J. Dairy Sci. 2008. 91:1433-1437. doi:10.3168/jds.2007-0567
© 2008 American Dairy Science Association ®
Short Communication: Prepartal Concentration of Estradiol-17β in Heifers with Stillborn Calves
U. S. Sorge*,1,
D. F. Kelton* and
R. Staufenbiel
* Department of Population Medicine, University of Guelph, Guelph, Ontario, N1G 2W1 Canada
Klinik für Klauentiere, Freie Universität Berlin, 14163 Berlin, Germany
1 Corresponding author: usorge{at}uoguelph.ca
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ABSTRACT
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This study was conducted to investigate hormonal imbalances preceding stillbirths and dystocia in primiparous heifers. The study was conducted between 2003 and 2004 on a German dairy farm, including 433 heifers. Starting 3 wk before calving, a weekly blood sample was collected. At calving, another blood sample was obtained, and the calving ease (grade 0 = unassisted to grade 2 = heavy pull with mechanical calf puller), sex, birth weight, as well as vitality status (stillborn, alive) of the calf were recorded. The blood serum was analyzed for estradiol-17β and progesterone concentration. At parturition, the measured estradiol-17β concentration was greater in heifers delivering bulls than in those with female calves and was increasing with greater birth weight of the calf and increasing calving difficulty score. Already 2 wk before calving, the serum estradiol-17β concentration was significantly smaller in heifers with stillborn than live calves. On the other hand, the progesterone concentration was greater 2 wk before calving in heifers with stillborn calves, but it was unaffected by the birth weight or sex of the calf or the calving difficulty score. Stillborn and live calves did not differ in birth weight or pregnancy duration. The smaller estradiol-17β concentrations of the heifers with stillborn calves could indicate an abnormality of the placenta or an abnormality of hormonal signals from the calf to the placenta in the weeks before the calving.
Key Words: hormone • estradiol-17β • stillbirth • heifer
In recent years, the percentage of stillborn dairy calves has increased (TopAgrar, 2001; Kornmatitsuk et al., 2004). Stillbirths are defined as the death of a calf after 260 d of pregnancy, before, during, or within the first 24 h postnatum. Stillbirth can be caused by multiple reasons. The main factor leading to stillbirths is dystocia, accounting for approximately two-thirds of the perinatal losses. Heifers are particularly prone to dystocia because of a disproportion of maternal pelvis and calf size (Hickson et al., 2006). Nevertheless, approximately one-third of stillborn calves are born without assistance and seem grossly normal. Previous studies have shown hormonal imbalances in the weeks before calving in cows with dystocia (O’Brien and Stott, 1977) or stillborn calves (Kornmatitsuk et al., 2003, 2004). The objective of this study was to examine the hormonal profile of Holstein heifers prepartum in regards to dystocia and stillbirths.
The study was conducted between November 2003 and October 2004 on 1 German dairy farm. The heifers on this farm were AI using high-calving ease bulls, and the herd average age at first calving was 25.6 ± 1.0 mo.
In the last 3 wk of pregnancy, 433 Holstein heifers were bled once per week (at the morning feeding) as well as within 30 min after calving. Nine milliliters of blood was collected from the coccygeal vein/artery into a serum syringe (9 mL Z, S-Monovette, Sarstedt, Germany; needle: BD Microlance 3; 18 G; 1.5; 1.2 x 40 TW.PM.; BD Drogheda, Ireland). The blood samples were centrifuged within 1 h of sampling (4,000 g/min for 15 min.), and the serum was subsequently frozen at –20°C until further analysis. Serum was analyzed for estradiol-17β and progesterone using radioimmunoassay test kits with 125I-marked estradiol-17β and progesterone, respectively (ImmuChem Double Antibody Estradiol-17β and ImmuChem Double Antibody Progesterone, MP Biomedicals Germany GmbH, Eschwege), in accordance with the instructions of the manufacturer. The cross-reactivity of the antiserum against estradiol-17β was as follows: estradiol-17β 100%, estrone 20%, estriol 1.15%, estradiol-17
0.68%, and androstenedione, progesterone, and testosterone smaller than 0.01%. According to the manufacturer, the detection limit of the kit is 7.2 pg/mL. The intraassay and interassay variation of the test lie between 4.7 and 10.7% and 5.9 and 11.9%, respectively. The cross-reactivity of the antiserum against progesterone was as follows: progesterone 100%, 20-dihydroprogesterone 5.41%, desoxycorticosterone 3.80%, corticosterone 0.7%, 17 -hydroxyprogesterone 0.67%, pregnenolone 0.41%, androstenedione 0.23%, testosterone 0.16%, and cholesterol and estrogens smaller than 0.1%.
Heifers close to calving were monitored hourly, 24 h a day, by experienced and obstetrically trained herdsmen. The herdsmen provided obstetrical assistance if there was no obvious progress in the birth of the calf for 30 to 50 min, in the absence of visible abnormal presentation. Assistance was given earlier if an abnormal presentation of the calf was diagnosed. The calving difficulty score was 0 = unassisted calvings, 1 = easy pull, 2 = heavy pull, use of mechanical calf puller required, and 3= cesarean section or fetotomy. At birth, the calving difficulty score, sex of the calf, its birth weight, and vitality status (stillborn/alive) were recorded. The serum samples were not available for analysis from approximately 40 animals at calving (Table 1
).
The statistical analyses were carried out utilizing the software SPSS (SPSS for Windows, Release 15.0., SPSS Inc., Chicago, IL). The association between categorical variables was examined utilizing exact 
2 test. Differences in hormonal profile, birth weight of the calf, and pregnancy duration between the categories of the dichotomous variables sex of the calf, and occurrence of stillbirths (yes/no) were analyzed utilizing the Student t-test. To assess correlations between the continuous variables, we used Spearman rank correlation.
Twenty-eight calves of the 433 calves were stillborn (Table 2). Stillbirth was not correlated to the sex of the calf (P > 0.4). The serum estradiol-17β concentration of the heifers increased exponentially in the last 3 wk of pregnancy and was positively correlated to the birth weight of the calf in the last 20 d of pregnancy (20 to 16 d: r = 0.246, P= 0.003; 15 to 11 d: r = 0.143, P= 0.025; 10 to 6 d: r = 0.229, P = 0.000; 5 to 1 d: r = 0.143, P = 0.000; parturition: r = 0.217, P = 0.000). No correlation was found between the progesterone concentration and the birth weight of the calf (P 
0.1).
The sex of the calf influenced the maternal serum estradiol-17β concentration. Heifers carrying bull calves had greater serum estradiol-17β concentration than those with female calves (P 
0.009). For example, at calving, the mean estradiol-17β concentration was 871 ± 457 pg/mL and 722 ± 343 pg/mL for heifers with male and female calves, respectively. Furthermore, the ease of calving seemed to be associated with different prepartum estradiol-17β concentrations. Grade 3 calvings were not observed. At parturition, the greater the calving difficulty score, the greater the measured estradiol-17β concentration (Figure 1). Heifers calving with a grade 2 dystocia (n = 22) had greater estradiol-17β concentrations than heifers with unassisted (n = 193; P = 0.002) or light pull calvings (n = 177; P = 0.09), which differed as well (P = 0.02). Of grade 2 calvings, 43.5% of the calves were born with breech presentation, and 43.5% of the heifers had reduced relaxation of soft birth canal. Breech presentations and poor soft tissue relaxation accounted for 25.0 and 21.4% in parturitions with stillborn calves and 6.9 and 13.8% in those with liveborn calves, respectively (P 0.002).
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Figure 1. Prepartum estradiol-17β concentrations of heifers with different calving ease scores (calving ease: 0 = unassisted; 1 = light pull; 2 = heavy pull, use of mechanical calf puller;  = outliers, * = extreme values; a,b: P = 0.02; a,c: P = 0.002; b,c: P = 0.09).
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In the last 2 wk of pregnancy, heifers with stillborn calves had smaller estradiol-17β concentrations (P 0.026; Figure 2), whereas the prepartum progesterone concentration was greater (d 15 to 11 prepartum) than in heifers with live calves (P = 0.028). But neither the birth weight nor the duration of the pregnancy differed between stillborn (38.8 ± 5.5 kg; 278.3 ± 7.0 d) and calves born alive (38.9 ± 3.8 kg; 279.4 ± 4.7 d; P 0.4). At calving, the serum estradiol-17β concentration of heifers with stillborn calves (n = 24) was 531 ± 381 pg/mL compared with 821 ± 412 pg/mL in those with liveborn calves (n = 368; P < 0.001). No difference was detected in the progesterone concentration at calving. Therefore, the estradiol-17β:progesterone ratio differed between heifers with stillborn and calves born alive at calving (P = 0.002). Yet, this difference was not detected in the days preceding calving (P > 0.28).
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Figure 2. Prepartum estradiol-17β concentrations of heifers with stillborn and live calves ( = outlier, * = extreme value; a,b: P = 0.026; c,d: P = 0.002; e,f: P < 0.001).
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Two weeks before calving, heifers with stillborn calves expressed smaller serum estradiol-17β concentrations. Similar observations were made by Kornmatitsuk et al. (2003, 2004), who found smaller estrone sulfate concentrations in heifers with stillborn calves prepartum. The serum estradiol-17β concentration was correlated to the sex and birth weight of the calves, the latter being correlated to the size of the placenta (Alexander, 1964; Mellor, 1987). Also, male calves were significantly heavier than female calves. In late gestation, estrogens are produced in the bovine fetoplacental unit (Echternkamp and Hruska, 1984; Döcke, 1994). The sex of the calf was, however, evenly distributed between dead and live calves, and the birth weight also did not differ between these 2 groups. Therefore, a smaller placenta as the reason for the lower serum estradiol-17β concentration (Zdunczyk, 1991) or an in-sufficiency in the placental nutrient supply to the calf seem unlikely. Significant variations were also observed in the serum estradiol-17β concentrations with different calving difficulty scores. Based on in vitro observations, Leung and Wathes (2000) postulated that progesterone functions as an inhibitor, whereas estradiol triggers oxytocin receptor synthesis in the endometrium.
The statistical analysis of the data generated by this project was limited by the general lack of statistical power. Although we were able to generate hormonal profiles for 393 heifers, only 24 of these animals produced stillborn calves, limiting our effective sample size to 24. Given these circumstances, a multivariable analysis was not recommended. Nevertheless, an attempt was made to fit 2 logistic models to investigate the ability of prepartum concentrations of estradiol and progesterone to predict stillbirth. Included in these models were the calving difficulty score, birth weight, and sex of the calf. But in neither case was the model fit deemed to be good enough to stand up to scrutiny.
Prepartal hormonal imbalances (estradiol-17β to progesterone concentration) still could have led to the expression of less oxytocin receptors in the uterus and an impaired preparation of the soft tissues for the parturition (Shah et al., 2006). This would subsequently lead to weaker bouts of uterine contractions to correctly position the calf in the birth canal and inhibit the correct expulsion of the calf and relaxation of the soft birth canal. This might explain the observed duration of the third stage of labor being 108 ± 57.2 min and 79 ± 33.6 min for stillborn and calves born alive (P = 0.038), respectively. Furthermore, the duration of the second stage of parturition was not monitored so that we do not know whether crucial delays occurred during this period as observed by Carrier et al. (2006). But, it has to be emphasized that one-third of the stillborn calves were born without assistance. Nevertheless, the difference in the duration of the stage 3, large percentage of breech presentations, and missing relaxation of the soft tissue of the birth canal in grade 2 calvings and stillborn calvings seem to support the possibility of weaker bouts. But, without close inspection of the fetal membranes or a postmortem examination of the stillborn calves, it is impossible to draw further conclusions regarding whether the lower estradiol-17β concentrations of the heifers with stillborn calves indicate an abnormality of the placenta as suggested by Shah et al. (2007) or an abnormality of hormonal signals from the calf to the placenta as early as 2 wk before calving. Risk factors leading to this phenomenon of stillborn calves are not known. In this study, it seems unlikely that management or nutrition affected the placental function or stillbirth rate.
All heifers were homebred, raised under the same management conditions with the same nutritional regimen, and bred to bulls that were tested and selected for light calving ease. But, a genetic component cannot be ruled out completely. Further studies are needed examining the composition of the placenta of stillborn calves and investigating a possible heritability of stillbirth. Given the small prevalence of stillbirth, future studies will need to be significantly larger to allow for a meaningful statistical analysis.
Received for publication August 1, 2007.
Accepted for publication December 19, 2007.
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