Blood lipid, glucose, and insulin concentrations in Morgan horses and Thoroughbreds

Scientific Papers Blood Lipid, Glucose, and Insulin Concentrations in Morgan Horses and Thoroughbreds REFEREED

Jenifer A. Nadeau,a Nicholas Frank,b Satyender R. Valipea, and Sarah B. Elliotb

ABSTRACT Insulin resistance has been detected in obese Morgan horses and it has been suggested that horses of this breed are predisposed to this condition. The objective of this study was to determine whether blood lipid, glucose, and insulin concentrations differed between Morgan horses and Thoroughbreds housed at the same facility. Fourteen Morgan horses (five mares, nine geldings) ranging in age from 4 to 14 years were compared with 21 Thoroughbreds (11 mares, 10 geldings; age range 7–20 years) from the same herd. A single blood sample was collected from each horse after grain was withheld overnight. Variables were compared between breed groups and breed-specific reference ranges were calculated. Triglyceride, cholesterol, nonesterified fatty acid, glucose, and insulin concentrations did not differ between breeds of horse in this study. This may be because horses included in this study did not suffer from obesity and were regularly exercised.

to equine metabolic syndrome (EMS), which has been associated with development of insulin resistance (IR) and laminitis.2 Because the predisposition to IR may be influenced by breed, blood lipid, glucose, and insulin concentrations may serve as indicators of breed-related differences in energy metabolism. This theory is supported by results of a study performed by Robie et al.1 in 1975 in which significantly higher serum total lipid concentrations were detected in Morgan horses. Thoroughbreds were selected for comparison as representatives of a hard-to-maintain breed. However, only eight Morgan horses and seven Thoroughbreds were compared in the aforementioned study and serum insulin concentrations were not measured. The objective of the study reported here was therefore to compare blood lipid, glucose, and insulin concentrations between Morgan horses and Thoroughbreds kept at the same location under similar conditions. MATERIALS AND METHODS

Key words: Insulin resistance; equine; Morgan; Thoroughbred; energy metabolism INTRODUCTION Morgan horses have been described as easy-tomaintain horses or “easy keepers” because they appear to require fewer calories to maintain body weight.1,2 Horses of this breed are speculated to be predisposed From the University of Connecticut, Department of Animal Science, Storrs, CTa; and the University of Tennessee, Department of Large Animal Clinical Sciences, Knoxville, TN.b Reprint requests: Jenifer A. Nadeau, University of Connecticut, Department of Animal Science, Unit 4040, 3636 Horsebarn Hill Rd. Ext., Storrs, CT 06269-4040. 0737-0806/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2006.07.005

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Selection of Horses The study protocol was approved by the University of Connecticut Animal Care and Use Committee. A total of 35 horses used for polo or moderate riding were studied. Fourteen Morgan horses (five mares, nine geldings) and 21 Thoroughbreds (11 mares, 10 geldings) were included in the study. Horses were either housed in stalls with turnout time (light riding horses) or stalls without consistent turnout time (polo horses). At the time of the study, 15 Thoroughbreds were included in the university’s polo program and were not being exercised regularly, whereas 14 Morgans and six Thoroughbreds were involved in the university’s riding program and were regularly exercised. All horses were normally fed orchard grass hay (6.5 kg/d) and a 14% protein concentrate (4.5 kg/d).

401

Table 1

Grain Hay, as fed

Composition of grain and hay fed to horses in this study

14

DE, Mcal/lb

% CP

% Ca

%P

1.34 0.76

14 9.32

0.6 0.41

0.5 0.19

12 10 8 6

Physical Measurements Body weights were estimated using a standard weight tape and body condition scores (BCS) were assigned using the 0 to 9 scale developed by Henneke et al.3 Ages were obtained from medical records.

4 2 0

Morgan

Blood Collection Horses were placed in box stalls bedded with pine shavings at 5:00 pm the night before sampling. Riding horses were exercised until 8:30 pm the night before samples were collected. Grain was withheld but grass hay was fed as normal (Table 1 lists the composition of grain and hay). Before blood collection, the horse’s body weight was determined using a Nutrena Horse and Pony Height–Weight Tape. For blood collection, horses were restrained in the stall with a halter and lead rope. Blood samples were collected between 7:00 am and 9:00 am by inserting a 1.5-inch 18-gauge needle into the jugular vein. Sixty milliliters of blood was withdrawn and then immediately transferred to blood tubes. Blood tubes were placed in crushed ice for 10 minutes and then transferred to a cooler with ice packs. Samples were transported to the laboratory within 2 hours and then centrifuged at 1000g in a refrigerated centrifuge for 20 minutes. Plasma or serum was transferred to plastic tubes and stored at ⫺20°C until analyzed.

Analysis of Plasma Lipids Concentrations of triglyceride (TG) and cholesterol were measured in plasma using enzymatic colorimetric reagents (Wako Chemicals, Richmond, VA) in an automated discrete analyzer (Cobas Mira, Roche Diagnostic Systems Inc, Somerville, NJ). Plasma nonesterified fatty acid (NEFA) concentrations were measured using an in vitro enzymatic colorimetric test kit (Wako) and a microtiter plate procedure.

Thoroughbred Breed

Figure 1. Scatterplot of serum insulin concentrations in 14 Morgan horses (5 mares, 9 geldings) and 21 Thoroughbreds (11 mares, 10 geldings).

insulin.4 Duplicate assays were performed on each sample and intra-assay coefficients of variation less than 5% were required for acceptance of results from both assays. The limit of detection for insulin was 1.2 ␮IU/ml. The reciprocal inverse square of basal insulin (RISQI) and the modified glucose-to-insulin ratio (MIRG) were calculated as previously described.5

Statistical Analysis Breed groups were compared using analysis of variance and a software program (SAS, version 9.1, SAS Institute Inc, Cary, NC ). Residuals were tested for normality, and a log transformation was used where needed. Least square means of the untransformed variables were compared with Fisher’s least significant difference (LSD). Reference ranges were determined nonparametrically as the 2.5th to 97.5th percentile. Significance was defined as P ⬍ .05.

Plasma glucose concentrations were measured using a colorimetric assay (Glucose, Roche Diagnostic Systems Inc) on the same analyzer. Insulin concentrations were determined using a radioimmunoassay (RIA; Coat-A-Count Insulin, Diagnostic Products Corp, Los Angeles, CA) previously validated for equine

RESULTS Breed groups differed (P ⬍ .001) with respect to age but not body weight or BCS (Table 2). Breed groups differed in exercise (P ⬍ .001) in that Morgans were exercised more than Thoroughbreds. Plasma or serum concentrations of TG, cholesterol, NEFA, glucose, and insulin concentrations did not differ (P ⬎ .05) between breed groups (Table 3). Serum insulin concentrations did not differ between breed groups (Fig. 1). RISQI and MIRG did not differ (P ⬎ .05) between breed groups (Table 4).

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Journal of Equine Veterinary Science

Measurement of Glucose and Insulin Concentrations

September 2006

Table 2

Physical measurements in 14 Morgan horses (5 mares, 9 geldings) and 21 Thoroughbreds (11 mares, 10 geldings)

Variable

Group

Mean

Median

Range

P

Age (years)

Morgan (n ⫽ 14) (n ⫽ 21)

8 13

6 13

4–14 7–20

⬍.001

Body weight (kg)a

Morgan (n = 14) (n = 21)

490 497

484 490

423–570 445–578

.544

BCSb

Morgan (n = 14) (n ⫽ 21)

4–6 4–6

.591

5.3 5.3

5.5 5.5

aEstimated by using Nutrena Horse and Pony Height-Weight Tape, 1978, Coburn Co. Inc. and formula lb body weight ⫽ [heart girth (in)2 ⫻ length (in)]/330. bBCS

⫽ body condition score (0 to 9 scale)3.

Table 3

Plasma lipid and glucose concentrations, and serum insulin concentrations for 14 Morgan horses (5 mares, 9 geldings) and 21 Thoroughbreds (11 mares, 10 geldings)

Variable

Group

Mean

Median

95% Reference Rangea

P

Triglyceride (mg/dl)

Morgan (n ⫽ 14) (n ⫽ 21)

17.5 16.5

17.0 16.7

11.1–31.6 7.4–25.8

.601

Cholesterol (mg/dl)

Morgan (n ⫽ 14) (n ⫽ 21)

98.6 99.0

97.4 96.0

78.1–125.8 78.8–124.5

.940

NEFAb (␮mol/l)

Morgan (n ⫽ 14) (n ⫽ 20)

106.2 150.5

94.4 133.4

8.8–259.0 6.3–551.3

.297

Glucose (mg/dl)

Morgan (n ⫽ 14) (n ⫽ 21)

71.5 74.8

73.8 73.8

59.8–82.0 66.6–87.6

.138

Insulin (␮U/ml)

Morgan (n ⫽ 14) (n ⫽ 21)

4.5 4.1

3.4 2.9

2.7–8.2 2.3–12.0

.593

aReference bNEFA

range determined nonparametrically as the 2.5th to 97.5th percentile.

⫽ nonesterified fatty acids.

Table 4

Calculated reciprocal inverse square of basal insulin (RISQI) and the modified glucose-to-insulin ratio (MIRG) for 14 Morgan horses (5 mares, 9 geldings) and 21 Thoroughbreds (11 mares, 10 geldings)

Calculation

Group

Mean

Median

95% Reference Rangea

P

RISQI

Morgan (n ⫽ 14) (n ⫽ 21)

0.49 0.54

0.52 0.59

0.35–0.61 0.29–0.66

.184

MIRG

Morgan (n ⫽ 14) (n ⫽ 21)

4.41 3.70

4.02 3.20

2.47–6.92 2.65–9.14

.152

aReference

range determined nonparametrically as the 2.5th to 97.5th percentile.

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DISCUSSION Morgan horses and Thoroughbreds did not differ with respect to plasma or serum concentrations of TG, cholesterol, NEFA, glucose, and insulin concentrations obtained from a single blood sample. RISQI and MIRG did not differ between breeds. Results do not provide evidence to support the hypothesis that these breeds of horse differ with respect to glucose or lipid metabolism. Horses included in this study were not obese (BCS range, 4–6) according to the BCS cutoff of 7 or less that has been used by others6 to determine obesity and some horses were being regularly exercised. These factors may have significantly impacted results. Obesity has previously been associated with insulin resistance in horses6 and insulin sensitivity improves in obese mares subjected to short-term exercise, independent of changes in body weight.7 In a recent study of obese insulin-resistant horses, three of the seven horses examined were of the Morgan horse breed, but none of the horses was regularly exercised.8 Serum insulin concentrations in single blood samples collected after overnight feed deprivation did not differ between breed groups. Differences in serum insulin concentrations may not have been detected when breed groups were compared because this measurement is a nonspecific indicator of insulin sensitivity in the horse.9 Specific measurements of insulin sensitivity could have been obtained using either the euglycemic hyperinsulinemic clamp (EHC) procedure or frequently sampled intravenous tolerance test (FSIGT). However, both of these tests are technically challenging and require a large number of samples.9 The combined glucose-insulin test (CGIT) provides a simpler nonspecific test for IR, but this test was not used in this study.10 Insulin sensitivity (SI) was estimated using proxies developed with SI values obtained from minimal model analysis of FSIGT data.5 RISQI and MIRG proxies are based on plasma glucose and serum insulin concentrations derived from data collected from 46 horses, including weanlings, geldings, and pregnant mares of the Thoroughbred or Arabian breeds.5 RISQI is an indicator of insulin sensitivity and MIRG is an indicator of insulin response. A low RISQI indicates low insulin sensitivity, whereas a low MIRG indicates a reduced capacity for ␤-cell secretion.5 Treiber et al5 reported a mean RISQI of 0.39 (mU/L)-0.5 and mean MIRG of 4.05 mUinsulin2/[10•L•mgglucose] for the 46 horses included in the study. Mean RISQI and MIRG values for horses in our study fell within the 5th and 3rd quintiles, respectively, of the reported quintiles. Plasma lipid concentrations did not differ between breed groups in this study and this finding contrasts

with results of a study reported by Robie et al.1 in which serum cholesterol and total lipid concentrations were higher (P ⬍ .05) in Morgan horses when compared with Thoroughbreds.1 However, these differences could be explained if Morgan horses included in the previous study were insulin resistant. Plasma TG concentrations are positively correlated (n ⫽ 31; r ⫽ 0.55; P ⫽ .002) with resting insulin concentrations in donkeys with hyperlipidemia.1,11 Some evidence suggests that the Morgan horses studied by Robie et al.1 were insulin resistant because they exhibited a delayed return of blood glucose concentrations to the preinjection level during intravenous glucose tolerance tests. Unfortunately, no further comparisons can be made because serum insulin concentrations were not reported. Reference ranges for plasma lipids calculated in this study compare favorably with previously reported mean plasma lipid concentrations. In a previous study, mean ⫾ SE plasma TG concentrations in four mares (Quarter Horse [n ⫽ 2], Thoroughbred, and Arabian) fed grass hay or feed-deprived for 36 hours and sampled every 4 hours were 57.1 ⫾ 1.4 mg/dl and 67.0 ⫾ 3.5 mg/dl, respectively.12 Mean NEFA concentrations of 46.4 ⫾ 2.1 ␮mol/l and 299.9 ⫾ 19.9 ␮mol/l, respectively, were detected in the same study. In another study, mean ⫾ SD plasma TG concentrations of 26.4 ⫾ 17 mg/dl, 87.1 ⫾ 111 mg/dl, 59.0 ⫾ 74 mg/dl, and 24.2 ⫾ 21 mg/dl were detected after 16 hours of feed deprivation in nonobese ponies, obese ponies, obese ponies with laminitis, and nonobese Standardbreds, respectively.13 Finally, in a study of mixed breed Quarter Horse type mares that were placed on different diets, baseline (week 0) mean plasma NEFA, TG, and cholesterol concentrations of 184.5 µmol/l, 33.7 mg/dl, and 79.0 mg/dl were detected after overnight feed deprivation.14 Our results do not provide evidence to support the hypothesis that Morgan horses differ from Thoroughbreds with respect to their glucose and lipid metabolism, but conclusions are limited by the subjects examined and study design. Obese or untrained horses were not included in this study and these animals are likely to differ with respect to their glucose and lipid metabolism from the horses studied here. It also should be recognized that insulin sensitivity was not directly measured in this study, so differences in glucose dynamics may be detected between breeds if a challenge test such as the FSIGT is used.

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Journal of Equine Veterinary Science

ACKNOWLEDGMENTS The authors acknowledge Dr. Arnold Saxton from the Department of Animal Science at the University of Tennessee for his assistance with the statistical analysis of these results.

September 2006

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