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* Department of Population Medicine, and
Department of Animal and Poultry Science, University of Guelph, Ontario, Canada, N1G 2W1
1 Corresponding author: rwalsh{at}uoguelph.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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1,000 µmol/L in the first week postpartum were less likely to be diagnosed pregnant after first insemination. In the second week postpartum, the cows with circulating BHBA concentrations 1,400 µmol/L were significantly less likely to be pregnant after first artificial insemination. A dose response relationship was found when a comparison of the probability of pregnancy after first insemination and duration of elevated circulating ketone bodies was investigated. The probability of pregnancy was reduced by 20% in cows diagnosed subclinically ketotic in either the first or second week postpartum. Nevertheless, cows above the subclinical ketosis threshold in both the first and second week postpartum were 50% less likely to be pregnant after first insemination. Similarly, the median time to pregnancy increased in cows experiencing elevated BHBA concentrations in either (124 d) or both (130 d) the first and second week postpartum relative to cows never experiencing elevated BHBA concentrations (108 d). To further investigate this, the effect of elevated circulating BHBA was permitted to vary with time. The effect decreased with time, such that the daily probability of pregnancy increased similar to nonsubclinically ketotic cows by approximately 160 d in milk. From this analysis, both the relative circulating concentration of BHBA and the duration of elevated circulating BHBA were negatively associated with the probability of pregnancy at first service.
Key Words: subclinical ketosis • ß-hydroxybutyrate • time to pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Cows experiencing delayed onset of luteal activity postpartum typically have lower DMI, produce less milk, and subsequently lose more body condition early in lactation (Staples et al., 1990). At the whole-animal level, the magnitude and duration of negative energy balance are influenced by feed intake and milk production. At the organ and molecular level, variation in energy utilization efficiency, ranging from rumen function to the ability of the liver to repartition mobilized fat, affect energy balance status.
Previous research has reported an association between calculated negative energy balance and reproductive traits, including days to first luteal activity, first service conception risk, days from calving to AI, and days from calving to conception (Villa-Godoy et al., 1988; Staples et al., 1990; Reist et al., 2003b). The physiological state of energy deficiency impairs hypothalamic responsiveness to circulating estradiol-17ß, resulting in reduced GnRH pulse frequency and concomitant reduction in LH required for follicular deviation and eventual ovulation (Jolly et al., 1995; Dawuda et al., 2002; Butler, 2003).
Energy balance is dependent on many factors. Various metabolic and endocrine blood and milk measures such as NEFA, ketone bodies, glucose, insulin, milk fat, or fat:protein ratio are indirect measures of energy balance. Circulating ketones increase when the maximal ability of the liver to oxidize NEFA and store triacylglycerol is exceeded. Associations between elevated circulating ketone concentration and periparturient uterine disease were described by Reist et al. (2003a); however, investigation of thresholds was based on the distribution of circulating ketone concentrations or anecdotal experience rather than the associated change in the risk of disease (Duffield et al., 1998). Similarly, the effect of postpartum circulating ketone concentration on PR/AI, time to commencement of luteal activity, and time to pregnancy has been described later in lactation with variable results (Andersson and Emanuelson, 1984; Cook et al., 2001). A circulating BHBA concentration threshold immediately postpartum, defined by demonstrable reduction in the PR/AI, has not been validated.
The objectives of this retrospective analysis were to investigate relationships between the magnitude and duration of increased serum BHBA measured at –3 wk and in each of the first, second, third, sixth, and ninth weeks postpartum and the PR/AI, the time from calving to first AI, and time from calving to pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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2.5 thin; 2.75 to 3.5 fair; 3.75 fat); and collected blood samples. Each blood sample was collected from a coccygeal blood vessel into a 10-mL vacuum tube (Monoject red stopper blood collection tubes; Sherwood Medical, St. Louis, MO). Blood was collected at the time of monensin CRC or placebo oral administration 3 wk before calving and at wk 1, 2, 3, 6, and 9 postpartum. Blood samples were stored, on ice, in an insulated cooler from time of collection until sample processing occurred. Within 5 h postcollection, blood samples were centrifuged at 733 x g for 10 min. The samples of serum were submitted to the Clinical Pathology Laboratory (Department of Pathobiology, Ontario Veterinary College) for the measurement of Ca, P, total protein, urea, glucose, and BHBA concentrations and determination of aspartate aminotransferase activity using an automated analyzer (Dacos 2 analyzer; Coulter Electronics, Hialeah, FL). Peripartum disease information including dystocia (veterinary-assisted parturition), retained placenta (failure to pass the fetal membranes within 24 h), milk fever (veterinary diagnosed), metritis (inflammation of the uterus <15 DIM), clinical ketosis, displaced abomasum (left or right displacement diagnosed by a veterinarian), respiratory illness, and lameness was captured in on-farm data sheets, veterinary records, and on-farm computer record systems (Duffield et al., 1999). Reproductive data, including DIM at first insemination, conception date, number of inseminations, herd removal date, and pregnancy status at removal, were recorded using the same methods. There was minimal use of ovulation synchronization protocols. Pregnancy diagnosis was performed at least 40 d after insemination by rectal palpation. Five herds had first lactation animals excluded, because they had been exposed to a monensin premix within 4 wk of parturition.
Data Management and Statistical Analysis
Statistical analysis was performed using Intercooled Stata 9.1 (StataCorp LP, College Station, TX) and SAS (SAS Inst. Inc., Cary, NC). Descriptive statistics were used to characterize animals diagnosed pregnant after first insemination, relative to animals diagnosed not pregnant. Data were collected as part of a clinical trial; therefore, treatment remained in all analyses.
The shape of the BHBA curve between weeks was assessed using PROC MIXED in SAS (SAS Institute, 2004), accounting for the random effect of herd and repeated measures of cows within herd. After transforming the BHBA data into the natural logarithm scale, variables offered to the model included treatment, parity, periparturient disease, the pregnancy status diagnosed after first AI, and all 2-way interactions. Residual analysis was performed at the cow level. The distribution of residuals was symmetrical with long tails, thus violating the normality of residual assumption. To investigate the effect of influential observations (extreme values in the independent or dependent variables), they were removed from the analysis. Removal of influential observations improved the normality of residual assumption without affecting the direction of the reported coefficients indicating that the observed effect was not solely due to the effect of extreme observations. Final models included all observations. Least squares means of weekly BHBA concentration were estimated for pregnant and non-pregnant cows at each weekly measurement. To appropriately account for the repeated measures within cow, a postestimation adjustment was performed on the reported probability value using the Bonferroni-Sidak adjustment to account for correlation between sampling intervals.
The effect of BHBA concentration at each week on probability of pregnancy at first insemination was assessed using multivariable logistic regression accounting for correlation of cows within herd using a generalized estimating equation with an exchangeable correlation structure. Cow level variables offered to the model included parity, season of calving, initial BCS, BCS at 60 DIM, change in BCS, season of calving, and peripartum disease information. Herd level variables included farm type (free stall vs. tie stall) and herd size. Treatment with monensin was retained in all models, because the data were generated as part of a monensin clinical trial. Initial screening of variables was performed using a manual backward stepwise procedure with significant variables (P < 0.1) remaining in the model. This procedure was repeated for each week post-partum in which serum BHBA measurements were available. Initially, serum BHBA was offered as a continuous variable. Subsequently, based on the appearance of a smoothed scatterplot of serum BHBA and probability of pregnancy at first AI, realistic thresholds were created at 200 µmol/L increments from 1,000 to 1,600 µmol/L. Ultimately, sequential multivariable logistic regression, accounting for the effect of clustering at the herd level, was applied based on the predicted thresholds to evaluate their potential significance.
The effect of prolonged exposure to elevated ketone bodies on PR at first insemination was investigated by generating a composite variable representing the additive effect of the number of weeks with serum BHBA 1,000 µmol/L in the first week and 1,400 µmol/ L in the second week.
Finally, time from calving to first insemination and time to pregnancy were modeled using semiparametric survival analysis. This method accounted for animals that did not become pregnant. Robust standard errors were generated to account for clustering of animals within herds. Plotting the cumulative hazard relative to modified Cox-Snell residuals assessed goodness of fit. Animals with survival times less than 30 DIM were excluded from this analysis, because they were not at risk for pregnancy, and they did not complete the sampling protocol. The proportionality assumption was assessed graphically by plotting the logarithm of the hazard function against the logarithm of time. The Akaike information criterion was used to assess the overall fit of the final model. Delta-beta-like residuals were used to identify points of leverage, whereas deviance residuals were calculated and sorted to identify possible outliers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The data and outcome of first insemination were recorded for 796 cows. The PR/AI at first insemination was 34.8%. Further characterizations of animals based on pregnancy result to first AI are presented in Table 1
. Median time from calving to first insemination was 74 d, ranging from 31 to 243 d. Days in milk at first AI, parity, season of calving, and periparturient disease were significant in a model to determine risk factors for PR/AI. A BCS of 3.25 to 3.75 was assigned to 86% of animals independent of treatment. Treatment did not affect the probability of pregnancy at first AI (monensin-treated group, 35.2%; placebo group 34.5%; OR = 1.03; P = 0.84).
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). Treatment with the monensin CRC reduced the BHBA estimate at each sampling point (P < 0.001). Periparturient disease did not significantly alter the shape of the BHBA curve. Neither initial BCS, final BCS (thin, fair, fat), nor BCS change (1 unit) was significant in a model of factors affecting the probability of pregnancy at first AI.
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. Serum BHBA in wk 3, 6, and 9 were not significantly associated with PR.
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. From these models, cows with serum BHBA 1,000 µmol/L in wk 1 or 1,400 µmol/L in wk 2 were significantly less likely to be diagnosed pregnant after first insemination. There were a total of 288 animals with serum BHBA 1,000 µmol/L in wk 1, and 150 cows were 1,400 µmol/L in wk 2. A total of 104 cows were classified as SCK in both wk 1 and 2.
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).
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). Further, median time to first AI was delayed (P < 0.01) for cows calving in the spring relative to cows calving in the fall [median, 95% confidence interval (CI) spring = 81 d (95% CI = 78 to 85 d; fall 70 d, 68 to 72 d)].
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). The effect of elevated BHBA concentration remained until approximately 140 DIM, after which the daily probability of pregnancy, measured using the adjusted hazard ratio (HR), between SCK and non-SCK cows was equivalent (Table 6; Figure 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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There was a tendency for increased circulating BHBA concentrations in non-pregnant cows after first AI from 3 wk before calving to 9 wk after calving relative to cows diagnosed pregnant. The delayed effect of negative energy balance or hyperketonemia on reproductive performance is well documented. Both the duration and magnitude of negative energy balance are associated with reduced hypothalamic GnRH pulse frequency and perpetuation of an inappropriate negative feedback loop between estradiol concentration and the required LH release necessary to support follicular deviation and ovulation. This failure of communication along the hypothalamic-pituitary-ovarian axis is the most common physiologic lesion associated with negative energy balance and leads to delayed resumption of luteal activity (Reist et al., 2000; Butler, 2003).
Within individual weeks, the probability of pregnancy after first AI decreased linearly with increasing circulating BHBA concentration in both wk 1 and 2. A 3% reduction in probability of pregnancy for each 100 µmol of BHBA/L increase was significant in wk 2 (Figure 2
). This difference is relatively small; however, the range of values observed in the second week makes this finding very large at BHBA concentrations >1,400 µmol/L. Pregnancy risk at first insemination was not significantly affected by BHBA concentrations in wk –3, 3, 6, or 9. A strong association between elevated circulating ketone concentration in the first week post-partum and risk of uterine disease was reported (Reist et al., 2003a). Significant associations between proportion of lactating cows cycling by 30 DIM, PR/AI, and proportion pregnant by 80 DIM have been reported with hyperketonemia from the second to fifth week after calving (Plym Forshell et al., 1991; Reist et al., 2000; Koller et al., 2003).
Within individual weeks and using thresholds, cows with circulating BHBA concentrations 1,000 µmol/L (40% of cows) in the first week and 1,400 µmol/L (19% of cows) in the second week were at significantly increased risk of non-pregnancy after first insemination (Table 2). In the first week after calving, a 25% reduction in the probability of pregnancy was consistently predicted independent of threshold used. This association was significant at 1,000 µmol/L of BHBA. The low number of cows above the higher threshold increases the likelihood of a type II error in this analysis. In the second week after calving, the probability of pregnancy decreased with increasing BHBA concentration thresholds, with a 40 to 50% reduction at thresholds 1,400 µmol/L.
The duration of exposure to elevated circulating BHBA affects the PR/AI. Cows identified as SCK in either of the first 2 wk were 17% less likely to be pregnant after first AI, whereas cows that remained above the SCK thresholds in both weeks were 53% less likely to be pregnant. The probability of pregnancy after first insemination in cows classified as SCK in either week was not different (P = 0.28) from cows never classified as SCK but was greater than cows classified as SCK in both weeks (P = 0.02; Table 3). There are few other studies with which to compare these findings. The magnitude of and the rate of recovery from negative energy balance were associated with time to pregnancy (Reist et al., 2003a). Similarly, most of the variation in onset of ovarian activity and time to pregnancy was attributed to the maximum ketone concentration recorded, independent of the week postpartum when it occurred (Reist et al., 2000; Koller et al., 2003).
Time from calving to first insemination is a reflection of management practice regarding the voluntary waiting period and estrus detection efficiency and accuracy. Subclinically ketotic cows in either of the first 2 wk were inseminated 8 d later than animals that were never SCK. Delayed insemination between cows classified as SCK in either week relative to those classified as SCK in both weeks was not anticipated. This outcome may reflect a differential effect on reproductive efficiency for cows that experience a first elevation in circulating ketone concentrations in the first week (increased risk of uterine disease; Reist et al., 2003a) and second week (increased risk of delayed ovulation; Reist et al., 2000) relative to cows that recover from hyperketonemia before the second week of lactation (Table 3).
There was a significant effect of SCK on time from calving to pregnancy. Concentrations of BHBA that were 1,000 µmol/L in wk 1 and 1,400 µmol/L in wk 2 were associated with increased time to pregnancy. This effect was not consistent over time. Animals that were SCK in either week had an adjusted HR of 0.55 at 60 DIM and 0.75 at 120 DIM. Animals SCK in both weeks had an associated HR of 0.52 at 60 DIM and 0.84 at 120 DIM. In other words, the negative effect of SCK early in lactation appears to diminish as cows progress through lactation. Moreover, it is important to note that there was no significant difference in time to pregnancy between cows classified as SCK in either week relative to those classified as SCK in both weeks (P = 0.28; Table 5).
Despite reports on the association between energy balance and commencement of luteal activity, PR/AI, and time to first insemination and to pregnancy, the underlying mechanisms are still not fully clear. The results of the current analysis provide further evidence of residual effects of health problems in the transition period on subsequent reproductive performance. Specifically, SCK in early lactation is associated with impaired reproductive performance 50 to 100 d later. This delayed effect on reproductive traits has been attributed to increased risk of delayed commencement of ovulation (Butler, 2003). More recently, Leroy et al. (2006) found that in vitro maturation of follicles in media conditions analogous to those follicular fluid concentrations of glucose and BHBA experienced during SCK significantly reduced the ability of fertilized ova to become morula and hatched blastocysts, indicating a direct toxic effect of BHBA and NEFA on maturing ova.
The peak herd level prevalence of SCK has been reported from the second to fifth week postpartum (Andersson and Emanuelson, 1984). Attempts have been made to understand factors that influence when the peak level of SCK occurs within individual herds. One such study by Heuer et al. (2001) reported that season of calving and pen number (specifically management practices such as time away from feed) explained 17% of the variation in milk acetone concentrations. Thus, application of this data would best identify herds at risk for reproductive failure rather than cows.
Similar to the present study, others reported no association of BCS at calving, or change in BCS early in lactation, with any reproductive parameters (Ruegg and Milton, 1995). The distribution of BCS and BCS loss in this data set was similar between animals that were pregnant to first insemination and those that were not pregnant. Insufficient variation in body condition data has been previously cited as a partial explanation for the inconsistent association between BCS and energy balance (Heuer et al., 2000).
A treatment effect of Na monensin on time to pregnancy has not been reported. Further, the CRC formulation was not associated with alterations in commencement of luteal activity, days to first insemination, or days to conception, despite consistent reductions in serum BHBA (Abe et al., 1994; Lean et al., 1994; Duffield et al., 1999). No previous study, investigating the effect of monensin, has included ketone information in the same model. Inclusion of BHBA in the same model removes the effect on PR/AI achieved through the improvement of energy status that monensin treatment permits and assigns it to BHBA concentration. Thus, the underlying mechanism to explain this treatment effect is unknown. Monensin (22 mg/kg of DM) decreases days to first ovulation and decreases the number of large follicles before 30 DIM with no change in first service conception risk (Tallam et al., 2003). Both clinical and subclinical endometritis increases time to pregnancy (LeBlanc et al., 2002). This association may represent the link between monensin treatment and a reduced daily probability of pregnancy (HR).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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1,000 µmol/L in the first week postpartum or 1,400 µmol/L in the second week postpartum, were 20% less likely to be pregnant at first insemination. The probability of pregnancy was reduced by 50% in cows experiencing SCK in both of the first 2 wk. Using time to pregnancy as the outcome, SCK in either the first 2 wk postpartum decreased the daily probability of pregnancy (HR) until approximately 165 d postpartum.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication August 28, 2006. Accepted for publication January 31, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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