Evaluation of plasma ACTH, α-melanocyte–stimulating hormone, and insulin concentrations during various photoperiods in clinically normal horses and ponies and those with pituitary pars intermedia dysfunction

Jill Beech Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA 19348.

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 VMD, DACVIM
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Raymond C. Boston Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA 19348.

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 PhD
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Dianne McFarlane Department of Physiological Sciences, Center of Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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Sue Lindborg Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA 19348.

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Abstract

Objective—To measure plasma ACTH, D-melanocyte–stimulating hormone (D-MSH), and insulin concentrations during various photoperiods between February and October in horses and ponies with and without pituitary pars intermedia dysfunction (PPID).

Design—Cohort study.

Animals—13 clinically normal (control) ponies, 14 clinically normal (control) horses, 7 ponies with PPID, and 8 horses with PPID.

Procedures—Blood samples were collected from February through October during 8 photoperiods: 1, February 13 through March 2; 2, April 4 through 6; 3, June 19 through 22; 4, August 6 through 7; 5, August 14 through 17; 6, September 4 through 6; 7, September 26 through 28; and 8, October 16 through 18. Plasma ACTH, D-MSH, and insulin concentrations at each photoperiod were compared among groups.

Results—Log ACTH concentration was increased during photoperiod 4 through 8, compared with photoperiod 1 through 3, in all groups. In photoperiod 3 through 7, log ACTH concentrations were higher in horses and ponies with PPID, compared with values for control horses and ponies. D-Melanocyte–stimulating hormone (log and raw value) concentration was higher in photoperiod 2 through 8, compared with photoperiod 1, in control horses and ponies. In horses and ponies with PPID, log D-MSH concentration was higher in photoperiod 3 through 8, and D-MSH concentration was higher in photoperiod 4 through 8, compared with photoperiod 1. In control horses and ponies, plasma insulin concentration was lower in photoperiod 3 than in photoperiod 1.

Conclusions and Clinical Relevance—Plasma D-MSH and ACTH concentrations increased as daylight decreased from summer solstice (maximum daylight hours) to 12 hours of daylight.

Abstract

Objective—To measure plasma ACTH, D-melanocyte–stimulating hormone (D-MSH), and insulin concentrations during various photoperiods between February and October in horses and ponies with and without pituitary pars intermedia dysfunction (PPID).

Design—Cohort study.

Animals—13 clinically normal (control) ponies, 14 clinically normal (control) horses, 7 ponies with PPID, and 8 horses with PPID.

Procedures—Blood samples were collected from February through October during 8 photoperiods: 1, February 13 through March 2; 2, April 4 through 6; 3, June 19 through 22; 4, August 6 through 7; 5, August 14 through 17; 6, September 4 through 6; 7, September 26 through 28; and 8, October 16 through 18. Plasma ACTH, D-MSH, and insulin concentrations at each photoperiod were compared among groups.

Results—Log ACTH concentration was increased during photoperiod 4 through 8, compared with photoperiod 1 through 3, in all groups. In photoperiod 3 through 7, log ACTH concentrations were higher in horses and ponies with PPID, compared with values for control horses and ponies. D-Melanocyte–stimulating hormone (log and raw value) concentration was higher in photoperiod 2 through 8, compared with photoperiod 1, in control horses and ponies. In horses and ponies with PPID, log D-MSH concentration was higher in photoperiod 3 through 8, and D-MSH concentration was higher in photoperiod 4 through 8, compared with photoperiod 1. In control horses and ponies, plasma insulin concentration was lower in photoperiod 3 than in photoperiod 1.

Conclusions and Clinical Relevance—Plasma D-MSH and ACTH concentrations increased as daylight decreased from summer solstice (maximum daylight hours) to 12 hours of daylight.

During the past few years, there has been increasing recognition of the need for accurate endocrine tests for early detection of PPID in horses and ponies and also for determination of the influence of season on these test results.1–3 The usefulness of measuring ACTH or α-MSH concentrations to monitor status of disease, progression, or response to treatment has not been clearly defined, and consideration of any seasonal influence on hormones is of paramount importance when interpreting whether ACTH or α-MSH concentrations are within reference range or signify pituitary dysfunction.

The dexamethasone suppression test was originally considered the gold standard for diagnosis, but it is less reliable than initially reported and also can be affected by season.1,3,4,a Although results of 1 study3 reveal abnormal dexamethasone suppression test results only in horses with PPID, results of another study1 reveal abnormal results in healthy ponies and horses in September. In the latter study,1 all horses with cortisol concentrations ≥ 1 μg/dL following dexamethasone administration ranged in age from 13 to 25 years; therefore, it is possible that age or subclinical pituitary dysfunction could have affected results. In contrast, 2 semiferal ponies in the same study1 with abnormal seasonal responses were only 3 and 4 years old. In that study,1 a correlation was found between age of the horse and plasma cortisol concentration at the beginning and end of the dexamethasone suppression test; several horses were confirmed as having PPID within several years following the study.

Increasingly, plasma ACTH concentration has been used to screen horses for PPID, but the variability of results of single samples from PPID-affected horses and seasonal effects can lead to misinterpretation.1,3 Higher ACTH concentrations were reported for semiferal ponies and horses (at pasture with access to housing) in September, compared with those in January and May, and in some ponies, ACTH concentrations were greater than the reference range.1 Seasonal variations can affect not only the use of baseline ACTH as a diagnostic test but also its usefulness for monitoring horses with PPID. Although ACTH response to thyrotropin-releasing hormone stimulation is affected by season, seasonal effects did not change the classification of individual horses as clinically normal or as having PPID; however, this test is more cumbersome than use of basal samples.3

It has been suggested that measuring plasma α-MSH concentration may be superior to measuring plasma ACTH concentration to detect PPID because the former is primarily a product of the pars intermedia and ACTH is secreted primarily from the pars distalis.5,6,b Immunohistochemical staining has resulted in the detection of ACTH and α-MSH in the pars intermedia of clinically normal horses and those with PPID.7–10 α-Melanocyte–stimulating hormone, but not ACTH, is secreted from pars intermedia explants in response to thyrotropin-releasing hormone in vitro.6 A sensitivity of 88% and specificity of 85% were reported when a concentration of α-MSH ≥ 90 pmol/L was used as a cutoff value for clinically normal horses and PPID-affected horses, as defined by dexamethasone suppression test results.b However, the reliability of the dexamethasone suppression test has since been questioned,1,3 and to our knowledge, there have been no studies performed to determine whether α-MSH concentration is more sensitive or specific than ACTH concentration in the detection of PPID in horses. Like ACTH, α-MSH appears to be affected by season. This has been shown for stabled horses and semiferal ponies, with the latter being more affected.2 It is not clear whether the difference between horses and ponies is the result of a difference in management, body condition, location, or reproductive status or whether it reflects a true pony-versus-horse difference.

High plasma insulin concentrations and insulin resistance can be seen in horses and ponies with PPID.11,12 Plasma insulin concentrations have been shown to fluctuate throughout the day in unaffected horses13 and in those with PPID,12 and concentrations of insulin in ponies have been reported to be higher than in horses.14,15 Study results indicate that insulin sensitivity differs among breeds15 and is influenced by obesity and diet.16,17 The effect of season itself on plasma insulin concentrations has not been reported for clinically normal horses and ponies, although insulin concentrations were significantly higher in laminitis-prone ponies, compared with clinically normal ponies, in summer but not in winter.18 The range in insulin concentration appeared greater in the clinically normal ponies in summer, compared with winter.18

The purpose of the study reported here was to evaluate plasma ACTH, α-MSH, and insulin concentrations in clinically normal horses and ponies and also in those with pituitary dysfunction kept under similar management conditions in the same geographic region during different photoperiods. Differences among groups, the magnitude of any seasonal effect and when it was greatest, and whether measurements were outside the reference range during any of these times were evaluated.

Materials and Methods

Animals—Horses and ponies used in this study were classified as clinically normal (n = 27) or as having PPID (15), according to previously published criteria.3 Briefly, clinically normal horses and ponies had no evidence of PPID or other disease on physical examination, and horses and ponies with PPID had at least 2 of the classical clinical signs of the disease. None of the horses and ponies in this study underwent necropsy, but pituitary hyperplasia has been reported for horses clinically classified as having PPID.3 The study included 13 clinically normal ponies (control group ponies; 4 castrated males, 1 sexually intact male, and 8 females), 14 clinically normal horses (control group horses; 8 castrated males and 6 females), 7 ponies with PPID (PPID group ponies; 2 castrated males and 5 females), and 8 horses with PPID (PPID group horses; 3 castrated males and 5 females). Ages (mean ± SD) were 7.1 ± 2.5 years, 6.6 ± 1.6 years, 21.3 ± 4.3 years, and 24.5 ± 5.2 years for control group ponies, control group horses, PPID group ponies, and PPID group horses, respectively. All procedures in this study were approved by an institutional animal care and use committee, and owners of ponies and horses provided informed consent for testing.

Procedures—As previous studies1,2 revealed stable ACTH and α-MSH concentrations during times when the photoperiod is < 10 hours and higher concentrations in September than in January or May, blood samples were not obtained when length of daylight was < 10 hours in this study. A Web site19 was consulted, and photoperiod and dates of interest were selected. Blood sample collection photoperiod number, dates, length of day in hours, and mean daily temperature in degrees Celsius (Farenheit) were as follows: photoperiod 1, February 13 through March 2 (approx 10.5 hours; 1°C [34°F]); photoperiod 2, April 4 through 6 (13 hours; 4°C [39°F]); photoperiod 3, June 19 through 22 (15 hours; 22°C [72°F]); photoperiod 4, August 6 through 7 (14 hours; 27°C [81°F]); photoperiod 5, August 14 through 17 (13.5 hours; 25°C [77°F]); photoperiod 6, September 4 through 6 (13 hours; 24°C [75°F]); photoperiod 7, September 26 through 28 (12 hours; 23°C [73°F]); and photoperiod 8, October 16 through 18 (11 hours; 19°C [64°F]). The frequent sample collection times between August and October were selected to identify when peak secretion of ACTH and α-MSH occurred.

All plasma samples were assayed for ACTH and α-MSH. Insulin concentration was measured on plasma samples from 6 control group horses, 6 control group ponies, 8 PPID group horses, and 4 PPID group ponies. Four of the PPID group horses and 4 of the PPID group ponies were being treated with pergolide. Pergolidec dose ranged from 0.5 mg a day in ponies to 3 mg a day in horses, PO. All horses and ponies, except 6 of the PPID group horses that were housed on pasture with shelters at the university, were privately owned pleasure horses and ponies in Chester County (latitude, 39.8°N; longitude, 75.7°W). Management conditions were similar for all horses and ponies, horses and ponies were fed hay and grain (with the exception of 2 horses and 7 ponies), and all were turned out in pasture for varying amounts of time depending on the season. None of the ponies was fed grain prior to obtaining blood samples. Of the control group horses, only 1 consistently received grain, and 12 were fed grain prior to blood collection only in photoperiods 1 and 2. Of the PPID group horses, 3 received grain. The height, body weight calculated from a horse height-weight tape,d and BCSs20 were recorded at photoperiod 5. Although horses and ponies were not evaluated by weight tape at every photoperiod, the BCS was evaluated by the same technician at each sample collection photoperiod and did not change during the study.

Blood samples were obtained between 8:00 AM and 11:00 AM by the same technician with the horses and ponies in their home environment. Blood samples were collected by jugular venipuncture into evacuated glass tubes containing EDTA as an anticoagulante and centrifuged within 2 hours, and the plasma was transferred to polypropylene containers and frozen at −70°C. For ACTH and insulin assays, frozen plasma samples were placed on ice packs and sent by overnight mail to the New York State Animal Health Diagnostic Center at Cornell University. A sequential immunometric assay that used chemiluminescence for signal generation was used to measure plasma ACTH concentrations.f The assay for ACTH is generally specific for ACTH fragment 1 through 39, but has approximately 12% to 14% cross-reactivity with fragment 18 through 39. For the α-MSH assay, frozen plasma samples were sent on dry ice to the laboratory and measured by use of a commercially available radioimmunoassayg with a sensitivity of 3 pmol/L and intra- and interassay variation of 5% for high and low concentrations.2 Insulin (expressed in μIU/mL) was measured by means of a double antibody assay with 125-iodine labeled human insulin, guinea pig anti-porcine insulin primary anti-serum, and goat anti–guinea pig IgG second antibody with polyethylene glycolh that was validated for use in horses.i Laboratory reference range values were 9 to 35 pg/mL for ACTH and 10 to 40 μIU/mL for insulin. For α-MSH, reference concentrations were considered to be < 90 pmol/L.b

Statistical analysis—For comparison of plasma ACTH, α-MSH, and insulin concentrations at the different photoperiods and among groups, regression analysis with clustering on horses to ease the assumption of independence and the Bonferroni correction for multiple comparisons were used. In regard to establishment of a P value for hypothesis testing, when the Bonferroni correction was applied, a value of P < 0.003 was used; otherwise, a value of P ≤ 0.05 was used. Robust variance analysis was used to compare plasma hormone concentrations among all horses and all ponies, and to compare plasma insulin concentrations between ponies and horses with the same clinical diagnosis at different seasons. Kruskal-Wallis equality of populations rank test was used to analyze percentage change in plasma α-MSH and ACTH concentration in the different photoperiods, compared with photoperiod 1, and to detect whether percentage change varied between control group and PPID group horses and ponies. Body condition scores were compared among the different groups by use of the Mann-Whitney test. All analyses were performed with a commercially available software program,j and for parametric analysis, because of the wide variation in concentrations, hormonal data were normalized by conversion to a log scale for analysis.

Results

Body condition scores—Body condition scores were significantly higher in control group ponies (6.2 ± 1.4), compared with control group horses (4.9 ± 0.4), but not significantly different between PPID group horses and PPID group ponies (5.4 ± 0.9 and 5.4 ± 1.2, respectively). The BCS was not significantly different between control group horses and PPID group horses or between control group horses and PPID group ponies. Although BCS alone was positively correlated with plasma log α-MSH, α-MSH, and log insulin concentrations, BCS was not correlated with plasma ACTH or insulin concentrations, and the association between BCS and plasma log α-MSH, α-MSH, and log insulin concentrations was lost when seasonal influence was considered and when all horses were compared with all ponies.

Plasma ACTH, α-MSH, and insulin concentrations in control horses and ponies—For control group horses and ponies, plasma ACTH and log ACTH concentrations were significantly higher in photoperiod 4 through 8, compared with photoperiod 1 through 3; higher in photoperiod 7 than in photoperiod 8; and lower in photoperiod 2 than in photoperiod 1 (Tables 1 and 2). Plasma α-MSH and log α-MSH concentrations were significantly higher in photoperiods 2 through 8 than in photoperiod 1, higher in photoperiods 4 through 8 than in photoperiods 2 and 3; higher in photoperiods 6 and 7 than in photoperiod 4; higher in photoperiod 7 than in photoperiod 5; and higher in photoperiods 6 and 7 than in photoperiod 8. There were no significant seasonal changes in plasma insulin concentration except that log insulin concentration was lower in photoperiod 3 than in photoperiod 1. No significant differences in plasma hormone concentrations were seen between castrated males and sexually intact females except that, in photoperiod 3, the log ACTH concentration was lower in females. Baseline plasma concentrations for ACTH were within reference range (≤ 35 pg/mL) for all control group horses except for 1 horse in photoperiod 7 (58.1 pg/mL). Values were > 35 pg/mL in 1 control group pony in photoperiods 4 through 8 (65.3, 60.2, 64.8, 44.5, and 43.5 pg/mL, respectively); in another control group pony in photoperiods 4, 6, and 7 (60.9, 70.6, and 43 pg/mL, respectively); and in 1 control group pony in photoperiods 4 and 6 (38.6 and 87.3 pg/mL, respectively). In clinically normal horses and ponies, plasma α-MSH concentration appeared to have a more robust change with photoperiod, compared with plasma ACTH concentration (Figure 1). Plasma concentration of α-MSH was > 90 pmol/L in 1 control group horse in photoperiod 7, whereas concentrations > 90 pmol/L were seen in 3 control group ponies in photoperiod 4, 1 control group pony in photoperiod 5, 4 control group ponies in photoperiod 6, 5 control group ponies in photoperiod 7, and 3 control group ponies in photoperiod 8. Of the 16 plasma samples from the 8 control group ponies where plasma α-MSH concentration was > 90 pmol/L, the plasma ACTH concentration was > 36 pg/mL in only 9 samples. In the control group horse with a high plasma α-MSH concentration of 126.1 pmol/L, the plasma ACTH concentration was increased to 58.1 pg/mL.

Table 1—

Mean ± SD plasma ACTH (pg/mL), α-MSH (pmol/L), and insulin (MIU/mL) concentrations during various photoperiods in control horses and ponies and in horses and ponies with PPID.

VariableNo.Photoperiods*
12345678
Control
   ACTH2719.4 ± 5.7a17 ± 5.6b17.9 ± 6.1a,b26.6 ± 12.7c,d25.6 ± 9.2c,d32 ± 17c,d30.4 ± 11.2d24.3 ± 6.7c
   α-MSH275.5 ± 3.5a6.9 ± 3.4b9.9 ± 7.3b42.7 ± 31.7c,e42.9 ± 22.6c,e,f65.1 ± 44.3d,f78.2 ± 58.1d40.7 ± 27.4c
   Insulin1235 ± 29.332.8 ± 32.619 ± 18.137.2 ± 39.732.4 ± 34.225.4 ± 28.234.5 ± 32.131.4 ± 32.1
PPID
   ACTH1539.0 ± 38.7a34.1 ± 27.2a45.9 ± 31.1a,c93.7 ± 137.3a,b,c95.3 ± 105.3a,c180.6 ± 158.4b,d149.7 ± 140.2b,c67.7 ± 55.8b,c
   α-MSH1520.4 ± 26.2a20.2 ± 29.0a,c41.7 ± 46.9a,c,d112.4 ± 157.1b,c,d,e,f,g134.1 ± 109.8b,e236.4 ± 152.7f,g236.2 ± 161.6e,g123.5 ± 110.2b,d,f
   Insulin1233.8 ± 24.253.8 ± 52.345.4 ± 27.838.0 ± 29.540.1 ± 17.844.2 ± 32.043.0 ± 39.737.9 ± 21.7

See Materials and Methods section for blood sample collection dates, hours of daylight, and environmental temperature for each photoperiod. Absence of a shared superscript letter indicates a significant difference within a row. Rows without superscripts have no significantly different concentrations.

No. = Number of horses and ponies.

Table 2—

Mean ± SD plasma log ACTH (pg/mL), log α-MSH (pmol/L), and log insulin (MIU/mL) concentrations during various photoperiods in control horses and ponies and in horses and ponies with PPID.

VariableNo.Photoperiods*
12345678
Control
   Log ACTH272.93 ± 0.26a2.78 ± 0.32b2.83 ± 0.33a,b3.19 ± 0.41c,d3.19 ± 0.31c,d3.36 ± 0.43c,d3.35 ± 0.35d3.15 ± 0.27c
   Log α-MSH271.52 ± 0.63a1.82 ± 0.47b2.09 ± 0.66b3.49 ± 0.76c,e3.62 ± 0.54c,e,f4.02 ± 0.52d,f4.11 ± 0.72d3.49 ± 0.69c
   Log insulin123.20 ± 0.92a3.12 ± 0.87a,b2.64 ± 0.79b3.11 ± 1.05a,b3.07 ± 0.92a,b2.91 ± 0.76a,b3.23 ± 0.81a,b3.00 ± 0.98a,b
PPID
   Log ACTH153.35 ± 0.78a3.29 ± 0.69a3.62 ± 0.70a,c4.07 ± 0.86b,e,f4.20 ± 0.79b,e,f4.84 ± 0.89d4.69 ± 0.84d3.99 ± 0.69b,c,e
   Log α-MSH152.46 ± 1.05a2.35 ± 1.06a,b3.17 ± 1.11b4.21 ± 0.92d,f,g4.60 ± 0.80d,f,g5.22 ± 0.77e5.21 ± 0.81e,g4.48 ± 0.87d,f
   Log insulin123.27 ± 0.773.63 ± 0.883.62 ± 0.693.40 ± 0.703.57 ± 0.563.52 ± 0.823.41 ± 0.883.37 ± 0.88

See Table 1 for key.

Figure 1—
Figure 1—

Changes in mean plasma log MSH and log ACTH concentrations in control horses and ponies (n = 27) and horses and ponies with PPID (15) during various photoperiods. Plasma log α-MSH concentration was significantly (P < 0.05) different at all photoperiods between groups. Plasma log ACTH concentration was significantly (P < 0.05) different between the control group and PPID group in photoperiod 3 through 8.

Citation: Journal of the American Veterinary Medical Association 235, 6; 10.2460/javma.235.6.715

Plasma ACTH, α-MSH, and insulin concentrations in horses and ponies with PPID—For PPID group horses and ponies, plasma ACTH concentration was significantly higher in photoperiods 6 through 8, compared with photoperiod 1, and was less in photoperiods 3 and 5, compared with photoperiod 6 (Tables 1 and 2). The plasma log ACTH concentration was significantly higher in photoperiods 4 through 8 than in photoperiods 1 and 2; higher in photoperiods 4 through 7 than in photoperiod 3; higher in photoperiod 6 than in photoperiods 4, 5, and 8; and higher in photoperiod 7 than in photoperiod 8. The plasma α-MSH concentration was higher in photoperiods 4 through 8 than in photoperiod 1, higher in photoperiods 5 through 8 than in photoperiod 2, higher in photoperiods 5 through 7 than in photoperiod 3, higher in photoperiod 6 than in photoperiod 5, and higher in photoperiod 7 than in photoperiod 8. The plasma log α-MSH concentration was higher in photoperiods 3 through 8 than in photoperiod 1, higher in photoperiods 4 through 8 than in photoperiods 2 and 3, higher in photoperiods 6 and 7 than in photoperiods 4 and 8, and higher in photoperiod 6 than in photoperiod 5. Plasma insulin concentration did not significantly change with photoperiod. Of 67 samples from 12 horses and ponies with plasma ACTH concentrations exceeding the reference range by > 2 pg/mL, 42 samples had a plasma α-MSH concentration that was also high (1 of the 67 samples did not have an α-MSH measurement for comparison). There were too few horses and ponies and unequal sex distribution to adequately compare hormone concentrations of sexually intact females with those of castrated males.

Comparison of control group and PPID group horses and ponies—Plasma log ACTH concentration was significantly lower in control group horses and ponies than in PPID group horses and ponies in photoperiods 3 through 8. The plasma log α-MSH concentration was significantly lower in control group horses and ponies, compared with PPID group horses and ponies, in all photoperiods. Evaluation of control group and PPID group horses and ponies revealed that the plasma log α-MSH concentration increased relative to the plasma log ACTH value just prior to photoperiod 4 and remained higher through photoperiod 8 (Figure 1). When percentage changes in plasma α-MSH, ACTH, log α-MSH, and log ACTH concentrations were examined for the different photoperiods, despite wide variation among individual percentage changes, PPID group horses and ponies had significantly greater percent increases in plasma log ACTH and ACTH concentrations than control group horses and ponies in photoperiods 3 through 8. When compared with photoperiod 1, median percent change in plasma ACTH concentration between photoperiods 3 through 8 ranged from −7% to 48% for control horses and ponies and from 32% to 274% for PPID group horses and ponies. The median percent change in plasma log ACTH concentration ranged from −2% to 15% for the control group horses and ponies and from 11% to 40% for the PPID group horses and ponies in photoperiods 3 through 8. At no time did the percentage change in plasma α-MSH and log α-MSH concentrations differ between the 2 groups. When data were stacked to compare the relationship among groups over photoperiods, plasma log insulin concentrations were higher in PPID group horses and ponies, compared with control group horses and ponies, only in photoperiod 3.

Differences in plasma hormone concentrations between horses and ponies—When all ponies and all horses were compared, regardless of clinical status, the only significant differences were lower plasma ACTH and log insulin concentrations in photoperiod 1 and higher plasma α-MSH and log insulin concentrations in photoperiod 7 in ponies, compared with horses. Compared with control group horses, control group ponies had significantly higher plasma log α-MSH and α-MSH concentrations in photoperiods 4 through 8 and higher plasma ACTH and log ACTH concentrations in photoperiods 4 and in 6 through 8 (Table 3). In control group horses, plasma insulin and log insulin concentrations were significantly lower in photoperiods 4 and 6, compared with photoperiod 1, and lower than in control group ponies in photoperiod 3. When individual photoperiods were examined, no other significant differences were seen between horses and ponies despite a higher plasma insulin concentration in ponies. Plasma insulin concentrations were more variable in ponies than in horses. In 1 control group pony (BCS, 5), the plasma insulin concentration was ≥ 90 μIU/mL at every photoperiod. Plasma log insulin concentrations were higher in PPID group horses than control group horses overall and in all photoperiods except photoperiods 1 and 7. There was no difference in plasma insulin or log insulin concentrations between PPID group ponies and control group ponies.

Table 3—

Comparison of mean ± SD plasma ACTH (pg/mL), α-MSH (pmol/L), and insulin (MIU/mL) concentrations between control horses and ponies and horses and ponies with PPID during various photoperiods.

VariableNo.Photoperiods*
12345678
ACTH
   Cont horses1419.4 ± 4.216.5 ± 4.517.8 ± 5.121.9 ± 4.9a23.7 ± 5.224.8 ± 6.1a25.9 ± 10.6a21.4 ± 2.6a
  (2.9 ± 0.2)(2.8 ± 0.3)(2.8 ± 0.3)(3.1 ± 0.2a)(3.1 ± 0.2)(3.2 ± 0.3a)(3.2 ± 0.3a)(3.0 ± 0.1a)
   Cont ponies1319.9 ± 7.217.5 ± 6.718.1 ± 7.331.6 ± 16.5b27.8 ± 12.239.7 ± 21.4b35.2 ± 10.0b27.2 ± 8.2b
  (2.9 ± 0.33)(2.8 ± 0.4)(2.8 ± 0.4)(3.3 ± 0.5b)(3.2 ± 0.4)(3.5 ± 0.5b)(3.5 ± 0.3b)(3.2 ± 0.3b)
   PPID horses856.2 ± 47.245.5 ± 33.261.8 ± 33.6141.6 ± 178.5136.1 ± 132.9247.5 ± 170.8208.6 ± 169.694.1 ± 65.2
  (3.8 ± 0.8)(3.6 ± 0.8)(4 ± 0.6)(4.5 ± 0.9)(4.6 ± 0.8)(5.3 ± 0.7)(5.11 ± 0.7)(4.4 ± 0.5)
   PPID ponies719.4 ± 7.821.1 ± 8.227.8 ± 14.739.1 ± 16.948.6 ± 22.4104.0 ± 107.982.4 ± 48.937.6 ± 17.9
  (2.9 ± 0.5)(3 ± 0.4)(3.2 ± 0.6)(3.6 ± 0.5)(3.8 ± 0.5)(4.3 ± 0.8)(4.2 ± 0.7)(3.5 ± 0.6)
α-MSH
   Cont horses145.7 ± 2.86 ± 2.48.5 ± 8.725.3 ± 12.8a29.2 ± 11.6a38.5 ± 7.3a43.2 ± 28.4a24.5 ± 9.6a
  (1.6 ± 0.5)(2.7 ± 0.4)(1.9 ± 0.7)(3.1 ± 0.6a)(3.3 ± 0.4a)(3.6 ± 0.2a)(3.6 ± 0.5a)(3.1 ± 0.6a)
   Cont ponies135.2 ± 4.27.8 ± 4.111.5 ± 5.461.4 ± 35.5b58.8 ± 21.9b93.6 ± 49.9b115.9 ± 58.9b58.2 ± 29.7b
  (1.4 ± 0.5)(1.9 ± 0.4)(2.3 ± 0.7)(3.9 ± 0.6b)(4.0 ± 0.4b)(4.4 ± 0.2b)(4.6 ± 0.5b)(3.9 ± 0.6b)
   PPID horses831.5 ± 32.631.9 ± 36.548.8 ± 32.8168.0 ± 212.9167.0 ± 127.7306.4 ± 155.1279.1 ± 190.2164.3 ± 134.5
  (3 ± 1.1)(2.8 ± 1.2)(3.5 ± 0.9)(4.5 ± 1.1)(4.8 ± 0.9)(5.6 ± 0.7)(5.4 ± 0.7)(4.8 ± 0.8)
   PPID ponies77.7 ± 3.96.8 ± 3.733.5 ± 61.156.7 ± 30.496.5 ± 77.6156.3 ± 111.2187.3 ± 116.076.8 ± 49.8
  (1.9 ± 0.7)(1.8 ± 0.5)(2.7 ± 1.2)(3.9 ± 0.6)(4.4 ± 0.7)(4.8 ± 0.7)(5 ± 0.9)(4.1 ± 0.8)
Insulin
   Cont horses616.0 ± 7.814.5 ± 7.316.0 ± 5.6a10.8 ± 5.515.8 ± 6.812.5 ± 4.717.3 ± 12.113.7 ± 8.0
  (2.7 ± 0.5)(2.6 ± 0.5)(2.7 ± 0.4)(2.3 ± 0.5)(2.6 ± 0.5)(2.5 ± 0.4)(2.7 ± 0.6)(2.4 ± 0.7)
   Cont ponies653.9 ± 31.151.0 ± 38.422 ± 25.8b63.5 ± 42.149.0 ± 43.238.4 ± 36.351.8 ± 37.549.1 ± 38.0
  (3.7 ± 1)(3.7 ± 0.8)(2.5 ± 1.1)(4 ± 0.7)(3.5 ± 1.1)(3.4 ± 0.8)(3.8 ± 0.6)(3.6 ± 0.9)
   PPID horses839.3 ± 27.369.3 ± 58.354.3 ± 29.443.6 ± 33.742.4 ± 17.349.1 ± 34.641.9 ± 44.447.3 ± 18.5
  (3.4 ± 0.8)(3.9 ± 0.8)(3.8 ± 0.7)(3.5 ± 0.8)(3.6 ± 0.5)(3.7 ± 0.7)(3.4 ± 0.9)(3.7 ± 0.7)
   PPID ponies422.8 ± 13.022.8 ± 13.727.7 ± 14.027.0 ± 16.835.5 ± 20.334.3 ± 27.845.3 ± 34.219.3 ± 15.0
  (3 ± 0.7)(3 ± 0.7)(3.2 ± 0.6)(3.2 ± 0.5)(3.4 ± 0.7)(3.2 ± 1.0)(3.5 ± 1.0)(2.7 ± 0.8)

Values within parentheses are log concentration values.

Different superscript letters indicate a significant (P < 0.05) difference for that photoperiod between horses and ponies within their respective group (PPID or control).

Cont = Control group.

See Table 1 for remainder of key.

When ponies and horses with PPID were evaluated separately, ponies overall had lower plasma log ACTH and log α-MSH concentrations than horses, but when each of the different photoperiods was examined individually, no difference was seen between horses and ponies (Table 3). Both PPID group horses and ponies had an increased plasma log α-MSH concentration in photoperiods 3 through 8 and plasma log ACTH concentration in photoperiods 4 through 8, compared with photoperiod 1. There were no significant differences in plasma insulin concentrations between horses and ponies with PPID at any time. In the PPID group horses and ponies, plasma ACTH concentration was > 35 pg/mL by > 2 pg/mL in 52 of 64 samples from horses and 18 of 32 samples from ponies; plasma α-MSH concentration was > 90 pmol/L by > 2 pmol/L in 28 of 63 samples from horses and 19 of 32 samples from ponies.

Effect of treatment with pergolide—Of the PPID group horses and ponies, plasma ACTH and α-MSH concentrations were compared between horses and ponies receiving pergolide and not receiving pergolide. Horses and ponies receiving pergolide, compared with those not receiving pergolide, had significantly less of an increase in plasma log α-MSH concentration in photoperiods 7 and 8, significantly lower plasma log ACTH concentration during photoperiod 8, and a lower (but not significantly [P = 0.056] lower) value in photoperiod 7.

Discussion

Results of this study indicated that seasonal changes in plasma α-MSH and ACTH concentrations occured in clinically normal horses and ponies and in those with PPID. It was not possible to separate influence of environmental temperature from photoperiod, as its increase was associated with the photoperiod; however, there is no basis to suspect that environmental temperature would be a major influence. Although changes were greater in control group ponies, compared with control group horses, as previously reported,1,2 in this study, both plasma α-MSH and ACTH concentrations were greater than reference range values in fewer ponies and horses during September and to a lesser extent than in earlier reports.1,2 Although the reasons for the discrepancies are speculative, age, reproductive status, types of horses and ponies in the studies, management, and time of sample collection could all be contributing factors. In an earlier study,2 blood samples were obtained between 9:00 AM and 3:00 PM, whereas in this study, they were collected between 8:00 AM and 11:00 AM. Pulsatile secretion of α-MSH has been found for dogs, and circadian periodicity has been reported for cats.21–23 In horses, no significant differences have been measured in samples collected at 8:00 AM, 12:00 noon, or 4:00 PM,2 but pulsatile secretion of ACTH and α-MSH in a highly correlated manner has been detected in pituitary venous effluent of horses.24 Also, intensified rates of jugular venous sample collection in horses might reveal secretion pulses, as the former has been shown to unmask high-frequency proopiomelanocortin-peptide pulses in sheep.25 Although clinically normal horses and ponies in the current study were not screened by endocrinologic testing or necropsy examination to confirm a histologically normal pituitary, they were all younger than 11 years old, had no history to suggest endocrine dysfunction, and had no clinical signs of endocrine dysfunction at any time during the study. It was therefore considered unlikely that they had subclinical PPID. Ponies were mainly Welsh or Welsh crosses with access to pasture and stabling, thus differing from 2 previous reports1,2 in which ponies were semiferal and living with a herd at pasture. Although it had been anticipated that photoperiod might have a greater effect on hormone secretion in horses and ponies kept at pasture continuously, a seasonal effect was observed in ponies in the current study. Although the sex distribution of horses and ponies differed between this and the 2 earlier studies,1,2 it is unlikely to explain the differences in findings between studies, as no differences were seen between sexes in any of the studies. The BCS of the semiferal pony herd in the previous studies1,2 was not substantially different than that of the clinically normal ponies in this study and unlikely to explain differences in results.k Although an effect of BCS could potentially be an influence in our study, as the BCS of the clinically normal ponies was significantly higher than that of clinically normal horses, evaluation of a larger number of ponies and horses with low and high BCSs would be needed to ascertain whether BCS plays a role or whether the seasonal change is the result of some other factors peculiar to ponies.

Seasonal effects on α-MSH and ACTH appear to vary among species. Reports26,27 on seasonal effects in women have been inconsistent. Seasonal effects reported for horses have been limited to single samples obtained during long or short days, and measurements during potential transition photoperiods have not been reported. In contrast to studies in the United States, in a study from England,18 ponies in summer generally had ACTH concentrations within reference range but in winter had values above reference range.

In 1 study,28 Camarque horses had a significant increase in α-MSH concentrations as they aged, but in other breeds, no correlation with age or coat color was seen. Our study was not designed to evaluate the influence of age or body condition; in fact, we deliberately did not choose aged clinically normal horses or ponies, as we wished to avoid the inclusion of horses and ponies with possible subclinical pituitary dysfunction.

In horses and ponies with PPID and clinically normal horses and ponies in our study, plasma α-MSH concentration was lower than plasma ACTH concentration early in the year, but had a greater increase relative to ACTH just before photoperiod 4, which is just after the summer solstice (maximum daylight hours) and when daylight hours start to wane. Although findings of other studies1,2 reveal higher ACTH and α-MSH concentrations in September, our study is the first to evaluate hormone concentrations during multiple photoperiods between February and October and compare them with concentrations during 1 or 2 time points earlier in the year. Results of our study revealed that α-MSH and ACTH concentrations start increasing prior to August, peaking in the latter part of August and early to mid-September, and declining by October.

The reasons plasma α-MSH and ACTH concentrations are highest when length of daylight is waning from its longest period and then decrease and remain low when the photoperiod is short are unknown; whether the duration or amplitude of the melatonin signal during darkness is a factor is also unknown. Although there have been studies23,25–27 on melatonin signaling in other species, including sheep, studies29,30 on seasonal and photoperiod effects in horses are limited. It is possible that the increase in plasma ACTH and α-MSH concentrations results from decreasing hypothalamic dopaminergic inhibitory control. A decrease in periventricular dopaminergic neurons and a decrease in dopamine and its metabolites in the pars intermedia in horses with PPID have been reported and postulated to be important in explaining high proopiomelanocortin concentrations.31,32 Rams that have undergone surgical manipulation to disconnect the hypothalamus from the pituitary have hypersecretion of α-MSH thought to be attributed to loss of dopaminergic regulation by the hypothalamus.33,34 Whether a similar loss is occurring in horses and ponies is speculative. Results of a recent study30 reveal that plasma concentrations of dopamine are lower at certain times of the day in cushingoid horses, compared with age-matched clinically normal horses in March, June, and September but not in December. However, in our study, significant increases in plasma α-MSH and ACTH concentrations were not seen until after June 22, and this in addition to the seasonal changes seen in the clinically normal horses and ponies suggested that the circulating concentration of dopamine is not the only factor regulating hormone secretion.

It was of interest that direction of daylight change (ie, periods of daylight becoming shorter) and not length of photoperiod per se was the factor associated with changes in plasma ACTH and α-MSH concentrations in this study. In studies35,36 on goats, cortisol secretion was affected by the direction of the photoperiod change, and despite equal photoperiods, cortisol concentrations were higher in autumn than in spring, and the melatonin secretion rhythm was also slightly different.

Interestingly, the patterns of increase in plasma α-MSH and ACTH concentrations were similar in both horses and ponies with PPID and in clinically normal horses and ponies; the photoperiod regulation of the neuroendocrine axis appeared conserved in the horses and ponies with PPID. In another study,30 melatonin concentrations measured during different seasons and at different times of day also were not different between cushingoid and clinically normal horses. In our study, although the increase in plasma α-MSH concentration was initially more rapid in horses with PPID, compared with clinically normal horses, plasma α-MSH concentrations were greater than plasma ACTH concentrations at the same time in clinically normal horses and ponies and in horses and ponies with PPID (Figure 1). For other species, correlation and lack of correlation between α-MSH and ACTH concentrations have been reported.22,23,37

Results of our study indicated that insulin concentrations are minimally or not influenced by photoperiod. However, interpretation should be cautious, as numbers of horses and ponies in our study were small, and multiple factors affect insulin sensitivity in horses, including weight loss and exercise, which also may be affected by season.38 Also, only single samples were obtained, and single samples may not represent daily serum insulin concentrations. Daily fluctuations have been reported both for clinically normal horses and in horses with equine Cushing's syndrome, with the lowest concentration reported to be at 8:00 AM and peak at 12:00 noon in both groups.12,13 Larger numbers of horses with multiple samples collected daily with identical and controlled husbandry would probably be required to clearly define whether there is an effect. However, within the limits of this study, no major seasonal changes in plasma insulin concentrations were seen, which was clearly different from the pattern found for ACTH and α-MSH secretion.

Dopamine agonists have been documented to decrease secretion of α-MSH and ACTH in species, including horses.5,39,40 However, it is now recognized that season can influence results of monitoring ACTH concentrations in horses being treated, and this could confound evaluation of drug effect. The current study was not designed to evaluate the effect of pergolide, but the data suggested it affects the seasonal increase in α-MSH and ACTH in horses and ponies with PPID, and further study is warranted.

Results of this study indicated that seasonal variation in plasma ACTH and particularly α-MSH concentrations may confound monitoring horses and ponies for PPID. On the basis of these data, the reference range limits for plasma α-MSH concentration probably require better definition, with larger numbers of horses and ponies confirmed as clinically normal with other endocrine testing and by necropsy examination at different seasons. Until now, most reports of reference range values for hormones in horses and ponies have not specified season. It is conceivable that 90 pmol/L may be too high a value for plasma α-MSH concentration for distinguishing between clinically normal and affected horses and ponies during some seasons. Results of this study also indicated that reference range values for plasma ACTH, α-MSH, and insulin concentrations may be different for horses and ponies, and investigators should consider seasonal adjustment for ACTH and α-MSH. Further investigation is needed to understand the neuroendocrine regulation of pituitary hormone secretion in clinically normal horses and ponies and those with PPID.

ABBREVIATIONS

α-MSH

α-Melanocyte–stimulating hormone

BCS

Body condition score

PPID

Pituitary pars intermedia dysfunction

a.

Miesner TJ, Beard LA, Schmall SM, et al. Results of overnight dexamethasone suppression test repeated over time in horses suspected of having equine Cushing's disease (abstr). J Vet Intern Med 2003;17:420.

b.

Horowitz ML, Neal L, Watson JL. Characteristics of plasma adrenocorticotropin, B-endorphin and α-melanocyte stimulating hormone as diagnostic tests for pituitary pars intermedia dysfunction in the horse (abstr). J Vet Intern Med 2003;17:386.

c.

Wickliffe Pharmaceuticals, Lexington, Ky.

d.

The Coburn Co Inc, Whitewater, Wis.

e.

Becton-Dickinson, Franklin Lakes, NJ.

f.

Diagnostic Products Corp, Los Angeles, Calif.

g.

Euria α-MSH RIA, American Laboratory Products Co, Windham, NH.

h.

Linco/Millipore Corp, Billerica, Mass.

i.

Place N, Schanbacher BJ, Lamb S, Animal Health Diagnostic Center, Cornell University, Ithaca, NY: Unpublished data, 2007.

j.

Stata 9.2, StataCorp, College Station, Tex.

k.

Sue McDonnell, Adjunct Professor in Behavior and Reproduction, University of Pennsylvania, Kennett Square, Pa: Personal communication, 2008.

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  • Figure 1—

    Changes in mean plasma log MSH and log ACTH concentrations in control horses and ponies (n = 27) and horses and ponies with PPID (15) during various photoperiods. Plasma log α-MSH concentration was significantly (P < 0.05) different at all photoperiods between groups. Plasma log ACTH concentration was significantly (P < 0.05) different between the control group and PPID group in photoperiod 3 through 8.

  • 1.

    Donaldson MT, McDonnell SM, Schanbacher BJ, et al. Variation in plasma adrenocorticotropic hormone concentration and dexamethasone suppression test results with season, age, and sex in healthy ponies and horses. J Vet Intern Med 2005;19:217222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    McFarlane D, Donaldson MT, McDonnell SM, et al. Effects of season and sample handling on measurement of plasma D-melanocyte-stimulating hormone concentrations in horses and ponies. Am J Vet Res 2004;65:14631468.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Beech J, Boston R, Lindborg S, et al. Adrenocorticotropin concentration following administration of thyrotropin-releasing hormone in healthy horses and those with pituitary pars intermedia dysfunction and pituitary gland hyperplasia. J Am Vet Med Assoc 2007;231:417425.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Dybdal NO, Hargreaves KM, Madigan JE, et al. Diagnostic testing for pituitary pars intermedia dysfunction in horses. J Am Vet Med Assoc 1994;204:627632.

    • Search Google Scholar
    • Export Citation
  • 5.

    Orth DN, Holscher MA, Wilson MG, et al. Equine Cushing's disease: plasma immunoreactive proopiolipomelanocortin peptide and cortisol levels basally and in response to diagnostic tests. Endocrinology 1982;110:14301441.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    McFarlane D, Beech J, Cribb A. Alpha melanocyte stimulating hormone release in response to thyrotropin releasing hormone in healthy horses, horses with pituitary pars intermedia dysfunction and equine pars intermedia explants. Domest Anim Endocrinol 2006;30:276288.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Okada T, Shimomuro T, Oikawa M, et al. Immunocytochemical localization of adrenocorticotropic hormone-immunoreactive cells of the pars intermedia in Thoroughbreds. Am J Vet Res 1997;58:920924.

    • Search Google Scholar
    • Export Citation
  • 8.

    Yoshikawa H, Oishi H, Sumi A, et al. Spontaneous pituitary adenomas of the pars intermedia in 5 aged horses: histopathological, immunohistochemical and ultrastructural studies. J Equine Sci 2001;12:119126.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Orth DN, Nicholson WE. Bioactive and immunoreactive adrenocorticotropin in normal equine pituitary and in pituitary tumors of horse with Cushing's disease. Endocrinology 1982;111:559563.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Boujon CE, Bestetti GE, Meier HP, et al. Equine pituitary adenoma: a functional and morphological study. J Comp Pathol 1993;109:163178.

  • 11.

    Van der Kolk JH, Wensing T, Kalsbeek HC, et al. Laboratory diagnosis of equine pars intermedia adenoma. Domest Anim Endocrinol 1995;12:3539.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    McGowan CM, Frost R, Pfeiffer DV, et al. Serum insulin concentrations in horses, with equine Cushing's syndrome: response to a cortisol inhibitor and prognostic value. Equine Vet J 2004;36:295298.

    • Search Google Scholar
    • Export Citation
  • 13.

    Evans JW, Thompson PG, Winget CM. Glucose and insulin biorhythms in the horse. J S Afr Vet Assoc 1974;45:317329.

  • 14.

    Jeffcott LB, Field JR. Glucose tolerance and insulin sensitivity in ponies and Standardbred horses. Equine Vet J 1986;18:97101.

  • 15.

    Firshman AM, Valberg SJ. Factors affecting clinical assessment of insulin sensitivity in horses. Equine Vet J 2007;39:567575.

  • 16.

    Freestone JF, Beadle R, Shoemaker K, et al. Improved insulin sensitivity in hyperinsulinemic ponies through physical conditioning and controlled feed intake. Equine Vet J 1992;24:187190.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Hoffman RM, Boston RC, Stefanovski D, et al. Obesity and diet affect glucose dynamics and insulin sensitivity in Thoroughbred geldings. J Anim Sci 2003;81:23332342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Bailey SR, Habershon-Butcher JL, Ransom KJ, et al. Hypertension and insulin resistance in a mixed-breed population of ponies predisposed to laminitis. Am J Vet Res 2008;69:122129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Weather Underground Web site. Available at: www.wunderground.com. Accessed Mar 2007.

  • 20.

    Henneke DR, Potter GD, Krieder JL, et al. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Vet J 1983;15:371372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Kooistra HS, Greven SH, Mol JH, et al. Pulsatile secretion of D-MSH and the differential effects of dexamethasone and haloperidol on the secretion of D-MSH and ACTH in dogs. J Endocrinol 1997;152:113121.

    • Crossref
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