Musculoskeletal injuries are a major cause of morbidity and death in racehorses1,2; in our experience, such injuries are responsible for approximately 50% of days lost from training among Thoroughbreds in flat-race training in the United Kingdom. The main problems affecting bones include fractures,3 disease of the dorsal aspect of the third metacarpal bone (also known as sore shins),4 and joint disease.5 These are essentially exercise-related injuries that occur because bone is insufficiently robust to withstand the loads engendered by training and racing. Fractures are the most serious problem and have the same underlying pathogenesis as fatigue fractures in human athletes6; results of a recent UK study3 indicatethat the incidence of nontraumatic fracture in flat-racing horses is 1.15/100 horse months. Several factors may influence fracture risk in horses, including sex,7–10 and among military recruits, women are at greater risk of sustaining a stress fracture.11
In humans, sex steroids are responsible for establishing dimorphism in the skeleton, and both initiate the pubertal growth spurt and end it by inducing growth plate closure.12,13 After skeletal maturation, sex hormones continue to affect the balance of bone remodelling, with estrogen acting to suppress bone turnover and maintain balanced rates of bone formation and resorption.14 It is also well established that estrogen deficiency at menopause results in increased bone turnover in women,15,16 and estrogen depletion following ovariectomy results in bone loss in several species including sheep,17 goats,18 rats,19 and mice.20 Circulating concentrations of estrogen fluctuate significantly throughout the estrous cycle, and studies21,22 in women and cynomolgus monkeys have determined that these changes are associated with altered bone turnover.
Although little information is available as to whether changes in circulating concentrations of sex hormones influence bone cell function in horses, we have recently determined that there are sex-associated differences in bone turnover in 2-year-old Thoroughbreds, with males (colts) having higher serum concentrations of biochemical markers of bone formation and resorption than females (fillies).23 In the same study, serum concentrations of PICP, a marker of type I collagen formation, were higher in fillies than in colts in late spring and early summer. Therefore, we hypothesized that this may be attributable to the influence of sex hormones because this period coincides with the start of the breeding season.
The natural breeding season of mares lasts from early spring to late summer, and the normal estrous cycle of 21 days is defined as the interval between ovulations when ovulation is accompanied by behavioral estrus or circulating progesterone concentration < 1 ng/mL.24 The period of estrus normally lasts 7 days; serum estrogen concentrations start to increase 2 or 3 days before this and peak near the middle of estrus.25 This is followed by a 14-day luteal phase when serum progesterone concentrations are > 1 ng/mL.25,26
Measurement of circulating progesterone concentration is therefore a useful way of determining when mares are in the luteal phase of the cycle.24
The purpose of the study reported here was to investigate the relationship between stage of estrous cycle and bone cell activity in Thoroughbreds. Our hypothesis was that fluctuations in sex hormone concentrations associated with the estrous cycle alter bone turnover in mares and that this is a source of uncontrollable variability when measuring serum bone marker concentrations in female horses, as it is in female humans. Concentrations of biochemical markers in serum were used to assess bone cell activity, and stage of estrous cycle was determined by measuring serum progesterone concentration. The biochemical markers measured were osteocalcin (a noncollagenous bone matrix protein that is released into the circulation following synthesis by osteoblasts), PICP (which provides a measure of newly synthesized type I collagen), and ICTP (a type I collagen degradation product that reflects osteoclastic resorption of bone matrix).27 In recent years, several studies28–31 have described the measurement of bone markers in horses, and currently, this is the only method for noninvasive assessment of changes in bone cell activity in vivo.
Materials and Methods
Horses and blood samples—Blood samples were collected at approximately 30-day intervals (in April through September 2002) from forty-seven 2-year-old Thoroughbred mares that were undergoing training with 7 commercial flat racing trainers. Informed consent was obtained from the trainers for all horses included in the study. The study was undertaken as part of a larger project to determine the relationship between serum bone marker concentrations and risk of injury or training in racehorses. On each occasion, blood samples were collected via jugular venipuncture, before exercise, and at the same time of the day (5 to 7 AM). The samples were then allowed to clot for 1 hour and were centrifuged at 2,000 Xg for 10 minutes. Samples were stored at −80°C until the end of the study, when all measurements were carried out.
Osteocalcin assay—Serum osteocalcin concentrations were measured by use of a commercially available competitive ELISAa that has been previously validated for use in horses.b Because the assay is calibrated for human use and concentrations in 2-year-old Thoroughbreds are markedly higher than those in human adults, all serum samples were diluted 1:2 with the wash solution provided with the kit to ensure that concentrations were within the expected range of the assay (0 to 32 ng/mL). The limit of sensitivity of the assay is 0.45 ng/mL. In this study, the intra-assay coefficient of variation for osteocalcin was 5.5% and the corresponding interassay variation was 5.6%.
PICP assay—The concentration of serum PICP was determined by use of a competitive radioimmunoassayc that has been previously validated for use in horses.29 Serum was diluted 1:5 with PBS solution when concentrations were greater than the upper assay limit of 500 μg/L. The limit of sensitivity of the assay is 1.2 μg/L. The intra-assay coefficient of variation was 5.6% in the concentration range in this study; the corresponding interassay variation was 6.7%.
ICTP assay—Concentrations of ICTP were measured in serum samples by use of a commercially available competitive radioimmunoassayc that has been previously validated for use in horses.29 The limit of sensitivity of the assay is 0.5 μg/L. The intra-assay coefficient of variation was 4.3% in the concentration range in this study; the corresponding interassay variation was 7.2%.
Progesterone assay—Serum progesterone concentrations were determined by use of an indirect competitive ELISA developed for use with bovine serumd; the ELISA was modified to use standards that were prepared in progesterone-free serum from colts that had previously been gelded. A serum progesterone concentration ≥ 1 ng/mL was considered an indication that the mare was in the luteal phase; a concentration < 1 ng/mL was considered an indication that the mare was in another stage of estrus. To confirm that the assay was valid for use in horses, progesterone concentrations in 23 samples from the April time point were also assessed by use of an immunometric assaye at a commercial laboratory.f The intra-assay coefficient of variation for the competitive ELISA was 8.9%. The corresponding interassay variation was 8%.
Statistical analysis—A mixed-model analysis of the data was performed by use of computer software.g The procedure fits mixed linear models to balanced or unbalanced data and estimates fixed effects and variance components by use of the restricted maximum likelihood method. The effect of stage of estrous cycle and month on serum marker concentrations was evaluated by including these as main and first-order interaction effects in the model, with serum osteocalcin, ICTP, and PICP concentrations as outcome variables. To take account of the repeated measures, month of sample was included as a repeated effect. Training yard was included as a random effect in the model because it was likely to account for variation between horses. Different covariance structures were assessed for use in modelling the dependence between observations, and a compound symmetry covariance structure was used in the final model. Statistical significance was achieved at a value ofP ≤ 0.05. Different models were compared with the Akaike information criterion. Differences between mares in the luteal phase and those at other stages of estrus at each monthly time point were identified by use of Bonferroni corrected post hoc tests. Agreement between the 2 progesterone assays in assigning horses to either the luteal-phase or other stage of estrus group was assessed by use of Cohen's kappa.
Results
Validation of serum progesterone ELISA—The indirect, competitive ELISA used was validated by comparing results with those of an immunometric assay used routinely by a commercial laboratory to measure progesterone concentration in equine serum. When serum progesterone concentrations ≥ 1 ng/mL were considered indicative of the luteal phase and those < 1 ng/mL were considered indicative of other stages of estrus, 21 of 23 horses were identically categorized by the 2 assays. Agreement between the 2 assays (as measured with Cohen's kappa) was 0.83 (95% confidence interval, 0.6 to 1.0; P < 0.001). There was a significant positive correlation between the 2 assays (R = 0.9; P < 0.001). Mean serum progesterone concentration for the samples assessed was 3.99 ng/mL for the ELISA and 2.71 ng/mL for the immunometric assay.
Reproductive status—Serum progesterone concentrations were used to establish whether the mares were in the luteal phase of the cycle (serum progesterone concentration ≥ 1 ng/mL) or at other stages of the estrous cycle (serum progesterone concentration < 1 ng/mL; Figure 1). Among the 47 mares, the number in the luteal phase was 22 (46.8%) in April and 31 (66%) in May; this increase reflected the onset of reproductive activity at this time of year. The number of mares in the luteal phase increased to a peak in August (36/47 [76.6%] mares) and then decreased again in September (29/47 [61.7%] mares), which is consistent with horses being seasonal, long-day breeders. All mares in the study population had a serum progesterone concentration > 1 ng/mL at 1 or more time points during the study period.
Percentage of Thoroughbred mares (n = 47) in the luteal phase of the estrous cycle (ie, had evidence of reproductive activity; gray bars) from April through September, compared with mares at other stages of estrus (white bars).
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Percentage of Thoroughbred mares (n = 47) in the luteal phase of the estrous cycle (ie, had evidence of reproductive activity; gray bars) from April through September, compared with mares at other stages of estrus (white bars).
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Percentage of Thoroughbred mares (n = 47) in the luteal phase of the estrous cycle (ie, had evidence of reproductive activity; gray bars) from April through September, compared with mares at other stages of estrus (white bars).
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Relationship between stage of estrous cycle and serum bone marker concentrations—Via a mixedmodel analysis, stage of the cycle was found to have a significant effect on serum osteocalcin and ICTP concentrations (P < 0.001 and P = 0.01, respectively). Mean ± SD serum osteocalcin and ICTP concentrations (37.49 ± 7.7 ng/mL and 10.89 ± 2.87 μg/L, respectively) were higher in mares in the luteal phase, compared with mares at other stages of estrus (35.78 ± 7.04 ng/mL and 10.71 ± 2.48 μg/L, respectively). No significant (P = 0.51) effect on serum PICP concentration was detected. When bone marker concentrations for mares in the luteal phase and those at other stages of the estrous cycle were compared in individual months, osteocalcin concentrations were found to be higher in mares during the luteal phase in April (P = 0.036), May (P = 0.022), and July (P = 0.004) and serum ICTP concentrations were significantly (P = 0.027) higher in May (Figure 2).
Serum concentrations of osteocalcin (A), ICTP (B), and PICP (C) in Thoroughbred mares (n = 47) in the luteal phase (gray bars) and other stages of estrus (white bars) from April through September. Compared with mares at other stages of estrus, serum osteocalcin and ICTP concentrations were significantly (P < 0.001 and P= 0.01, respectively) higher in mares in the luteal phase. The number (N) of mares in each group is given under each bar. Boxes represent the interquartile range containing 50% of all values. The whiskers extend to the highest and lowest values, excluding outliers. The horizontal line across each box represents the median value. *Serum osteocalcin concentrations were significantly higher in mares in the luteal phase in April (P = 0.036), May (P = 0.022), and July (P = 0.004), compared with mares at other stages of estrus. •Serum ICTP concentration was significantly (P = 0.027) higher in mares in the luteal phase in May, compared with mares at other stages of estrus. NS = Serum PICP concentration was not significantly (P > 0.05) different between mares in the luteal phase and mares at other stages of estrus.
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Serum concentrations of osteocalcin (A), ICTP (B), and PICP (C) in Thoroughbred mares (n = 47) in the luteal phase (gray bars) and other stages of estrus (white bars) from April through September. Compared with mares at other stages of estrus, serum osteocalcin and ICTP concentrations were significantly (P < 0.001 and P= 0.01, respectively) higher in mares in the luteal phase. The number (N) of mares in each group is given under each bar. Boxes represent the interquartile range containing 50% of all values. The whiskers extend to the highest and lowest values, excluding outliers. The horizontal line across each box represents the median value. *Serum osteocalcin concentrations were significantly higher in mares in the luteal phase in April (P = 0.036), May (P = 0.022), and July (P = 0.004), compared with mares at other stages of estrus. •Serum ICTP concentration was significantly (P = 0.027) higher in mares in the luteal phase in May, compared with mares at other stages of estrus. NS = Serum PICP concentration was not significantly (P > 0.05) different between mares in the luteal phase and mares at other stages of estrus.
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Serum concentrations of osteocalcin (A), ICTP (B), and PICP (C) in Thoroughbred mares (n = 47) in the luteal phase (gray bars) and other stages of estrus (white bars) from April through September. Compared with mares at other stages of estrus, serum osteocalcin and ICTP concentrations were significantly (P < 0.001 and P= 0.01, respectively) higher in mares in the luteal phase. The number (N) of mares in each group is given under each bar. Boxes represent the interquartile range containing 50% of all values. The whiskers extend to the highest and lowest values, excluding outliers. The horizontal line across each box represents the median value. *Serum osteocalcin concentrations were significantly higher in mares in the luteal phase in April (P = 0.036), May (P = 0.022), and July (P = 0.004), compared with mares at other stages of estrus. •Serum ICTP concentration was significantly (P = 0.027) higher in mares in the luteal phase in May, compared with mares at other stages of estrus. NS = Serum PICP concentration was not significantly (P > 0.05) different between mares in the luteal phase and mares at other stages of estrus.
Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1527
Discussion
In the present study, increases in serum concentrations of biochemical markers of bone formation and resorption were detected during the luteal phase of the estrous cycle in Thoroughbreds. To the authors' knowledge, this is the first report of findings that indicate that bone cell activity is influenced by stage of reproductive cycle in mares; the data not only provide basic information on biological processes in equine bone, but also form the basis for future studies to establish the clinical relevance of this association. Furthermore, our data indicated that the stage of estrus must be considered as a source of uncontrollable variability in serum bone marker concentrations in female horses, as it is in female humans.
The finding that serum osteocalcin and ICTP concentrations were significantly higher during the luteal period, compared with other stages of estrus, is consistent with results of several similar studies32–34 in humans. Although serum PICP concentration in mares of the present study was not significantly affected by stage of the estrous cycle, PICP is a less specific marker for bone, and type I collagen synthesis in other tissues may contribute to circulating PICP concentrations.35,36 In cynomolgus monkeys, serum osteocalcin and urinary CTx (a marker of bone resorption) concentrations are also inversely correlated with circulating estrogen concentration during the estrous cycle.22 Our data are also consistent with findings in postmenopausal women and ovariectomized animals that have indicated that the effect of estrogen withdrawal is an increase in bone turnover.16,37-39 However, it was not possible to obtain an immunoassay that could reliably measure low serum concentrations of estrogen in horses for use in the present study, and the relationship between bone turnover and serum estrogen concentration in horses still needs to be established. To provide more information on the net balance between bone formation and bone resorption at different stages of estrus, concentrations of bone markers, progesterone, and estrogen should be measured longitudinally (eg, every other day) in a group of mares throughout an estrous cycle. Obviously, this approach was not feasible in the present study because the mares used were not experimental animals but were being maintained in a commercial training environment.
When bone marker concentrations in mares in the luteal phase and those at other stages of the estrous cycle were compared by individual months, serum osteocalcin concentrations during the luteal phase were significantly higher in April, May, and July. For ICTP, serum concentrations were also significantly higher in mares in the luteal phase in May. In a previous pilot study performed by our group, significantly higher serum bone marker concentrations were also detected in mares in the luteal phase during April, which suggests that bone cells may be particularly sensitive to changes in concentrations of circulating sex hormones that occur in late spring with the onset of estrous cycling. In addition, because mares included in the present study were only 2 years old, it is likely that this would be their first estrous cycle and thus the first time the skeleton had been exposed to the bone-modulating effects of sex hormones, which may have enhanced the potential effect on bone turnover.
It must also be considered that other factors, including intercurrent disease, drug administrations, or exercise (all of which could not be controlled in the present study), may have reduced the magnitude of the effect of sex steroids on serum bone marker concentrations.
Exercise affects bone cell function, and it has been determined that high-speed gallop training, which horses undergo increasingly throughout the training season, is inversely correlated with serum osteocalcin and ICTP concentrations in 2-year-old Thoroughbreds.h It is also that likely this accounts for the gradual decrease in serum marker concentrations over the period of the study, as we have previously determined that age and time of year (season) have no significant effect on serum bone marker concentrations in 2-year-old horses of either sex.23 It is therefore possible that the effects of exercise on bone marker concentrations could be a confounding factor in the present study and may explain why differences in serum bone marker concentrations between mares in the luteal phase and those at other stages of the estrous cycle were less apparent later in the breeding season. It is noteworthy that although it has been widely reported that intense exercise regimens can lead to amenorrhea and hypo-estrogenism in female athletes,40 results from the present study indicate that the intensive training regimens undertaken by 2-year-old racing Thoroughbred mares do not interfere with cyclicity. It must also be considered that the less apparent difference in serum bone marker concentrations between the luteal phase and other phases of the estrous cycle later in the breeding season may reflect a decrease in bone turnover at the end of puberty.
The findings of our study led us to hypothesize that increased bone turnover associated with changing sex steroid concentrations during the estrous cycle could place mares at increased risk of injury at specific times of year. Some epidemiologic studies8–10 have previously identified sex as a risk factor for injury in racing Thoroughbreds. Results of 1 study10 indicated that there was a significant association between sex and the probability of fatal musculoskeletal injury during racing, whereas for horses that had a fracture during training, females were generally younger and geldings were generally older than expected. In a more recent study,8 geldings were at higher risk of musculoskeletal injury during racing, compared with mares. In contrast, a recent UK epidemiologic study3 did not reveal that sex was a risk factor for fracture. However, as far as we are aware, no studies have specifically examined the relationship between stage of the estrous cycle or stage of the breeding season and injury. Epidemiologic approaches clearly provide a method that could be used in future studies to assess the relationship between stage of estrus and risk of injury. Another way of testing the clinical importance of this relationship would be to determine whether bone mass (and thus bone strength) changes in mares between the first and last estrous cycles in a breeding season. Clearly, such a study would have to be done in an experimental situation and involve mares that were not in training because exercise is known to result in changes in bone mineral density.41,42 Furthermore, dual energy X-ray absorptiometry and quantitative computed tomography (methods that can be used to determine changes in bone mineral density in horses ex vivo43,44) cannot currently be used in standing, conscious horses.
Overall, our data have indicated that bone cell activity in Thoroughbred mares fluctuates during the estrous cycle and that the luteal period is associated with an increase in serum concentrations of biochemical markers of bone formation and bone resorption. Further studies are now required to determine whether these changes in bone cell activity associated with stage of estrus are of clinical relevance.
ABBREVIATIONS
| PICP | Carboxy-terminal propeptide of type I collagen |
| ICTP | Cross-linked carboxy-terminal telopeptide of type I collagen |
Metra Osteocalcin, Quidel Corp, San Diego, Calif.
Hoyt S, Siciliano PD. A comparison of ELISA and RIA techniques for the detection of serum osteocalcin in horses, in Proceedings (abstr). 16th Equine Nutr Physiol Symp 1999; 351–352.
Orion Diagnostica, Espoo, Finland.
Ridgeway Science, St Briavels, Gloucestershire, UK.
Immulite Progesterone, Diagnostic Products Corp, Los Angeles, Calif.
Beaufort Cottage Laboratories, Newmarket, Suffolk, UK.
SAS for Windows, version 8.1, SAS Institute Inc, Cary, NC.
Jackson BF, Lonnell C, Verheyen K, et al. Biochemical markers of bone turnover in racehorses are influenced by training and gender (abstr). J Bone Miner Res 2002;17:1334.
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