Abstract
Determining if ovariectomy will abolish unwanted behaviors can be challenging in mares without ultrasonographic ovarian abnormalities and/or endocrine values not consistent with a granulosa cell tumor. The objective of the study was to identify mares that have a positive improvement in unwanted behaviors after elective standing laparoscopic ovariectomy and to determine if the change is associated with any pre- and post-operative endocrine profiles and/or with ovarian histological findings. During the study period 27 mares underwent a bilateral standing laparoscopic ovariectomy. Pre- and post-operative questionnaires and endocrine profiles, along with ovarian histopathology were performed and data were analyzed. Eighty-nine percent of clients were satisfied with the change in behavior after surgery. There were no significant associations between specific elevated hormones or absent luteal tissue with any specific unwanted behaviors. Pre-operative endocrine values were not predictive of post-operative owner satisfaction but bilateral ovariectomy for the treatment of unwanted behaviors in mares had a high likelihood of achieving owner satisfaction.
Résumé
Histopathologie ovarienne, analyse endocrinienne pré- et post-opération et modifications du comportement chez 27 juments soumises à une ovariectomie bilatérale laparoscopique en position debout. Déterminer si une ovariectomie éliminera les comportements non-désirés peut représenter un défi chez les juments sans anomalie ovarienne détectable par échographie et/ou ayant des valeurs endocrines non cohérentes avec une tumeur des cellules de la granulosa. L’objectif de la présente étude était d’identifier les juments avec une amélioration positive des comportements non-désirés à la suite d’une ovariectomie élective par laparoscopie en position debout et de déterminer si le changement était associé avec un quelconque profil endocrinien pré- et post-opération et/ou avec des trouvailles histologiques dans les ovaires. Durant la période d’étude 27 juments ont été soumises à une ovariectomie laparoscopique bilatérale en position debout. Des questionnaires pré- et post-opération ainsi que des profils endocriniens et des analyses histopathologiques des ovaires furent réalisés et les données analysées. Quatre-vingt-neuf pourcents des clients étaient satisfaits avec le changement dans le comportement après la chirurgie. Il n’y avait pas d’association significative entre des niveaux élevés d’hormones spécifiques ou l’absence de tissu lutéal et des comportements non-désirés spécifiques. Les valeurs endocrines pré-opération n’étaient pas prédictives de la satisfaction post-opération des propriétaires mais l’ovariectomie bilatérale pour le traitement de comportements non-désirés chez les juments avait un grand potentiel d’obtenir la satisfaction du propriétaire.
(Traduit par Dr Serge Messier)
Introduction
Mares are commonly presented to veterinarians with the complaint of unwanted behaviors including aggression, training issues, disagreeable demeanor, excitability, and frequent urination (1). Frequent use of hormonal supplements for behavior modification, and increasing number of owners and referring veterinarians interested in surgical options to abolish these unwanted behaviors indicate that there is a demand for medical and surgical intervention in these mares. Ovariectomies are traditionally performed for treatment of ovarian neoplasms (2–4) and ovarian or estrus-related medical issues (1,3,5–7); however, bilateral ovariectomies have become more frequently performed for treatment of undesired behaviors in mares (1,5,7). This treatment for unwanted behaviors was first described by Hooper et al (5), and a more recent study by Kamm and Hendrickson (1) found an 83% improvement in behavior, from the clients perspective, after ovariectomy in mares. These studies, however, did not include any hormonal analysis or histopathology results.
Mares with granulosa cell tumors (GCT) can display prolonged anestrus, aggressive or undesired stallion-like behavior, persistent estrus-like behaviors, or nymphomania, which are characteristic for the condition (2–4,8–10). Mares demonstrating unwanted behaviors, such as aggression and persistent estrus, in which there are no ovarian abnormalities identified on transrectal ultrasound and/or in the serum endocrine profile or have an abnormal endocrine profile that is not consistent with a GCT are diagnostically challenging. It is difficult for the veterinarian to determine if an ovariectomy will abolish the unwanted behaviors in these mares. Determining which of these mares would respond positively with improvement in behavior post-ovariectomy could allow for a more accurate and appropriate case selection. The objectives of this study were to determine the proportion of mares that have a positive improvement in unwanted behaviors after laparoscopic ovariectomy and if the change in behavior can be associated with the pre- and post-operative endocrine profiles and/or with ovarian histological findings.
Materials and methods
Case selection and pre-operative management
Mares admitted to Colorado State University Veterinary Teaching Hospital for a bilateral ovariectomy because of unwanted behavior were included in this study. Routine signalment and history were obtained and owners completed a pre-operative questionnaire which included the reason for pursuing the ovariectomy, description of any specific unwanted behaviors, and duration of the unwanted behaviors. Initial physical examination parameters and pre-operative blood analysis [including at a minimum blood packed cell volume and total protein values, or a complete blood (cell) count (CBC) and serum chemistry] values were recorded before surgery in all mares. A pre-operative blood sample (30 mL) was obtained from the jugular vein. Serum was separated from the samples, aliquoted into cryovials, and stored at −20°C for future endocrine panel analysis. Samples from all patients were stored frozen and the analysis was performed at the end of the study period for laboratory consistency. A pre-operative transrectal ultrasound of all mares was performed and ovarian size and appearance were recorded. Feed was withheld for 12 h before surgery, but mares had access to water until the time of surgery.
Surgical procedure
An IV catheter was placed in the left or right jugular vein and the mare was positioned in standing stocks. Pre-operative penicillin G potassium (Pfizerpen; Pfizer, New York, New York, USA), 22 000 IU/kg body weight (BW), IV, and flunixin meglumine (Prevail; VetOne, Boise, Idaho, USA), 1.1 mg/kg BW, IV, were administered approximately 30 min before the start of surgery. Sedation was initially achieved with detomidine hydrochloride (Dormosedan; Zoetis, Kalamazo, Michigan, USA), 0.01 mg/kg BW, IV, and butorphanol tartrate (Torbugesic; Zoetis), 0.01 mg/kg BW, IV. Throughout the procedure a level plane of sedation was maintained by a continuous infusion of detomidine hydrochloride (20 mg; Zoetis) and butorphanol tartrate (10 mg; Zoetis) in 1 L 0.9% sodium chloride (at a rate adjusted to effect). The surgical sites were routinely clipped, aseptically prepared, draped, and then blocked with local anesthetic infiltration at the determined portal sites using mepivacaine hydrochloride (Carbocaine-V; Zoetis), 0.01 to 0.05 mL/kg BW per site. Three laparoscopic portals were created in bilateral paralumbar fossas as previously described (11,12). The peritoneal space was insufflated with carbon dioxide to 10 to 12 mmHg and maintained throughout the procedure. Mepivacaine hydrochloride (Carbocaine-V; Zoetis), 0.01 to 0.05 mL/kg BW, was injected into the mesovarium, mesosalpinx, and proper ligament of the ovary using a laparoscopic injection needle (12). All ovariectomies were performed using laparoscopic graspers and a vessel-sealing device (LigaSure Atlas; Medtronic, Minneapolis, Minnesota, USA) for ovarian pedicle hemostasis and tissue transection as previously described (12). After ovarian pedicle transection, both ovaries were removed from the left paralumbar fossa. The incisions were closed in a conventional manner (11). All ovaries were submitted for histopathologic assessment and review by a Board-certified pathologist.
Post-operative management and follow-up
After surgery, the mares were monitored for any complications during the hospitalization period. Patients received flunixin meglumine (Prevail; VetOne), 1.1 mg/kg BW, IV, at 12 h after surgery and then transitioned to a course of oral phenylbutazone (VetriBute; VetOne), 2.2 mg/kg BW, q12h for 4 d. No post-operative antibiotics were administered. At a time between 60 and 90 d after surgery, owners completed a follow-up questionnaire. A venous blood sample (30 mL) was obtained at this time and the serum was aliquoted into cryovials and stored at −20°C for future endocrine panel analysis.
Histopathologic evaluation
Ovaries were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 to 5 μm, and stained with hematoxylin and eosin (H&E) for histologic examination. Representative sections determined by pathologist discretion, from each submitted ovary were examined by a Board-certified pathologist. Other stains were used when indicated. No attempt was made to distinguish between GCT and granulosa-theca cell tumors.
Hormone profile analysis
Pre- and post-operative serum samples were submitted to the University of California, Davis Clinical Endocrinology Laboratory and analyzed for levels of testosterone, inhibin, estrone sulfate, anti-Müllerian hormone (AMH), and progesterone. These tests are validated for use in the horse. Reference ranges of this laboratory were used to determine normal versus abnormal values.
Statistical analysis
Mare age and weight were reported as ranges and means. Duration of unwanted behavior, reported specific unwanted behaviors, elevated hormone levels, histopathological diagnosis of GCT, and time until owners noticed a change in behavior were all reported as frequencies and percentages. When analyzing the association between specific unwanted behaviors and endocrine elevations or diagnosis of CGT on histopathology, if the continuous data did not meet normality assumption, the data were log-transformed before analysis. If the numbers were low, then a non-parametric approach of the Kruskal-Wallis test was performed to analyze continuous data, whereas Fisher’s exact test was used to analyze categorical data. A P-value of 0.05 was considered statistically significant. SAS v9.4 (SAS Institute, Cary, North Carolina, USA) was used for all statistical analyses.
Results
Over an 18-month period, 27 mares with unwanted behaviors were presented to Colorado State University Teaching Hospital for a bilateral ovariectomy. Mare breeds included 13 American Quarter horses, 4 American Paint horses, and 10 mares belonging to 9 different breed types. Mares were between 3 and 24 y old (median: 10 y) and weighed from 327 to 650 kg (median: 475 kg).
The mares were presented for various unwanted behaviors and often more than 1 specific behavior per mare (Table 1). The unwanted behavioral issues had been present for < 6 mo in 26% (7/27), 6 to 12 mo in 11% (3/27), and > 12 mo in 63% (17/27) of mares. After surgery, there was ≥ 83% improvement in all specific unwanted behaviors and > 90% improvement in the behaviors including general disagreeable demeanor, aggression towards other horses, problems with training, and displaying constant estrus (Table 1).
Table 1.
Number of mares (%) presented for specific behavior issues and post-operative improvement in the unwanted behavior.
| Number of mares presenting for specific unwanted behavior | Post-operative improvement in specific unwanted behavior | |
|---|---|---|
| General disagreeable demeanor | 21/27 (78%) | 20/21 (95%) |
| Aggression towards other horses | 20/27 (74%) | 19/20 (95%) |
| Problems with training | 18/27 (67%) | 17/18 (94%) |
| General excitability | 18/27 (67%) | 16/18 (89%) |
| Stallion-like behavior | 18/27 (67%) | 16/18 (89%) |
| Displaying constant estrus | 14/27 (52%) | 13/14 (93%) |
| Frequent urination | 12/27 (44%) | 10/12 (83%) |
| Aggressiveness towards people | 7/27 (26%) | 6/7 (86%) |
Before surgery, rectal ultrasound of the ovaries was performed on all mares and appeared to be normal with no suspicion of a GCT and represented varying stages of the estrus cycle or anestrus. Ovary size ranged from 2.8 × 3.3 cm to 6.0 × 7.0 cm and appearance ranged from multiple small follicles to as large as a 50-mm follicle.
Pre-operative endocrine analysis was performed on 26 mares and post-operative endocrine analysis was performed on 25 mares. One pre-operative sample, which belonged to a mare with normal ovaries on histopathologic assessment, was misplaced. Two mares were euthanized before the 60-day post-operative blood was taken. In 1 of these cases the mare’s aggression towards humans was unmanageable and did not change after ovariectomy; the mare was euthanized 5 wk after surgery. The second mare was euthanized due to a humeral fracture (5 wk after surgery) unrelated to the ovariectomy. The number of mares with abnormal hormone concentrations prior to surgery for each group are reported in Table 2. Anti-Müllerian hormone was the most commonly increased endocrine value with 50% of all mares having concentrations above the cut-off value > 3.8 ng/mL and only 44% of mares with histologically confirmed GCT having an increase in AMH. A testosterone level > 45 pg/mL was the most consistent hormone elevated in 56% of mares diagnosed with a GCT. Post-operative serum concentrations of testosterone, AMH, and inhibin were within the normal reference ranges for all 27 mares. Concentrations of these 3 hormones were significantly decreased from the pre-operative serum concentration (P < 0.0001). No mares had post-operative progesterone concentrations > 0.5 ng/mL. Post-operative progesterone and estrone sulfate were significantly lower than pre-operative values, both with P-values < 0.0001. No significant associations were found between specific hormone concentrations and specific unwanted behaviors. Ovarian histological assessment confirmed bilateral normal ovaries in 67% (18/27), unilateral GCT in 19% (5/27), and bilateral GCTs in 15% (4/27). Diagnosis of a GCT was not associated with any specific pre-operative behavior.
Table 2.
Pre-operative endocrine profiles in mares undergoing bilateral ovariectomy.
| Testosterone | Inhibin > 0.7 ng/mL | Anti-Müllerian hormone (AMH) | Estrone sulfate > 6.0 ng/mL | Progesterone | |
|---|---|---|---|---|---|
| All mares (n = 27) | A 8/26 (31%) | 1/26 (4%) | C 13/26 (50%) | 3/26 (12%) | E 13/26 (50%) |
| B 1/26 (4%) | D 5/26 (19%) | F 14/26 (54%) | |||
| Histologically confirmed GCT (n = 9) | A 5/9 (56%) | 0/9 (0%) | C 4/9 (44%) | 1/9 (11%) | E 4/9 (44%) |
| B 1/9 (11%) | D 1/9 (11%) | F 4/9 (44%) | |||
| Histologically confirmed normal (n = 17) | A 3/17 (18%) | 1/17 (6%) | C 9/17 (53%) | 2/17 (12%) | E 9/17 (53%) |
| B 0/17 (0%) | D 4/17 (24%) | F 10/17 (59%) |
UC Davis Clinical Endocrinology Laboratory reference ranges at the time of sample submission:
Testosterone (non-pregnant): Normal: 20 to 45 pg/mL; marginally elevated: 45 to 70 pg/mL; elevated: 70 to 100 pg/mL; indicative of tumor: ≥ 100 pg/mL. A-values — > 45 pg/mL but < 100 pg/mL; B-values — > 100 pg/mL.
Inhibin (non-pregnant): Normal ranges: < 0.7 ng/mL; indicative of tumor: > 0.7 ng/mL. AMH (non-pregnant): Normal range: 0.1 to 6.9 ng/mL; marginally elevated: 7.0 to 7.9 ng/mL; concentrations consistent with granulosa cell tumor: > 8.0 ng/mL; C-values — > 3.8 ng/mL but < 8.0 ng/mL; D-values — > 8.9 ng/mL. Estrone sulfate (standard size mares): Non-pregnant or pregnant (first 45 days of pregnancy): 0.1 to 6.0 ng/mL; borderline: 6.1 to 10 ng/mL; pregnancy of 45 d or greater: > 10.0 ng/mL. Progesterone: Absence of active luteal tissue: 0.1 to 0.5 ng/mL; borderline for the presence of luteal tissue: 0.5 to 1.0 ng/mL; presence of luteal tissue: > 1.0 ng/mL; E-values — < 0.5 ng/mL; F-values — < 1.0 ng/mL but > 0.5 ng/mL.
Clients had a high level of satisfaction after surgery independent of the presenting complaint(s). Eighty-two percent (22/27) of owners were very satisfied, 7% (2/27) satisfied, 4% (1/27) partially satisfied, and 7% (2/27) were dissatisfied with the overall outcome with the change in unwanted behaviors post-ovariectomy. Eight percent (2/24) of owners noticed the change in behavior within 2 wk after surgery, 71% (17/24) between 2 and 4 wk after surgery, 13% (3/24) between 1 and 2 mo after surgery, and 8% (2/24) between 2 and 6 mo after surgery. For the 1 mare whose owner was partially satisfied and the 2 mares whose owners were dissatisfied with the procedure, the reason given was there was no change in the unwanted behaviors observed post-ovariectomy. In 1 of these cases the mare’s aggression towards humans was unmanageable and did not change post-ovariectomy. Due to the concern for human safety, 5 wk after surgery this mare was euthanized; she had bilateral GCT confirmed on histopathology.
Discussion
Some owners may discriminate against using intact mares for performance because of unwanted behaviors during estrus (5). It can also be a challenge to manage large groups of intact mares where aggressive behavior towards other horses can be detrimental to humans and horses, particularly in confined areas such as is the case for some guest ranches and rescues. Standing bilateral laparoscopic ovariectomy offers the client a minimally invasive and affordable technique to potentially eliminate unwanted behavior related to hormonal influences. However, questions remain on the justification and success for this procedure in mares when performed for the reason of behavior alone (13). Our results demonstrate a high (89%) overall owner satisfaction with the outcome in change in behavior after bilateral ovariectomy. Improvement was noted in all types of undesirable behaviors described by owners. This is consistent with findings from previous studies in which client satisfaction post-ovariectomy for the treatment of unwanted behaviors has been reported as 75% to 83% (1,5,14).
In this study there was no significant association between a specific elevated hormone (testosterone, inhibin, AMH, or estrone sulfate) or absent luteal tissue (low progesterone) with any specific unwanted behaviors. Results from this study support the conclusion that unwanted behavior in mares having normal appearing ovaries on ultrasound could be explained by having early stage GCTs, as 33% (9/27) of mares in this study had a confirmed GCT on histopathology. Bilateral GCTs may also be more common than previously thought and was recently reported to occur in 27% of mares diagnosed with GCT (15). Our results support that bilateral GCTs are underdiagnosed, as 44% (4/9) of mares in this study with a GCT diagnosed, undergoing bilateral ovariectomy for unwanted behaviors with little clinical suspicion of GCT, had confirmed bilateral GCT identified on histopathology.
Granulosa cell tumors are hormonally active (8,16) and reported hormone analysis suggestive of a GCT includes decreased serum progesterone, and increased serum testosterone (8,17,18), inhibin (17), and AMH (19–21). An increase in testosterone in GCT’s usually occurs if there is a significant thecal cell component to the tumor and results in typical stallion-like behavior (3,17). The pre-operative endocrine results in our study showed that testosterone was increased above the normal reference range in 56% of the mares in which a GCT was histologically confirmed. This is consistent with the percentages of 40% to 54% from previous reports (20–23).
Inhibin production from the GCT is responsible for the inactivity of the contralateral ovary via suppression of follicle-stimulating hormone release from the pituitary (3). Inhibin has been previously reported as elevated in 60% to 100% of cases with confirmed GCT (15,18,20–22). Our results were not consistent with previous reports as inhibin was increased in none of the mares with confirmed GCT. This finding in a subset of mares without having substantial ovarian enlargement or ultrasonographic abnormalities of the affected ovary before surgery, suggests tumor growth was still in the early stages. Concentrations of AMH and inhibin have been highly correlated in cases of confirmed GCTs, reflecting their production by the granulosa cells within the tumor (21). Anti-Müllerian hormone was > 3.8 ng/mL in 44% (4/9) and > 8 ng/mL in 11% (1/9) of mares with a confirmed GCT, which again is well below previous reports of 98% to 100% (19–21). As with the results for inhibin, these mares likely had early stage tumor growth and therefore lacked a substantial AMH concentration. Interestingly, AMH concentrations in this study were found to be > 3.8 ng/mL in 9 of 17 mares and > 8.0 ng/mL in 4 of those 9 mares with histologically normal ovaries. Reported values of AMH range between 0.22 to 2.94 ng/mL in normal cycling and pregnant mares while concentrations > 4 ng/mL to 4.7 ng/mL have been used in previous studies to diagnose GCTs; however, UC Davis laboratory suggested values for suspected GCT are > 8 ng/mL with a gray zone of 3.8 to 8.0 ng/mL.
Surgical removal is regarded as the treatment of choice for GCTs (2,4,9–10,15,17,24). Inhibin (17) and testosterone concentrations decreased to a basal level within 24 h after surgical removal of the GCT (8,16,23) and AMH concentrations decreased about 50% within 48 h after GCT surgical removal (20). The rapid decline in various hormone concentrations after surgery infers that the increased pre-ovariectomy concentrations are of tumor origin (20,23). In our study, concentrations of testosterone, AMH, inhibin, and progesterone had all significantly decreased to basal levels and were within normal reference ranges 60 d after surgery. This return to normal concentrations occurred in mares with both histologically diagnosed GCTs and normal ovaries.
More recently, in addition to removal of diseased ovaries, elective bilateral ovariectomies have increased in popularity for correction of unwanted behaviors (1,7,14). These behaviors can be dangerous to other horses or humans and cause enough frustration and/or fear in owners and handlers that euthanasia is considered and pursued in some cases. While there was substantial owner satisfaction in behavior after ovariectomy in this study, it has been previously reported that mares exhibiting extreme aggression may not be successfully treated by this method (5,25). There was one such case in this study, in which a mare with confirmed bilateral GCT was euthanized 5 wk after ovariectomy for continued extreme aggression towards humans. This may have been a learned behavior that may or may not have been related to the tumor. Unfortunately, this mare did not undergo endocrine testing before euthanasia to determine if hormonal concentrations had returned to baseline after surgery.
Post-operative improvement in behavior is not always obtained in cases with the absence of confirmed GCT (1,14). This leads to continued questions on the justification for performing bilateral ovariectomies on the grounds of unwanted behaviors, and whether these unwanted behaviors can actually be improved with surgery (13). Results from this study support that mares with unwanted behavior will subjectively improve following surgery. Improvement rates depend on specific unwanted behaviors, but in this study ranged from 83% to 95%. Mares lose the normal pattern of estrous cyclicity after ovariectomy but may still show variable signs of estrus (5,7,25–26). Consistent with previous reports, results from this study show a similar outcome with 11% of clients partially satisfied or dissatisfied with the outcome in changing the behavior (5). The reason estrus and unwanted behaviors can be observed after an ovariectomy is still controversial (1,5,14,25–27). Our study provides no strong evidence to support a given reason for the continued unwanted behaviors in a small subset of mares post-ovariectomy; however, it is confirmed that hormones are at basal levels after bilateral ovariectomy.
Limitations of the study include that behavior was subjectively assessed and based on the opinion of the owners/agents of the mares. This may have resulted in a placebo effect as clients were not blinded to the treatment. The ovariectomies resulted from clinical cases seen at our hospital and the procedures occurred at various times of the year. As horses are seasonal breeders and time of the year can have various effects on the estrous cycle, seasonal effects on the endocrine profiles cannot be ruled out. This was a prospective study; therefore, we were limited by case enrollment. This may have affected the determination of associations between pre-operative hormonal concentrations and unwanted behavior; however, testosterone, AMH, inhibin, and progesterone all returned to normal concentrations after surgery. Serum from mares was frozen and stored for varying lengths of time. While many clinical samples are performed on recently collected samples, many studies use frozen samples to evaluate hormonal concentrations. Additionally, human sex-steroid hormones including progesterone, testosterone, and estradiol have been shown to be preserved frozen for up to 22 y (28) and even repeated freezing and thawing has had no meaningful effects on serum hormone concentrations (29).
Although the case numbers in this report were not high, to the authors’ knowledge this is the largest prospective clinical laparoscopic ovariectomy study evaluating pre- and post-operative behavior with ovarian histological findings and pre- and post-operative endocrine profiles. Overall, in the present study, a lower percentage of mares with GCT confirmed on histopathology had increased hormone concentrations compared to previous reports. This is believed to be a result of the high number of early stage GCTs in this study. Aside from the low percentage of increased hormone concentrations in mares with confirmed GCT, there was also a high number of false positive AMH results in the pre-operative endocrine values in mares with histologically confirmed normal ovaries.
Pre-operative hormone levels were not predictive of likelihood in beneficial effects of bilateral ovariectomy on improvement in behavior after surgery. However, clients in this study reported an overall high level of satisfaction in specific unwanted behaviors in the mares. Mares undergoing ovariectomy through minimally invasive standing laparoscopy have minimal post-operative pain (12), and although serious complications can occur, minimal morbidity and low occurrence of complications is reported (6). With this low morbidity and complication rate and a client satisfaction in 89% of mares undergoing bilateral ovariectomies for unwanted behaviors, it appears to be justified to perform bilateral ovariectomies on the grounds of unwanted behaviors. However, setting realistic client expectations about the outcome of bilateral ovariectomy for treatment of unwanted behaviors is important and it should be discussed that in a small subset of mares the behaviors are unchanged and mares can continue to show signs of estrus or unwanted behaviors.
Acknowledgments
The authors gratefully acknowledge the Marion and Hadley Stuart Foundation for funding this project and all Colorado State University equine clinicians, technicians, and veterinary students for their assistance and care of the mares.
Footnotes
The authors declare no conflict of interest related to this report.
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (hbroughton@cvma-acmv.org) for additional copies or permission to use this material elsewhere.
Funding for the project was through a donation from Marion and Hadley Stuart Foundation. There was no meeting with discussion of the results.
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