta Parity 1 Parity 2 Parity ≥ 3 Metritis Endometritis1 Off feed2 Ketosis2 Displaced abomasum2 Lameness2 Parity 1 Parity 2 Parity ≥ 3 Mastitis2

6.0 15.0 9.4 15.9 18.1 3.0 16.3 1.9 3.4 4.9 1.6 0.6 0.8 2.6 9.4

5.7 14.4 15.0 8.3 17.8 3.0 14.0 1.7 3.1 4.5 3.1 0.6 5.1 3.5 10.1 Mean score

Linear score3

5.9

5.9

1

These cows were systematically examined, including vaginoscopy, between 20 and 33 DIM, as part of a separate clinical trial. 2 Diagnosed within 30 d after calving. 3 Measured between 0 and 7 d after calving.

(random and fixed effects) linear regression model was used to compare least squares means of serum α-T in response to treatment (the Mixed procedure in SAS with herd as the random effect). RESULTS The study was conducted between September 1998 and October 1999. In total, 1184 animals were enrolled. Seven cows died or were sold after enrolment. A further 35 cows (3%) in which the interval from treatment to calving was longer than 16 d were deleted. Therefore, data from 1142 cows from 21 herds were available for analysis. One herd exited the dairy industry in December 1998 and was not replaced in the study. Four herds, representing approximately 29% of the study animals, were housed in free stall barns, with the rest in tie stalls. Fourteen herds, representing 50% of the cows in the study, provided seasonal access to pasture, although no herds practiced management intensive grazing. The actual proportion of individual animals that had access to pasture during the study is unknown. Descriptive information about the study animals is presented in Table 2 and about the incidence of peripartum disease in Table 3. Random assignment to treatment resulted in 49.7% of cows receiving vitamin E and 50.3% receiving the placebo. There was no association of treatment assignment with parity group (P = 0.16).

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The target was to administer the injection 1 wk before calving. The actual mean (standard deviation) and median intervals from treatment to calving were 7.1 (± 4) and 7 d, respectively, with a minimum of zero and the maximum truncated at 16 d. No hypersensitivity reactions or any other adverse reactions were observed after treatment. The response to treatment in the concentration of vitamin E in serum is shown in Table 4. Pretreatment α-T was similar between treatment groups. Cows were sampled weekly after treatment until the first week postpartum, so the exact pharmacokinetics were not determined. However, following one s.c. injection of 3000 IU of vitamin E, the apparent peak in serum α-T:C concentration was reached by 1 wk. Serum α-T:C was declining, but still elevated over controls 2 wk after treatment, and returned to baseline concentration by 3 wk after injection. Although the interval from treatment to calving was variable, injection with vitamin E increased serum α-T and α-T:C over controls in the last 3 d before calving, at calving, and for 5 to 7 d after calving (Table 5). Irrespective of treatment, cows that calved in the summer had higher mean pre-treatment α-T:C. Overall, there was no effect of treatment with vitamin E on the incidence of RP, mastitis, or metritis. However, there were differences in the effect of treatment on the incidence of RP between parity groups (Table 3). There was little association between herd-level mean vitamin E status 1 wk prepartum and herd incidence of RP (Figure 1; r2 = 0.02). Likewise, when cows were stratified based on being in a herd with aboveaverage incidence of RP (over 15%), there was no overall benefit of treatment in high-risk herds [odds ratio (OR) = 0.87, 95% confidence interval 0.51 to 1.49, P = 0.61]. Similarly, there was no threshold of herd mean α-T:C from the lower to the upper quartiles that identified cows in which treatment had a significant benefit. However, at the individual level, cows that had serum α-T:C > 3.5 × 10-3 in the 7 d prepartum tended to be at lower risk of RP (OR = 0.78, P = 0.12, adjusted for the effects of herd, twins, parity group, and season of calving). Overall, 40.8% of cows had serum α-T:C > 3.5 × 10-3 in last week prepartum, but cows that received the vitamin E injection were almost twice as likely to be above this threshold (OR = 1.8, P = 0.02). An initial logistic regression model of risk of RP including all 1142 cows, not considering animals’ baseline vitamin E status, indicated that parity group, calving season, twins, milk fever, and the interval from enrolment to calving (roughly, whether animals calved before or after their expected date), but not treatment, were significantly associated with risk of RP. Cows that delivered twins, and (accounting for twins) cows Journal of Dairy Science Vol. 85, No. 6, 2002

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LEBLANC ET AL. Table 4. Least squares means of serum vitamin E following one subcutaneous injection of 3000 IU of vitamin E or placebo, 1 wk before expected calving.1 Days after injection Treatment Mean α-tocopherol (µg/ml) n Vitamin E Placebo P Mean α-tocopherol:cholesterol mass ratio (× 10-3) n Vitamin E Placebo P

0 (Pretreatment)

7

14

21

311 2.73 2.70 0.79

288 3.42 2.45 <0.0001

146 2.84 2.01 0.0001

38 2.62 1.96 0.09

310 3.28 3.16 0.47

284 4.39 3.11 <0.0001

144 3.72 2.66 0.0001

38 3.27 2.88 0.46

1

Adjusted for the fixed effects of parity group and season of calving and the random effect of herd.

that calved within 1 wk of enrollment (i.e., on average, earlier than expected) were at higher risk of RP. Cows that calved in the summer were at the lowest, and cows that calved in the winter at the highest risk of RP. There was no overall effect of treatment on risk of RP. However, there was a significant interaction of treatment with parity group: heifers that received the vitamin E injection had a reduced risk of RP. There was also a tendency (P = 0.07) for an interaction of treatment with the interval from treatment to calving: cows that calved within 7 d of injection tended to have a higher rate of RP than those that calved between 8 and 16 d after treatment. Therefore, the data were first stratified on parity group. A second set of initial logistic regression models of risk of RP was built including 310 cows that had serum α-T measurements. First, controlling for the significant effects of parity group, calving season, twins, and the random effect of herd, thresholds of pretreatment vitamin E status, measured by serum α-T from 1.5 to 4.0 µg/ml or serum α-T:C from 1.5 to 4.5 × 10-3, in 0.5-

unit increments, were tested for association with risk of RP. The best discrimination for increased risk of RP was found at a threshold of α-T < 2.0 µg/ml (OR = 1.9, P = 0.006) or α-T:C < 2.5 × 10-3 (OR = 1.6; P = 0.06). No other thresholds were significantly associated with risk of RP. The initial models including pretreatment α-T status again indicated no significant effect of treatment on risk of RP. There were significant main effects of parity group, calving season, twins, and pretreatment serum vitamin E status, but not treatment. There were still significant interactions of treatment with parity group and treatment with interval to calving. However, the effect of treatment depended on pretreatment α-T:C < 2.5 × 10-3 (interaction term P = 0.06), but not on pretreatment α-T (treatment × pretreatment α-T < 2.0 µg/ml interaction P = 0.48). Strictly, these findings imply that the data should be stratified on these three effect modifiers (Rothman and Greenland, 1998)—parity group (three levels), baseline αT:C < 2.5 (two levels) and interval from enrollment to calving (two levels)—resulting in 12 strata. The data

Table 5. Least-squares means of serum vitamin E in peripartum cows that received one subcutaneous injection of 3000 of IU vitamin E or placebo, 1 wk before expected calving.1 Day relative to calving (2-d increments) Treatment Mean α-tocopherol (µg/ml) n Vitamin E Placebo P Mean α-tocopherol:cholesterol mass ratio (× 10-3) n Vitamin E Placebo P 1

−11

−9

−7

−5

−3

−1

1

3

5

7

35 2.71 2.49 0.74

46 2.96 2.77 0.59

67 3.11 2.27 0.03

84 3.02 2.57 0.21

88 2.81 2.74 0.82

86 3.13 2.68 0.05

81 3.19 2.43 0.01

86 3.12 2.15 <.01

90 2.97 2.42 0.04

77 2.74 2.17 0.06

35 3.35 3.22 0.88

46 3.36 3.25 0.81

67 3.82 2.64 0.01

83 3.74 3.16 0.16

88 3.42 3.29 0.70

84 3.99 3.40 0.08

80 4.38 3.19 <.01

85 4.32 2.80 <.01

88 3.78 3.26 0.19

77 3.64 3.06 0.16

Adjusted for the fixed effects of parity group and season of calving, and the random effect of herd.

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Figure 1. The association between herd mean α-tocopherol: cholesterol ratio 1 wk before expected calving and herd lactational incidence rate of retained placenta in 18 dairy herds (311 cows) in Ontario, Canada.

including vitamin E measurements were too sparse to do so. Furthermore, parity group and baseline vitamin E status are more biologically intrinsic factors, therefore two approaches were taken. First, the full dataset was stratified on parity group and reanalyzed, while a parallel analysis of the subset that had vitamin E determinations were stratified at the threshold of pretreatment α-T:C < 2.5 × 10-3. The apparent crude association of treatment with an increased incidence of RP in second-lactation animals (Table 3) is not biologically sensible. In a logistic regression of risk of RP in second-lactation cows, including the effects of twins, season of calving and interval from enrollment to calving, the treatment effect became nonsignificant (P = 0.14). In fact, five of the seven second-parity animals that had twins received the vi-

tamin E treatment. However, models could not be fitted with all the parameters and interaction terms that were tested in models for first, or third and greater parity. Additionally, the least squares mean pretreatment α-T:C ratio was not different between animals in second, and third and greater parity groups (P = 0.46), but primiparous animals tended (P = 0.08) to have lower α-T:C than parity group three (Table 2). Therefore, subsequent analyses were stratified into primiparous and multiparous animals. Without regard to pretreatment vitamin E status, treatment tended to reduce the risk of RP in primiparous cows by over 40% (OR = 0.56, P = 0.07; Table 6). The effect of treatment did not depend on season of calving, interval from treatment to calving, or body condition. There was no effect of dystocia on the risk of RP, but the few (n = 10) heifers that were thin (BCS ≤ 3.0) immediately prepartum had an increased risk of RP. In multiparous cows, there was no effect (P = 0.38) of treatment on the probability of RP, even when controlling for twins and milk fever (Table 7). In the subset of the data in which pretreatment vitamin status was measured, animals were stratified into low and adequate vitamin E status using a threshold of serum α-T:C < 2.5 × 10-3 at enrollment. Among the one third of animals with low baseline vitamin E, treatment tended to decrease the risk of RP by over 50% (Table 8; OR = 0.46, P = 0.11). Among these animals, the effect of treatment did not depend on parity group. Conversely, among cows with baseline serum α-T:C ≥ 2.5 × 10-3, there was no significant effect of treatment (P = 0.43; not shown). A summary of the effect of treatment with vitamin E on risk of RP across the strata of effect modifiers is presented in Table 9. All models tested the main effect of treatment and controlled for herd clustering and the significant covariates noted. Because of the sparseness of the stratified data, models with all covariates could not be fitted. Among cows with serum α-T:C < 2.5 × 10-3 approxi-

Table 6. Logistic regression model of risk of retained placenta in 325 primiparous cows that received one subcutaneous injection of 3000 IU vitamin E or placebo at random, one week before expected calving.1 Parameter

Odds ratio

95% Confidence interval

P

Vitamin E treatment Season of Calving Fall Winter Spring Summer Interval from enrollment to calving 0 to 7 d 8 to 16 d BCS ≤ 3.0 at enrolment

0.56

0.30 – 1.05

0.07

1.60 3.22 2.53 ...

0.70 – 3.63 1.27 – 8.17 0.87 – 7.39 ...

0.26 0.01 0.09 ...

2.46 ... 4.22

1.28 – 4.71 ... 1.19 – 15.2

0.007 ... 0.03

1

Adjusted for intra-herd correlation with generalized estimating equations. Journal of Dairy Science Vol. 85, No. 6, 2002

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LEBLANC ET AL. Table 7. Logistic regression model of risk of retained placenta in 812 multiparous cows that received one subcutaneous injection of 3000 IU of vitamin E or placebo at random, 1 wk before expected calving.1 Parameter

Odds ratio

95% Confidence Interval

P

Vitamin E treatment Season of calving Fall Winter Spring Summer Interval from enrollment to calving 0 to 7 d 8 to 16 d Twins Milk fever

1.22

0.79 – 1.88

0.38

1.46 1.80 1.06 ...

0.97 – 2.16 1.05 – 3.13 0.61 – 1.82 ...

0.07 0.03 0.84 ...

1.52 ... 7.46 2.12

0.99 – 2.34 ... 3.82 – 14.56 1.30 – 3.49

0.05 ... 0.0001 0.003

1

Adjusted for intra-herd correlation with generalized estimating equations.

mately 1 wk before expected calving, treatment with one injection of vitamin E reduced the risk of RP. Among cows above this threshold, there was no reduction in the probability of RP associated with injection of vitamin E. DISCUSSION This study is the largest randomized clinical trial of parenterally administered vitamin E in transition dairy cows to date. Despite the statistical power of the study, there was only a conditional decrease in the incidence of RP, and no effect of treatment on other measures of health. Three percent of the animals enrolled did not calve within 16 d after treatment. This was longer than the

duration of effect of the treatment. Furthermore, the data from these cows that calved between 17 and 26 d after injection were too sparse for statistical analysis. This wide distribution in actual calving dates, relative to expected, underlines the difficulty in administering time-sensitive treatments to peripartum cows. The incidences of disease in this study were comparable to reports under similar management conditions, with the exception of RP (Lissemore et al., 1992). The 15% incidence of RP likely reflects more accurate reporting because of the intensive data collection scheme. The hypothesis underlying the study was that vitamin E injected at a critical time prepartum would mitigate the normal peripartum drop in serum α-T, reduce the impairment of neutrophil function, and thereby

Table 8. Logistic regression model of risk of retained placenta in 103 cows that had low vitamin E status (serum α-tocopherol:cholesterol mass ratio < 2.5 × 10-3) at the time of random treatment with either one subcutaneous injection of 3000 IU vitamin E or placebo, 1 w before expected calving.1 Parameter

Odds ratio

95% Confidence Interval

P

Vitamin E treatment Parity group 1 2 3 Season of Calving Fall Winter Spring Summer Interval from enrollment to calving 0 to 7 d 8 to 16 d Twins Milk Fever BCS ≤ 3.0 3.25 to 3.75 ≥ 4.0

0.46

0.18 – 1.20

0.11

1.52 1.13 ...

0.85 – 2.72 0.24 – 5.37 ...

0.15 0.88 ...

0.32 0.58 0.94 ...

0.10 – 1.04 0.20 – 1.68 0.26 – 3.39 ...

0.06 0.31 0.92 ...

0.37 ... 2.89 1.23

0.21 – 0.66 ... 0.55 – 15.2 0.24 – 6.39

0.0007 ... 0.21 0.81

2.95 1.6 ...

1.22 – 7.17 0.75 – 3.56 ...

0.02 0.22 ...

1

Adjusted for intra-herd correlation with generalized estimating equations.

Journal of Dairy Science Vol. 85, No. 6, 2002

PREPARTUM INJECTION OF VITAMIN E Table 9. Summary of logistic regression models of the effect of one subcutaneous injection of 3000 IU vitamin E one wk prior to expected calving on the risk of retained placenta, accounting for significant effect modifiers.1

Parity group Animals with pretreatment α-tocopherol:cholesterol mass ratio < 2.5 × 10-3 Primiparous2 Multiparous3 Animals with pretreatment α-tocopherol:cholesterol mass ratio ≥ 2.5 × 10-3 Primiparous4 Multiparous5

Odds ratio

95% Confidence interval

P

36 68

0.27 0.47

0.08 – 0.92 0.15 – 1.54

0.04 0.21

54 152

0.71 1.46

0.29 – 1.73 0.62 – 3.49

0.46 0.39

n

1 All models adjusted for intra-herd correlation with generalized estimating equations. 2 Adjusted for season of calving. 3 Adjusted for season of calving, interval from enrollment to calving, twins and milk fever. 4 Adjusted for the interval from enrolment to calving.

decrease the incidence of RP and early lactation clinical mastitis. The results indicate that only RP is susceptible to a reduction in incidence, under certain conditions, when vitamin E is administered as in the present study. Given the relatively short duration of action of injected vitamin E, it is not surprising that RP was the disease most responsive to vitamin E supplementation. It occurs soonest after treatment, and impaired neutrophil activity in the last week prepartum has been associated with subsequent RP (Gunnink, 1984b; Kimura et al., 2000). The lack of impact of vitamin E injection on the incidence of early lactation clinical mastitis is consistent with a similar study by Erskine et al. (1997), but in contrast to reports of oral supplementation through the dry period and early lactation (Smith et al., 1984; Weiss et al., 1997). However, the control groups in these studies received little or no supplemental dietary vitamin E, whereas in the present study and that of Erskine et al. (1997), the majority of the study animals had adequate serum vitamin E before injection. The lack of association of treatment with the incidence of metritis or endometritis is in contrast to results from a similar study (Erskine et al., 1997) in which vitamin E reduced the incidence of postpartum uterine disease. However, the definition of metritis used here was restricted to animals with systemic illness, whereas Erskine et al. (1997) used a less explicit and more subjective case definition. Others (Harrison et al., 1984) have found no effect of oral vitamin E supplementation on the incidence of endometritis, although with reported rates of 60 to 85%, their case definition may have lacked specificity.

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Several interesting observations may be drawn from effects of the covariates on the risk of RP. The seasonal effect (lowest incidence of RP in Summer) was more pronounced among heifers, which may be due to a greater likelihood for late-gestation heifers to be pastured than with cows. In contrast to numerous observational studies, there was no effect of dystocia on the risk of RP. This may be attributable to a more restrictive case definition that required veterinary intervention. Conversely, milk fever was associated with increased risk of RP in the full dataset (as has been reported in numerous studies), but not in the smaller dataset including serum α-T:C measurements. This may simply reflect a lack of statistical power, because there is no known direct physiologic link between vitamin E and calcium homeostasis. On the other hand, recent evidence suggests that uterine motility in general (Eiler, 1997) and calcium supplementation in particular (Hernandez et al., 1999) are not causal factors for RP. Although nutritional management of hypocalcemia is independent of vitamin E, serum vitamin E status may act as a surrogate variable that helps to control confounding by unmeasured management factors that share elements of common pathways to hypocalcemia and low serum α-T (i.e., poor transition period nutritional management). However, twinning was significantly associated with a markedly increased risk of RP in both models. This effect would not be directly influenced by management factors because in few, if any, cases would cows have been known to be carrying twins ahead of time. Cows that calved within 7 d of treatment were at higher risk of RP than cows that calved 8 to 16 d after treatment, despite the lack of significant interaction with the effect of treatment in the final models. That is, cows that calved, on average, prior to their due date were more likely to have RP, independent of the effect of treatment. The physiologic basis for this is unclear, but could be associated with spending less time receiving a close-up dry cow ration and more rapid changes from far-off dry-cow rations and environment to lactating cow conditions. Selecting cows in which to measure vitamin E and selenium in a case-control structure may have biased the estimates of vitamin E status in the study population downward. Cows with RP were likely to have lower pretreatment and immediately prepartum serum vitamin E concentrations; therefore, cows with relatively low vitamin E status were overrepresented in the subsample relative to the whole study population. However, the objective was to provide an explanation of the observed treatment effect, not to provide an accurate estimate of the population mean. Journal of Dairy Science Vol. 85, No. 6, 2002

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The mean and median pretreatment concentrations of serum α-T were 2.7 and 2.5 µg/ml, respectively, which are above the threshold of 2.0 µg/ml for deficiency of vitamin E (Herdt and Stowe, 1991). However, these were below the suggested minimum target of 3.0 to 3.5 µg/ml (Weiss, 1998), and below the optimum of 3.5 to 4.0 µg/ml based on intracellular killing activity by neutrophils (Hogan et al., 1993). The pretreatment serum concentrations of α-T observed in the present study are very similar to those reported in peripartum cows in both an experimental study (Hogan et al., 1992), and a field survey in Ohio (Miller et al., 1995). In a controlled experiment of dietary supplementation of vitamin E, cows fed 100 IU/d through the dry period had mean plasma α-T of 2.2 µg/ml 2 wk prepartum, whereas cows fed 1000 IU/d had a mean of 3.0 µg/ ml (Weiss et al., 1997). If extrapolated to the present study, this suggests that on average, cows were receiving supplemental vitamin E, but at less than 1000 IU/ d, as has been advocated (Hogan et al., 1993; Weiss, 1998). Absorption and transport of vitamin E in the body closely follow that of lipid. Essentially all circulating α-T is located in lipoproteins, primarily high-density lipoprotein (Herdt and Smith, 1996). To account for this, particularly in transition cows that are mobilizing triglycerides, the most accurate routinely available measure of vitamin E status is the α-tocopherol to cholesterol ratio in serum (Weiss et al., 1992; Herdt and Smith, 1996). The suggestion has been made (Weiss et al., 1992; Herdt and Smith, 1996; Weiss, 1998) that the ratio of vitamin E to cholesterol is a more physiologically accurate measure of vitamin E status in peripartum dairy cows. The present results indicate that either serum α-T < 2.0 µg/ml or serum α-T:C < 2.5 × 10-3 1 wk before expected calving are useful thresholds to discriminate cows at significantly increased risk of RP. However, with respect to identifying cows for supplementation to prevent RP, only αT:C < 2.5 × 10-3 was predictive of a beneficial response to parenteral vitamin E. This supports the notion that, in peripartum dairy cattle, serum α-T:C better reflects the ability of the body to move circulating vitamin E into tissues and into neutrophils (Weiss et al., 1992). The cut-point identified here is consistent with the threshold of α-T:C ≥ 2.5 × 10-3 suggested to reflect “adequate” vitamin E status (AHDL-MSU reference range). Based on reduction in risk of early lactation clinical mastitis, an α-T:C mass ratio of 4.0 × 10-3 or greater may be optimal in peripartum cows (Weiss et al., 1997). In the present study, the mean and median pretreatment ratios of serum α-T:C were 3.2 and 2.9 × 10-3, respectively. Journal of Dairy Science Vol. 85, No. 6, 2002

Although selenium supplementation does not change the concentration of circulating vitamin E and vice versa (Weiss et al., 1990), selenium status has a potentially significant impact on the effect of supplemental vitamin E (Weiss et al., 1997). The mean and median pretreatment concentration of serum selenium was 69 ng/ml. This means that half of the animals had circulating selenium below the suggested concentration of 70 ng/ml (Hogan et al., 1993; AHDL-MSU reference range). Having pretreatment serum selenium level below 70 ng/ml was not a significant factor in any models of the risk of RP, but there were only 87 animals with selenium measurements. Injection of 3000 IU of vitamin E SC 1 wk before expected calving raised serum α-T and α-T:C in the days before calving and for 3 to 5 d postpartum (Table 5). The effect of treatment on peripartum serum vitamin E concentrations was consistent with other studies (Weiss et al., 1992, 1997). Serum α-T concentration was maintained in treated cows but fell in control cows, whereas serum α-T:C ratio was markedly increased in treated cows, but was unchanged or slightly decreased in control cows. Cows that received the injection of vitamin E had significantly higher serum α-T and α-T:C at calving than control cows (2.96 vs. 2.46 µg/ml, and 3.99 vs. 2.99 × 10-3, respectively). The present data indicated that overall, cows with serum αT:C ≥ 3.5 in the last week prepartum were significantly less likely to have RP, and that treatment made achievement of this threshold almost twice as likely. Furthermore, cows that received the injection of vitamin E were, on average, above the threshold and approaching the apparent optimum level of serum α-T:C for immune function (Weiss et al., 1997), but control cows, on average, were not. The level of serum α-T attained in response to injection was very similar to those in studies in which cows received 3000 IU of vitamin E s.c. 10 and 5 d before calving (3.1 µg/ml; Hogan et al., 1992) or 5000 IU i.m. once 1 wk before calving (approximately 3.0 µg/ml; Politis et al., 1995). In contrast, cows that received 3000 IU of vitamin E i.m. 8 to 14 d before calving (Erskine et al., 1997) had a higher peak level of plasma α-T around the time of calving (3.97 µg/ml) than in the present study. The level of serum α-T:C attained at calving in response to one injection of 3000 IU vitamin E s.c. appears to be comparable to that achieved by consumption of 1000 to 2000 IU of dietary vitamin E through the dry period (Weiss et al., 1997; Baldi et al., 2000). However, pretreatment vitamin E status in these studies was variable or not reported, yet appear to influence the absolute level of α-T attained. Furthermore, the values reported here are least squares means, accounting for the effects of parity and season of calving, as well as

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herd clustering, whereas the values reported elsewhere are generally unadjusted means. Finally, the relationship of serum α-T to neutrophil α-T is not clear (Weiss et al., 1992). Plasma α-T in the peripartum period has been shown to be positively correlated with intracellular killing by neutrophils (Hogan et al., 1992). However, it is not clear whether elevated α-T at calving is sufficient to enhance neutrophil function with respect to detachment of the placenta, or if α-T must be elevated prepartum in order to charge neutrophils with vitamin E during their development or migration. There was little relationship at the herd-level between pretreatment serum α-T:C and the incidence of RP (Figure 1). Conversely, there were clear associations at the cow level between prepartum serum vitamin E and the risk of RP, and between pretreatment serum vitamin E and the effect of injection of vitamin E on the risk of RP. This contrast is logical because there was large intraherd as well as interherd variation in serum vitamin E concentrations. Interherd variation depends largely on the amount of dietary vitamin E supplementation, as well as access to and quality of pasture for close-up dry cows. Intraherd variation depends on whether individual cows actually received and consumed the intended diet. Treatment was beneficial in cows with marginal vitamin E status (baseline serum α-T:C < 2.5 × 10-3) approximately 1 wk before calving. This implies that vitamin E does play a role in determining the likelihood of RP in multiparous cows, as in primiparous cows. However, 40% of heifers but only 30% of multiparous cows were below the adequate pretreatment ratio of α-T:C. In the last 2 wk prepartum, heifers consume approximately 4 kg less DM/d than cows (Vandehaar et al., 1999). If prepartum dietary supplementation with vitamin E is based on expected DMI by cows, heifers are likely to consume less than the targeted nutrient intake. On some farms, heifers may not receive a vitamin-fortified “close-up” diet before calving, or heifers may suffer decreased DMI due to social competition with older cows in the precalving group. Given the increased risk of RP in all cows with marginal vitamin E status, but a significant reduction in risk only in treated heifers, it is possible that the dose used was too low for older, presumably larger cattle. With a lower homeorrhetic drive for milk production, heifers mobilize less lipid prepartum than cows (Vandehaar et al., 1999). Thus, in heifers, there may have been greater partitioning of nutrients for growth, and therefore a greater movement of lipoproteins and vitamin E into tissues than in older cattle, where the net flow of lipids would be markedly out of tissues in support of lactation. Further research is needed to describe the

mechanism of movement of vitamin E into tissues and neutrophils, and whether this is different in primiparous compared with multiparous cattle. Finally, negative energy balance was likely less severe in heifers than in multiparous animals, which would favor neutrophil chemotaxis and therefore may contribute to a beneficial effect of supplemental vitamin E. Selection of animals for treatment appears to be best made at the individual, not the herd level. Most precisely, the benefit of prepartum parenteral vitamin E for reduction of risk of RP can be expected in animals with marginal prepartum vitamin E status. Primiparous animals were most likely to be in this category. Unfortunately, there was little association between herd-level measures of either vitamin E status or risk of RP and the effect of treatment. CONCLUSIONS The results of this study suggest that only cows with marginal vitamin E status (serum α-T:C < 2.5 × 10-3) 1 wk before expected calving will have a reduction in risk of RP as a result of one s.c. injection of 3000 IU of vitamin E. However, determination of the vitamin E status of individual cows costs approximately five times more than the injection and requires up to 1 wk for results. The present results suggest that a herdlevel mean vitamin E measurement may not yield useful information if there is wide variation between cows. Therefore, practically, it may be reasonable to treat primiparous cows, but not multiparous cows in many herds. ACKNOWLEDGMENTS Financial support was provided by Schering-Plough Animal Health and Dairy Farmers of Ontario. Particular thanks are due to Jodi Wallace for excellent assistance with data management. Tom Herdt and Mary Ruppenthal were extraordinarily helpful and efficient in performing the vitamin assays and offering advice. REFERENCES Allison, R. D., and R. A. Laven. 2000. Effect of vitamin E supplementation on the health and fertility of dairy cows: A review. Vet. Rec. 147:703–708. Arnaud, J., I. Fortis, S. Blachier, D. Kia, and A. Favier. 1991. Simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by isocratic high-performance liquid chromatography. J. Chromatogr. 572:103–116. Baldi, A, G. Savoini, L. Pinotti, E. Monfardini, F. Cheli, and V. Dell’Orto. 2000. Effects of vitamin E and different energy sources on vitamin E status, milk quality and reproduction in transition cows. J. Vet. Med. A 47:599–608. Bondurant, R. H. 1999. Inflammation in the bovine female reproductive tract. J. Dairy Sci. 82(Suppl. 2):101–110. Journal of Dairy Science Vol. 85, No. 6, 2002

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Eiler, H. 1997. Retained placenta. Pages 340–348 in Current Therapy in Large Animal Theriogenology. R. S. Youngquist, ed. W. B. Saunders Co., Philadelphia, PA. Erskine, R. J., P. C. Bartlett, T. Herdt, and P. Gaston. 1997. Effects of parenteral administration of vitamin E on health of periparturient dairy cows. J. Am. Vet. Med. Assoc. 211:466–469. Ferguson, J. D., D. T. Galligan and N. Thomsen. 1994. Principal descriptors of body condition score in Holstein cows. J Dairy Sci. 77:2695–2703. Goff, J. P., and R. L. Horst. 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci. 80:1260–8. Gunnink, J. W. 1984a. Retained placenta and leucocytic activity. Vet. Q. 6:49–51. Gunnink, J. W. 1984b. Pre-partum leucocytic activity and retained placenta. Vet. Q. 6:52–54. Harrison, J. H., D. D. Hancock, and H. R. Conrad. 1984. Vitamin E and selenium for reproduction of the dairy cow. J. Dairy Sci. 67:123–132. Herdt, T. H., and H. D. Stowe. 1991. Fat-soluble vitamin nutrition for dairy cattle. Vet. Clin. North Am. Food Anim. Pract. 7:391–415. Herdt, T. H., and J. C. Smith. 1996. Blood-lipid and lactation-stage factors affecting serum vitamin E concentrations and vitamin E cholesterol ratios in dairy cattle. J. Vet. Diagn. Invest. 8:228–232. Hernandez, J., C. A. Risco, and J. B. Elliott. 1999. Effect of oral administration of a calcium chloride gel on blood mineral concentrations, parturient disorders, reproductive performance, and milk production of dairy cows with retained fetal membranes. J. Am. Vet. Med. Assoc. 215:72–76. Hogan, J. S., W. P. Weiss, D. A. Todhunter, K. L. Smith, and P. S. Schoenberger. 1992. Bovine neutrophil responses to parenteral vitamin E. J. Dairy Sci. 75:399–405. Hogan, J. S., W. P. Weiss, and K. L. Smith. 1993. Role of vitamin E and selenium in host defense against mastitis. J. Dairy Sci. 76:2795–2803. Kimura, K., J. Goff, and H. Tyler. 2000. Association between retained placenta and neutrophil function in dairy cattle. J. Dairy Sci. 83(Suppl.1):3. (Abstr.) Lissemore, K. D., K. E. Leslie, P. I. Menzies, S. W. Martin, A. H. Meek, and W. G. Etherington. 1992. Implementation and use

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